WorldWideScience

Sample records for cell blot technique

  1. Western Blot Techniques.

    Science.gov (United States)

    Kim, Brianna

    2017-01-01

    The Western blot is an important laboratory technique that allows for specific identification and characterization of proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins are electophoretically transferred to a polyvinylidene fluoride (PVDF) membrane which is then incubated with specific antibodies, then developed to show the protein of interest. Here, we describe the transfer and detection of Outer surface protein A (OspA), a protein only found on the surface of Borrelia burgdorferi, the bacteria responsible for Lyme disease.

  2. Single-cell western blotting.

    Science.gov (United States)

    Quadri, Syed M S

    2015-01-01

    Cell heterogeneity is a variation in cellular processes in functionally similar cells. Cells from the same tissue which are considered genetically identical may have difference in size, structure, and level of protein expression which can lead to major impact on the functions of cell leading to difference in physiological consequences. Single-cell proteome-wide studies are used to detect cell heterogeneity. Flow cytometry and immunocytochemistry do play an important role in evaluating cell heterogeneity. However, these methods are based on separation by antibodies with limited specificity. Cross-reactivity can occur leading to bias in result. Western blot is done to separate the proteins according to molecular weight. Therefore, off-target and on-target signals can be discriminated. Detection of protein expression from a tissue can be done with the help of western blot. However, it is unable to differentiate protein expression of individual cells. For detection of this cell-to-cell variation, a highly advanced technique termed "single-cell western blotting" is carried out. Single-cell western blot has enabled us to detect protein expression at cellular level at a fairly advanced high resolution using a western blot designed to assess cell heterogeneity.

  3. Western Blot: Technique, Theory, and Trouble Shooting

    OpenAIRE

    Mahmood, Tahrin; Yang, Ping-Chang

    2012-01-01

    Western blotting is an important technique used in cell and molecular biology. By using a western blot, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells. The technique uses three elements to accomplish this task: (1) separation by size, (2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody to visualize. This paper will attempt to explain the technique and theory behind western blo...

  4. Western blot: technique, theory, and trouble shooting.

    Science.gov (United States)

    Mahmood, Tahrin; Yang, Ping-Chang

    2012-09-01

    Western blotting is an important technique used in cell and molecular biology. By using a western blot, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells. The technique uses three elements to accomplish this task: (1) separation by size, (2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody to visualize. This paper will attempt to explain the technique and theory behind western blot, and offer some ways to troubleshoot.

  5. Protein purification and analysis: next generation Western blotting techniques.

    Science.gov (United States)

    Mishra, Manish; Tiwari, Shuchita; Gomes, Aldrin V

    2017-11-01

    Western blotting is one of the most commonly used techniques in molecular biology and proteomics. Since western blotting is a multistep protocol, variations and errors can occur at any step reducing the reliability and reproducibility of this technique. Recent reports suggest that a few key steps, such as the sample preparation method, the amount and source of primary antibody used, as well as the normalization method utilized, are critical for reproducible western blot results. Areas covered: In this review, improvements in different areas of western blotting, including protein transfer and antibody validation, are summarized. The review discusses the most advanced western blotting techniques available and highlights the relationship between next generation western blotting techniques and its clinical relevance. Expert commentary: Over the last decade significant improvements have been made in creating more sensitive, automated, and advanced techniques by optimizing various aspects of the western blot protocol. New methods such as single cell-resolution western blot, capillary electrophoresis, DigiWest, automated microfluid western blotting and microchip electrophoresis have all been developed to reduce potential problems associated with the western blotting technique. Innovative developments in instrumentation and increased sensitivity for western blots offer novel possibilities for increasing the clinical implications of western blot.

  6. Single cell-resolution western blotting.

    Science.gov (United States)

    Kang, Chi-Chih; Yamauchi, Kevin A; Vlassakis, Julea; Sinkala, Elly; Duncombe, Todd A; Herr, Amy E

    2016-08-01

    This protocol describes how to perform western blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state. Like conventional western blotting, single-cell western blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g., isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). The gel layer functions as both a molecular sieving matrix during PAGE and a blotting scaffold during immunoprobing. scWB involves five main stages: (i) gravity settling of cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. We also describe microdevice fabrication for both uniform and pore-gradient microgels. To extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. Once the microdevice has been fabricated, the assay can be completed in 4-6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. The technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine.

  7. Single-Cell Western Blotting.

    Science.gov (United States)

    Sinkala, Elly; Herr, Amy E

    2015-01-01

    Little headway has been made in single cell protein analysis, aside from tools that rely solely on antibody-probe based detection (i.e., flow cytometry, immunocytochemistry), which are limited by low specificity and multiplexing capabilities. To address these protein analysis gaps, we have introduced a single-cell western blot (scWestern). The protein assay is capable of highly specific analysis by coupling antibody-based detection with a polyacrylamide gel electrophoresis (PAGE) protein separation. Cells are settled via gravity into polyacrylamide (PA) microwells, chemically lysed in the wells, and then subjected to PAGE through the walls of the microwells and into the surrounding PA gel. Over a thousand single-cell separations are performed simultaneously, and multiple protein targets of interest are investigated. After PAGE separation, photo-immobilization of all proteins to the gel allows for antibody probing and lends to the archival quality of the scWestern assay where new proteins targets can be investigated months after the initial separations are performed.

  8. Subcellular western blotting of single cells.

    Science.gov (United States)

    Yamauchi, Kevin A; Herr, Amy E

    2017-01-01

    Although immunoassays are the de facto standard for determining subcellular protein localization in individual cells, antibody probe cross-reactivity and fixation artifacts remain confounding factors. To enhance selectivity while providing single-cell resolution, we introduce a subcellular western blotting technique capable of separately assaying proteins in the 14 pL cytoplasm and 2 pL nucleus of individual cells. To confer precision fluidic control, we describe a passive multilayer microdevice that leverages the rapid transport times afforded by miniaturization. After isolating single cells in microwells, we apply single-cell differential detergent fractionation to lyse and western blot the cytoplasmic lysate, whereas the nucleus remains intact in the microwell. Subsequently, we lyse the intact nucleus and western blot the nuclear lysate. To index each protein analysis to the originating subcellular compartment, we utilize bi-directional electrophoresis, a multidimensional separation that assays the lysate from each compartment in a distinct region of the separation axis. Single-cell bi-directional electrophoresis eliminates the need for semi-subjective image segmentation algorithms required in immunocytochemistry. The subcellular, single-cell western blot is demonstrated for six targets per cell, and successfully localizes spliceosome-associated proteins solubilized from large protein and RNA complexes, even for closely sized proteins (a 7 kDa difference). Measurement of NF-κB translocation dynamics in unfixed cells at 15-min intervals demonstrates reduced technical variance compared with immunofluorescence. This chemical cytometry assay directly measures the nucleocytoplasmic protein distribution in individual unfixed cells, thus providing insight into protein signaling in heterogeneous cell populations.

  9. Reliability of Blotting Techniques to Assess Contact Lens Water Content.

    Science.gov (United States)

    Cañadas, Pilar; López-Miguel, Alberto; Gómez, Alba; López-de la Rosa, Alberto; Fernández, Itziar; González-García, María J

    2018-02-15

    To determine the reliability of wet and modified dry blotting techniques used in the gravimetric method to assess contact lens (CL) water content (WC), the accuracy of both techniques in comparison with the nominal WC, and also their agreement. We evaluated hydrated and dry CL mass values and WC using the gravimetric method in 440 daily disposable CLs. Samples assessed corresponded to Dailies Total 1, Dailies AquaComfort Plus, 1-Day Acuvue TruEye, and Biotrue ONEday. Back vertex power ranged from +3.00 diopters (D) to -6.00 D. Within-subject coefficient of variation (CVw) and intraclass correlation coefficients were calculated. Bland-Altman analysis was also performed. The modified dry blotting technique yielded significantly (P≤0.0001) higher hydrated CL mass values. The wet blotting technique provided significantly (P≤0.04) better consistency than the modified dry one. Values of CVw for wet and modified dry blotting techniques ranged from 1.2% to 2.1% and from 3.7% to 5.4%, respectively. As for dry CL mass values, CVw values were not significantly different (P≥0.05) between wet (range: 1.1%-1.9%) and dry (range: 1.0%-5.1%) blotting techniques, except for Dailies AquaComfort Plus (P=0.03). Bland-Altman analysis showed poor agreement between the techniques. The wet blotting technique yielded WC values close (around 1%) to nominal ones, in contrast to modified dry blotting technique (≥2.5%). The wet blotting technique is not only more reliable than the modified dry one when obtaining hydrated CL mass but also provides more accurate nominal WC measurements. Agreement between the techniques was poor.

  10. A Study of Rubisco through Western Blotting and Tissue Printing Techniques

    Science.gov (United States)

    Ma, Zhong; Cooper, Cynthia; Kim, Hyun-Joo; Janick-Buckner, Diane

    2009-01-01

    We describe a laboratory exercise developed for a cell biology course for second-year undergraduate biology majors. It was designed to introduce undergraduates to the basic molecular biology techniques of Western blotting and immunodetection coupled with the technique of tissue printing in detecting the presence, relative abundance, and…

  11. Serological Diagnosis of Paracoccidioidomycosis through a Western Blot Technique

    Science.gov (United States)

    Perenha-Viana, M. C. Z.; Gonzales, I. A. A.; Brockelt, S. R.; Machado, L. N. C.

    2012-01-01

    Paracoccidioidomycosis (PCM) is a serious infectious disease that progresses toward death if untreated. Its confirmatory diagnosis is made by the detection of the fungus Paracoccidioides brasiliensis in a direct mycological examination or by histopathology. However, these techniques are of low sensitivity. Serological tests seem to be more promising. The objective of this study was to test Western blot (WB) analysis using sera from patients suspected of PCM to determine whether it represents a safe and sensitive serological technique for a rapid and effective diagnosis for this disease. Sera from 517 patients were analyzed through WB analysis and double-immunodiffusion (DID) techniques using a crude exoantigen of P. brasiliensis 339. DID gave positive reactions for 140 sera (27%) and WB for 250 sera (48.4%). All sera that had a positive reaction by DID also had a positive result with a 43-kDa glycoprotein by WB analysis. Among the 377 samples that were negative by DID, 29.1% were reactive in WB analysis. For the cutoff dilution used (1:400), a positive reaction was not observed with any of the 102 sera from patients with other diseases in regions where such diseases are endemic and 30 healthy individuals tested as negative controls. These results prove WB analysis to be a sensitive technique and suggest its inclusion among routine laboratory assays as a safe method for PCM diagnosis. PMID:22301695

  12. Serological diagnosis of paracoccidioidomycosis through a Western blot technique.

    Science.gov (United States)

    Perenha-Viana, M C Z; Gonzales, I A A; Brockelt, S R; Machado, L N C; Svidzinski, T I E

    2012-04-01

    Paracoccidioidomycosis (PCM) is a serious infectious disease that progresses toward death if untreated. Its confirmatory diagnosis is made by the detection of the fungus Paracoccidioides brasiliensis in a direct mycological examination or by histopathology. However, these techniques are of low sensitivity. Serological tests seem to be more promising. The objective of this study was to test Western blot (WB) analysis using sera from patients suspected of PCM to determine whether it represents a safe and sensitive serological technique for a rapid and effective diagnosis for this disease. Sera from 517 patients were analyzed through WB analysis and double-immunodiffusion (DID) techniques using a crude exoantigen of P. brasiliensis 339. DID gave positive reactions for 140 sera (27%) and WB for 250 sera (48.4%). All sera that had a positive reaction by DID also had a positive result with a 43-kDa glycoprotein by WB analysis. Among the 377 samples that were negative by DID, 29.1% were reactive in WB analysis. For the cutoff dilution used (1:400), a positive reaction was not observed with any of the 102 sera from patients with other diseases in regions where such diseases are endemic and 30 healthy individuals tested as negative controls. These results prove WB analysis to be a sensitive technique and suggest its inclusion among routine laboratory assays as a safe method for PCM diagnosis.

  13. Improved semiquantitative Western blot technique with increased quantification range.

    Science.gov (United States)

    Heidebrecht, F; Heidebrecht, A; Schulz, I; Behrens, S-E; Bader, A

    2009-06-30

    With the development of new interdisciplinary fields such as systems biology, the quantitative analysis of protein expression in biological samples gains more and more importance. Although the most common method for this is ELISA, Western blot also has advantages: The separation of proteins by size allows the evaluation of only specifically bound protein. This work examines the Western blot signal chain, determines some of the parameters relevant for quantitative analysis and proposes a mathematical model of the reaction kinetics. Using this model, a semiquantitative Western blot method for simultaneous quantification of different proteins using a hyperbolic calibration curve was developed. A program was written for the purpose of hyperbolic regression that allows quick determination of the calibration curve coefficients. This program can be used also for approximation of calibration curves in other applications such as ELISA, BCA or Bradford assays.

  14. Single-cell Western blotting after whole-cell imaging to assess cancer chemotherapeutic response.

    Science.gov (United States)

    Kang, Chi-Chih; Lin, Jung-Ming G; Xu, Zhuchen; Kumar, Sanjay; Herr, Amy E

    2014-10-21

    Intratumor heterogeneity remains a major obstacle to effective cancer therapy and personalized medicine. Current understanding points to differential therapeutic response among subpopulations of tumor cells as a key challenge to successful treatment. To advance our understanding of how this heterogeneity is reflected in cell-to-cell variations in chemosensitivity and expression of drug-resistance proteins, we optimize and apply a new targeted proteomics modality, single-cell western blotting (scWestern), to a human glioblastoma cell line. To acquire both phenotypic and proteomic data on the same, single glioblastoma cells, we integrate high-content imaging prior to the scWestern assays. The scWestern technique supports thousands of concurrent single-cell western blots, with each assay comprised of chemical lysis of single cells seated in microwells, protein electrophoresis from those microwells into a supporting polyacrylamide (PA) gel layer, and in-gel antibody probing. We systematically optimize chemical lysis and subsequent polyacrylamide gel electrophoresis (PAGE) of the single-cell lysate. The scWestern slides are stored for months then reprobed, thus allowing archiving and later analysis as relevant to sparingly limited, longitudinal cell specimens. Imaging and scWestern analysis of single glioblastoma cells dosed with the chemotherapeutic daunomycin showed both apoptotic (cleaved caspase 8- and annexin V-positive) and living cells. Intriguingly, living glioblastoma subpopulations show up-regulation of a multidrug resistant protein, P-glycoprotein (P-gp), suggesting an active drug efflux pump as a potential mechanism of drug resistance. Accordingly, linking of phenotype with targeted protein analysis with single-cell resolution may advance our understanding of drug response in inherently heterogeneous cell populations, such as those anticipated in tumors.

  15. Evaluation of an immunodot blot technique for the detection of antibodies against Taenia solium larval antigens.

    Science.gov (United States)

    Salazar-Anton, Fernando; Tellez, Aleyda; Lindh, Johan

    2012-06-01

    Immunodiagnostic tests represent an important tool for diagnosis of cysticercosis, the disease caused by cysticerci of Taenia solium. Accurate diagnosis of neurocysticercosis (NCC) requires costly neuroimaging techniques (magnetic resonance imaging and computed tomography), which are seldom affordable for people in endemic countries. Hence, new low-cost diagnostic methods offering good sensitivity and specificity are needed. Here, we studied four immunodiagnostic tests immunodot blot Tsol-p27, a commercial ELISA, and Western blot Tsol-p27/TsolHSP36, and compared them with a commercial enzyme-linked immunoelectrotransfer blot (EITB) that we regarded as the gold standard method. The analyzed serum samples were obtained from 160 patients: 94 epileptics suspected of NCC, six individuals confirmed NCC-positive, and 60 with positive (30) or negative (30) serology for Chagas diseases. Of the 100 serum samples from epileptic patients, 13 were positive and 87 negative by EITB. Compared to Western blot Tsol-p27, immunodot blot Tsol-p27 offered similar specificity (97.8% vs. 95.6%) but better sensitivity (86.7% vs. 76.4%). The ELISA was similar to the immunodot blot Tsol-p27 regarding both sensitivity and specificity. Western blot TsolHSP36 provided the lowest sensitivity (61.9%) and specificity (86.1%). None of the antibodies in the serum samples from the Chagas control groups were recognized by immunodot blot Tsol-p27. Our results indicate that the immunodot blot Tsol-p27 provides good sensitivity and specificity. Furthermore, considering the simplicity and low cost of this test, it might be preferable as a diagnostic method in poorly equipped laboratories in endemic countries.

  16. Validation of a Dot-Blot quantitative technique for large scale analysis of beef tenderness biomarkers.

    Science.gov (United States)

    Guillemin, N; Meunier, B; Jurie, C; Cassar-Malek, I; Hocquette, J-F; Leveziel, H; Picard, B

    2009-10-01

    Beef tenderness is a very complex and multifactorial sensorial meat quality trait, which depends partly on muscle characteristics. This tissue is very variable according to animal type (age, breed and sex) and rearing conditions. Consequently, beef tenderness exhibits a great variability. Different research programs have revealed several genes or proteins which could be good markers of beef tenderness. In order to validate the relation of these markers with beef tenderness on a large population of bovines, it is necessary to have a large-scale and trusty technique which can access different quantities of proteins related to tenderness. In this study we firstly compared Western-Blot and Dot-Blot. Secondly, we evaluated Dot-Blot technical and biological capabilities for the quantification of protein biomarkers. The results demonstrated that the Dot-Blot technique with fluorescence detection presents numerous interests. This technique allows a good reproducibility and permits the simultaneous analysis of a large number of samples. The Dot-Blot technique defined and validated in this study can be used for protein biomarkers analyses, notably to predict beef tenderness. Another major result of this study is that about 5 to 10 animals per group are required to detect large differences (>1.5) in biomarker expression between tender and tough beef, whereas much larger numbers of animals (10 to 30) are required to detect smaller differences (about 1.2 to 1.3) taking into account the biological variability of these markers.

  17. Western blot analysis of cells encapsulated in self-assembling peptide hydrogels.

    Science.gov (United States)

    Burgess, Kyle A; Miller, Aline F; Oceandy, Delvac; Saiani, Alberto

    2017-12-01

    Continuous optimization of in vitro analytical techniques is ever more important, especially given the development of new materials for tissue engineering studies. In particular, isolation of cellular components for downstream applications is often hindered by the presence of biomaterials, presenting a major obstacle in understanding how cell-matrix interactions influence cell behavior. Here, we describe an approach for western blot analysis of cells that have been encapsulated in self-assembling peptide hydrogels (SAPHs), which highlights the need for complete solubilization of the hydrogel construct. We demonstrate that both the choice of buffer and multiple cycles of sonication are vital in obtaining complete solubilization, thereby enabling the detection of proteins otherwise lost to SAP aggregation. Moreover, we show that the presence of self-assembling peptides (SAPs) does not interfere with the standard immunoblotting technique, offering the potential for use in more full-scale proteomic studies.

  18. Evaluation of an immunodot blot technique for the detection of antibodies against Taenia solium larval antigens

    OpenAIRE

    Salazar-Anton, Fernando; Tellez, Aleyda; Lindh, Johan

    2012-01-01

    Immunodiagnostic tests represent an important tool for diagnosis of cysticercosis, the disease caused by cysticerci of Taenia solium. Accurate diagnosis of neurocysticercosis (NCC) requires costly neuroimaging techniques (magnetic resonance imaging and computed tomography), which are seldom affordable for people in endemic countries. Hence, new low-cost diagnostic methods offering good sensitivity and specificity are needed. Here, we studied four immunodiagnostic tests immunodot blot Tsol-p27...

  19. Preparation of Cell Lysate from Mouse Oocytes for Western Blotting Analysis.

    Science.gov (United States)

    Marangos, Petros

    2016-01-01

    Western Blotting has been used extensively for the identification of the protein factors that regulate mammalian oocyte meiosis. However, the limitations in collecting sufficient numbers of oocytes can hinder the efficiency of the technique. Here we provide a detailed protocol for the accurate preparation of mouse oocyte samples for Western Blotting analysis.

  20. The Western blot is a highly sensitive and efficient technique in diagnosing allergy to wasp venom.

    Science.gov (United States)

    Zollner, T M; Spengler, K; Podda, M; Ergezinger, K; Kaufmann, R; Boehncke, W H

    2001-11-01

    life threatening disease, false-negative test results need to be minimized. Therefore, the superiority of the Western blot with regard to sensitivity, specificity and overall efficiency makes this technique a valuable tool for its diagnosis.

  1. A comparison of the immune parameters of dogs infected with visceral leishmaniasis using Western blot and neutralization techniques.

    Science.gov (United States)

    Nogueira, Yeda L; Odorizzi, Rosa M F N; Nakamura, Paulo M

    2007-01-01

    The Western blot technique was used to demonstrate the presence of antibodies in the blood of dogs that presented canine visceral leishmaniasis. This technique was used against some specific molecules present in the lysate of the promastigote form of Leshmania chagasi. Through the association of the results of the Western blot technique with the morphological alterations seen as a result of the serum neutralization technique performed in McCoy cells (which mimetizes the macrophage) it was possible to observe the role of some molecules of great relevance in determining the disease in symptomatic dogs as well as that of some other molecules associated with asymptomatic infected dogs that may become transmitters as well as differentiating them as asymptomatic resistant dogs. In the sera analyses carried out during the immunobloting a variation of 9 to 27 immunoreacting bands was observed, which were then compared using Dice's similarity coefficient. In the dendrogram constructed on the basis of the coefficient, 50% similarity was observed among the total number of reagent bands with the promastigote lysate, thus creating five groups. The main difference observed related to the clinical condition of the dogs: symptomatic and asymptomatic dogs were found in separate groups. The asymptomatic group of dogs was distributed in two different places in the dendrogram because they presented two different behavior patterns regarding the cellular morphology in the serum neutralization reaction: the presence or absence of cellular lysis. According to this analysis it is possible to evaluate the immune status and associate it with specific markers observed in the reaction found in the Western blot strips.

  2. HOPE technique enables Western blot analysis from paraffin-embedded tissues.

    Science.gov (United States)

    Uhlig, U; Uhlig, S; Branscheid, D; Zabel, P; Vollmer, E; Goldmann, T

    2004-01-01

    In contrast to the spectrum of biochemical analyses of fresh material, that of archived specimens is widely restricted. Fixation of specimens with formalin, the most commonly used fixative, usually prevents further molecular analysis, since it leads to degradation of nucleic acids and denaturation of the antigenic determinants of proteins. To overcome these problems, the Hepes-glutamic acid buffer mediated Organic solvent Protection Effect (HOPE)-fixation technique has been developed, which preserves nucleic acids and antigenic determinants of proteins, thus expanding the applicability of immunohistochemical methods. In this study, we investigated whether HOPE-fixed tissue can be analyzed by Western blotting. Furthermore, a comparison with conventionally fixed and frozen material was made. The specimens used were tumor-free and obtained from lobectomies for lung cancer. All four antibodies tested, i.e., antibodies specific for focal adhesion kinase, surfactant protein A, PI-3-kinase, and IKKalpha, worked well if used for immunoblotting of HOPE-fixed and frozen tissue. By contrast, these antibodies showed no or only very weak specific binding if formalin-fixed specimens were analyzed. Our findings show that HOPE fixation maintains the antigenicity of proteins better than formalin fixation. The possibility for performing Western blotting with archived paraffin-embedded specimens extends the options for diagnostic and scientific analyses of fixed tissues.

  3. A Western blot and molecular genetic investigation of the estrogen receptor beta in giant cell arteritis.

    Science.gov (United States)

    Larsson, K; Nordborg, C; Moslemi, A-R; Nordborg, E

    2006-01-01

    The epidemiology of giant cell arteritis (GCA) may indicate a pathogenetic relationship between GCA and female sex hormone metabolism; GCA is two to four times more common in women compared with men. Our previous analyses gave no support for the hypothesis that the pathogenesis of GCA should be related to somatic mutations in the estrogen receptor alpha (ERalpha) gene. The object of the present study was to investigate the size of the estrogen receptor beta (ERBeta), and the size and nucleotide sequence of the ERBeta gene in temporal arteries in GCA. The ERBeta protein was analyzed by Western blot technique and the ERBeta gene by RT-PCR and direct sequencing of the PCR product. Western blot analysis revealed an ERBeta of normal size. There were no aberrations in size or nucleotide sequence in the ERBeta gene in the GCA patients. The present observations gave no support for the hypothesis that somatic mutations in the ERBeta gene should be involved in the pathogenesis of GCA.

  4. Western blotting as a method for studying cell-biomaterial interactions : The role of protein collection

    NARCIS (Netherlands)

    van Kooten, T.G.; Klein, CL; Kirkpatrick, CJ

    2001-01-01

    Research of cell-biomaterial interactions is building on knowledge and methods available in cell and molecular biology. Western blotting is one of the options to characterize protein expression in cell populations. Method transfer to biomaterial model systems is not trivial because of the structure

  5. Identification of Yeast V-ATPase Mutants by Western Blots Analysis of Whole Cell Lysates

    Science.gov (United States)

    Parra-Belky, Karlett

    2002-11-01

    A biochemistry laboratory was designed for an undergraduate course to help students better understand the link between molecular engineering and biochemistry. Students identified unknown yeast strains with high specificity using SDS-PAGE and Western blot analysis of whole cell lysates. This problem-solving exercise is a common application of biochemistry in biotechnology research. Three different strains were used: a wild-type and two mutants for the proton pump vacuolar ATPase (V-ATPase). V-ATPases are multisubunit enzymes and the mutants used were deletion mutants; each lacked one structural gene of the complex. After three, three-hour labs, mutant strains were easily identified by the students and distinguished from wild-type cells analyzing the pattern of SDS-PAGE distribution of proteins. Identifying different subunits of one multimeric protein allowed for discussion of the structure and function of this metabolic enzyme, which captured the interest of the students. The experiment can be adapted to other multimeric protein complexes and shows improvement of the described methodology over previous reports, perhaps because the problem and its solution are representative of the type of techniques currently used in research labs.

  6. High-selectivity cytology via lab-on-a-disc western blotting of individual cells.

    Science.gov (United States)

    Kim, John J; Sinkala, Elly; Herr, Amy E

    2017-02-28

    Cytology of sparingly available cell samples from both clinical and experimental settings would benefit from high-selectivity protein tools. To minimize cell handling losses in sparse samples, we design a multi-stage assay using a lab-on-a-disc that integrates cell handling and subsequent single-cell western blotting (scWestern). As the two-layer microfluidic device rotates, the induced centrifugal force directs dissociated cells to dams, which in turn localize the cells over microwells. Cells then sediment into the microwells, where the cells are lysed and subjected to scWestern. Taking into account cell losses from loading, centrifugation, and lysis-buffer exchange, our lab-on-a-disc device handles cell samples with as few as 200 cells with 75% cell settling efficiencies. Over 70% of microwells contain single cells after the centrifugation. In addition to cell settling efficiency, cell-size filtration from a mixed population of two cell lines is also realized by tuning the cell time-of-flight during centrifugation (58.4% settling efficiency with 6.4% impurity). Following the upstream cell handling, scWestern analysis detects four proteins (GFP, β-TUB, GAPDH, and STAT3) in a glioblastoma cell line. By integrating the lab-on-a-disc cell preparation and scWestern analysis, our platform measures proteins from sparse cell samples at single-cell resolution.

  7. Use of a Western blot technique for the serodiagnosis of glanders

    OpenAIRE

    Elschner, Mandy C; Scholz, Holger C; Melzer, Falk; Saqib, Muhammad; Marten, Peggy; Rassbach, Astrid; Dietzsch, Michael; Schmoock, Gernot; de Assis Santana, Vania L; de Souza, Marcilia MA; Wernery, Renate; Wernery, Ulrich; Neubauer, Heinrich

    2011-01-01

    Abstract Background The in vivo diagnosis of glanders relies on the highly sensitive complement fixation test (CFT). Frequently observed false positive results are troublesome for veterinary authorities and cause financial losses to animal owners. Consequently, there is an urgent need to develop a test with high specificity. Hence, a Western blot assay making use of a partly purified lipopolysaccaride (LPS) containing antigen of three Burkholderia mallei strains was developed. The test was va...

  8. Use of a Western blot technique for the serodiagnosis of glanders

    Directory of Open Access Journals (Sweden)

    de Souza Marcilia MA

    2011-01-01

    Full Text Available Abstract Background The in vivo diagnosis of glanders relies on the highly sensitive complement fixation test (CFT. Frequently observed false positive results are troublesome for veterinary authorities and cause financial losses to animal owners. Consequently, there is an urgent need to develop a test with high specificity. Hence, a Western blot assay making use of a partly purified lipopolysaccaride (LPS containing antigen of three Burkholderia mallei strains was developed. The test was validated investigating a comprehensive set of positive and negative sera obtained from horses and mules from endemic and non endemic areas. Results The developed Western blot assay showed a markedly higher diagnostic specificity when compared to the prescribed CFT and therefore can be used as a confirmatory test. However, the CFT remains the test of choice for routine testing of glanders due to its high sensitivity, its feasibility using standard laboratory equipment and its worldwide distribution in diagnostic laboratories. Conclusions The CFT should be amended by the newly validated Western blot to increase the positive likelihood ratio of glanders serodiagnosis in non endemic areas or areas with low glanders prevalence. Its use for international trade of horses and mules should be implemented by the OIE.

  9. Use of a Western blot technique for the serodiagnosis of glanders.

    Science.gov (United States)

    Elschner, Mandy C; Scholz, Holger C; Melzer, Falk; Saqib, Muhammad; Marten, Peggy; Rassbach, Astrid; Dietzsch, Michael; Schmoock, Gernot; de Assis Santana, Vania L; de Souza, Marcilia M A; Wernery, Renate; Wernery, Ulrich; Neubauer, Heinrich

    2011-01-19

    The in vivo diagnosis of glanders relies on the highly sensitive complement fixation test (CFT). Frequently observed false positive results are troublesome for veterinary authorities and cause financial losses to animal owners. Consequently, there is an urgent need to develop a test with high specificity. Hence, a Western blot assay making use of a partly purified lipopolysaccaride (LPS) containing antigen of three Burkholderia mallei strains was developed. The test was validated investigating a comprehensive set of positive and negative sera obtained from horses and mules from endemic and non endemic areas. The developed Western blot assay showed a markedly higher diagnostic specificity when compared to the prescribed CFT and therefore can be used as a confirmatory test. However, the CFT remains the test of choice for routine testing of glanders due to its high sensitivity, its feasibility using standard laboratory equipment and its worldwide distribution in diagnostic laboratories. The CFT should be amended by the newly validated Western blot to increase the positive likelihood ratio of glanders serodiagnosis in non endemic areas or areas with low glanders prevalence. Its use for international trade of horses and mules should be implemented by the OIE.

  10. Evaluating cytoplasmic and nuclear levels of inflammatory cytokines in cancer cells by western blotting.

    Science.gov (United States)

    Gatla, Himavanth R; Singha, Bipradeb; Persaud, Valerie; Vancurova, Ivana

    2014-01-01

    Increased expression and cellular release of inflammatory cytokines, interleukin-8 (IL-8; CXCL8), and high mobility group box-1 (HMGB1) are associated with increased cell proliferation, angiogenesis, and metastasis during cancer progression. In prostate and ovarian cancer cells, increased levels of IL-8 and HMGB1 correlate with poor prognosis. We have recently shown that proteasome inhibition by bortezomib (BZ) specifically increases IL-8 release from metastatic prostate and ovarian cancer cells. In this chapter, we describe a protocol to analyze the cytoplasmic and nuclear levels of IL-8 and HMGB1 in prostate and ovarian cancer cells by western blotting. IL-8 is localized in the cytoplasm in both cell types, and its protein levels are significantly increased by BZ. In contrast, HMGB1 is localized in the nucleus, and BZ increases its nuclear levels only in ovarian cancer cells. The protocol includes isolation of cytoplasmic and nuclear extracts, followed by SDS electrophoresis and western blotting, and can be easily modified to analyze the cytoplasmic and nuclear cytokine levels in other cell types.

  11. Important considerations for protein analyses using antibody based techniques: down-sizing Western blotting up-sizes outcomes.

    Science.gov (United States)

    Murphy, Robyn M; Lamb, Graham D

    2013-12-01

    Western blotting has been used for protein analyses in a wide range of tissue samples for >30 years. Fundamental to Western blotting success are a number of important considerations, which unfortunately are often overlooked or not appreciated. Firstly, lowly expressed proteins may often be better detected by dramatically reducing the amount of sample loaded. Single cell (fibre) Western blotting demonstrates the ability to detect proteins in small sample sizes, 5-10 μg total mass (1-3 μg total protein). That is an order of magnitude less than often used. Using heterogeneous skeletal muscle as the tissue of representation, the need to undertake Western blotting in sample sizes equivalent to single fibre segments is demonstrated. Secondly, incorrect results can be obtained if samples are fractionated and a proportion of the protein of interest inadvertently discarded during sample preparation. Thirdly, quantitative analyses demand that a calibration curve be used. This is regardless of using a loading control, which must be proven to not change with the intervention and also be appropriately calibrated. Fourthly, antibody specificity must be proven using whole tissue analyses, and for immunofluorescence analyses it is vital that only a single protein is detected. If appropriately undertaken, Western blotting is reliable, quantitative, both in relative and absolute terms, and extremely valuable.

  12. Laser capture microdissection of pancreatic ductal adeno-carcinoma cells to analyze EzH2 by Western Blot analysis.

    Science.gov (United States)

    Qazi, Aamer M; Aggarwal, Sita; Steffer, Christopher S; Bouwman, David L; Weaver, Donald W; Gruber, Scott A; Batchu, Ramesh B

    2011-01-01

    Pure populations of tumor cells are essential for the identification of tumor-associated proteins for the development of targeted therapy. In recent years, laser capture microdissection (LCM) has been used successfully to obtain distinct populations of cells for subsequent molecular analysis. The polycomb group (PcG) protein, enhancer of zeste homolog 2 (EzH2), a methyl-transferase that plays a key role in -transcriptional gene repression, is frequently overexpressed in several malignant tumors. High levels of EzH2 are often associated with advanced disease stage in many solid tumors; however, its role in the pathogenesis of pancreatic ductal adeno-carcinoma (PDAC) is poorly understood. Because of the limited sample availability and the absence of in vitro amplification steps for proteins, the use of LCM for proteomics studies largely depends on highly sensitive protein detection methods. Here, we developed a faster and sensitive Western blot protocol and validated it for the detection of EzH2 in ∼2,000 cells. Initially, cultured PANC-1 cells were used to optimize protein electrophoresis and western blotting conditions. Gradient gel electrophoresis in combination with optimized antibody concentrations, and a sensitive chemiluminescent assay provided a strong signal. In order to further confirm the role of EzH2 in PDAC, employing siRNA-mediated gene silencing via long lasting plasmid vectors containing shRNA, we investigated the potential role of EzH2 gene silencing in pancreatic cancer regression. Positive correlation of EzH2 expression was observed with advanced stage, serous histology, and increasing grade in pancreatic cancer patient tissues. Further EzH2 knockdown resulted in decreased cell growth and invasiveness. The findings of this study emphasize that western blotting of a LCM-generated pure population of cancer cells may be a valuable technique for the study of tumor-specific proteins.

  13. Profiling protein expression in circulating tumour cells using microfluidic western blotting.

    Science.gov (United States)

    Sinkala, Elly; Sollier-Christen, Elodie; Renier, Corinne; Rosàs-Canyelles, Elisabet; Che, James; Heirich, Kyra; Duncombe, Todd A; Vlassakis, Julea; Yamauchi, Kevin A; Huang, Haiyan; Jeffrey, Stefanie S; Herr, Amy E

    2017-03-23

    Circulating tumour cells (CTCs) are rare tumour cells found in the circulatory system of certain cancer patients. The clinical and functional significance of CTCs is still under investigation. Protein profiling of CTCs would complement the recent advances in enumeration, transcriptomic and genomic characterization of these rare cells and help define their characteristics. Here we describe a microfluidic western blot for an eight-plex protein panel for individual CTCs derived from estrogen receptor-positive (ER+) breast cancer patients. The precision handling and analysis reveals a capacity to assay sparingly available patient-derived CTCs, a biophysical CTC phenotype more lysis-resistant than breast cancer cell lines, a capacity to report protein expression on a per CTC basis and two statistically distinct GAPDH subpopulations within the patient-derived CTCs. Targeted single-CTC proteomics with the capacity for archivable, multiplexed protein analysis offers a unique, complementary taxonomy for understanding CTC biology and ascertaining clinical impact.

  14. Protein blotting with direct blotting electrophoresis.

    Science.gov (United States)

    Beck, S

    1988-05-01

    Direct blotting electrophoresis, a method designed to be of general application for the separation and electroblotting of macromolecules, has been adapted to produce protein blots suitable for subsequent processing by standard techniques such as dye staining or immunological detection. After their separation in a very short gel the protein bands are electrophoresed out of the gel onto an immobilizing matrix. The matrix which is moved across the bottom of the gel by a conveyor belt binds these proteins with high affinity. Once the protein samples have been loaded onto the gel and electrophoresis has been started, no further intervention is needed until the blot is completed. The total expenditure of time for such a direct blot is less than 4 h for a mixture of proteins in the molecular weight range of 14-70 kDa. The staining sensitivity of directly blotted proteins is about 200 ng protein per band as revealed by India ink staining.

  15. [Western blot technique standardization of the diagnosis of human fasciolosis using Fasciola hepatica excreted-secreted antigens].

    Science.gov (United States)

    Escalante, Hermes; Davelois, Kelly; Ortiz, Pedro; Rodríguez, Hans; Díaz, Enrique; Jara, César

    2011-01-01

    To evaluate the performance of the enzyme-linked immunoelectrotransfer blot assay (EITB, Western blot) using excretory/secretory antigens from adult forms of Fasciola hepatica (Fh E/S Ag) for the diagnosis of human fasciolosis. Antigens were obtained after 18 hours of incubation in culture medium Minimum Essential Eagle, prepared at a protein concentration of 0.15 ug/uL and run against a pool of sera of patients with proven fasciolosis (confirmed by the finding of parasite eggs in the stool microscopy). Antigens of 10, 12, 17, 23, 27, 30, 36, 43, 66 and 136 kDa were detected and used to develop the Western blot technique. The sensitivity was evaluated using sera from 67 fasciolosis patients, and the specificity using sera from 57 patients with other parasitic diseases, and 10 from healthy individuals. Out of the 67 sera, 64 reacted with the 23 kDa band and 61 with the one of 17 kDa. These two bands were not detected in sera from patients with other parasitic diseases or in those from healthy volunteers and thus could be considered specific and diagnostic. The sensitivity of the test, using the bands of 17 and 23 kDa, was 95.5% for positive reactions to at least one of these two bands, being its specificity 100% with a positive predictive value of 100% and negative predictive value of 95.71%.

  16. [Western blot technique standardization for specific diagnosis of Chagas disease using excretory-secretory antigens of Trypanosoma cruzi epimastigotes].

    Science.gov (United States)

    Escalante, Hermes; Jara, César; Davelois, Kelly; Iglesias, Miguel; Benites, Adderly; Espinoza, Renzo

    2014-01-01

    Evaluate the effectiveness of Western Blot for the specific diagnosis of Chagas disease using excretory-secretory antigens of Trypanosoma cruzi epimastigotes. Antigens were obtained after twenty hours of incubation in Eagle’s Minimum Essential Medium, which were prepared at a protein concentration of 0.2 ug/uL to be faced with 10 mL pool of serum from patients with Chagas disease and a conjugated anti-IgG labeled with peroxidase. The presence of the following antigens was observed: 10, 12, 14, 15, 19, 20, 23, 26, 30, 33, 36, 40, 42, 46, 58, 63, 69, 91, 100, and 112 kDa; of which antigens of 10, 12, 14, 15, 19, 20, 23, and 26 kDa were considered to be specific using pools of serum from patients with other parasitosis and serum from people with no parasites. The sensitivity of the technique was assessed using individual serum from 65 patients with Chagas disease; and the specificity with serum from 40 patients with other parasitosis, and serums from five people who did not have parasites. The technique has a sensitivity of 95.4% in the detection of one to eight specific bands, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 93.7%. Western Blot technique with excretory-secretory antigens of T. cruzi epimastigotes is effective in the diagnosis of Chagas disease in Peru; therefore, it can be used as a confirmatory test.

  17. Glyceraldehyde-3-phosphate dehydrogenase: a universal internal control for Western blots in prokaryotic and eukaryotic cells.

    Science.gov (United States)

    Wu, Yonghong; Wu, Min; He, Guowei; Zhang, Xiao; Li, Weiguang; Gao, Yan; Li, Zhihui; Wang, Zhaoyan; Zhang, Chenggang

    2012-04-01

    In the current study, we examined the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in a number of organisms and the stability of GAPDH under various conditions. Our results revealed that GAPDH is present in multiple Escherichia coli strains, the yeast strain GS115, Caenorhabditis elegans, rat PC12 cells, and both mouse and rat brain. Furthermore, GAPDH was stably expressed under different concentrations of inducer and at different times of induction in E. coli (BL21) cells and yeast GS115 cells. Stable expression of GAPDH protein was also observed in C.elegans and PC12 cells that were treated with different concentrations of paraquat or sodium sulfite, respectively. In addition, we were able to detect and identify the endogenous gapA protein in E.coli via immunoprecipitation and MALDI-TOF-MS analysis. Endogenous gapA protein and exogenously expressed (subcloned) GAPDH proteins were detected in E. coli BL21 but not for gapC. With the exception of gapC in E. coli, the various isoforms of GAPDH possessed enzymatic activity. Finally, sequence analysis revealed that the GAPDH proteins were 76% identical, with the exception of E. coli gapC. Taken together, our results indicate that GAPDH could be universally used as an internal control for the Western blot analysis of prokaryotic and eukaryotic samples. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  18. Analytical technique for label-free multi-protein detection based on Western blot and surface-enhanced Raman scattering.

    Science.gov (United States)

    Han, Xiao X; Jia, Hui Y; Wang, Yan F; Lu, Zhi C; Wang, Chun X; Xu, Wei Q; Zhao, Bing; Ozaki, Yukihiro

    2008-04-15

    We have developed a new analytical procedure for label-free protein detection designated "Western SERS", consisting of protein electrophoresis, Western blot, colloidal silver staining, and surface-enhanced Raman scattering (SERS) detection. A novel method of silver staining for Western blot that uses a silver colloid, an excellent SERS-active substrate, is first proposed in the present study. During the process of silver staining, interactions between proteins and silver nanoparticles result in the emergence of SERS of proteins. In the present study, we use myoglobin (Mb) and bovine serum albumin (BSA) as model proteins. From different protein bands on a nitrocellulose (NC) membrane, we have observed surface-enhanced resonance Raman scattering (SERRS) spectra of Mb and SERS spectra of BSA. The proposed technique offers dual advantages of simplicity and high sensitivity. On one hand, after the colloidal silver staining, we can detect label-free multi-proteins directly on a NC membrane without digestion, extraction, and other pretreatments. On the other hand, the detection limit of the Western SERS is almost consistent with the detection limit of colloidal silver staining, and the SERRS detection limit of Mb is found to be 4 ng/band. This analytical method, which combines the technique of protein separation with SERS, may be a powerful protocol for label-free protein detection in proteomic research.

  19. The western blot

    Science.gov (United States)

    Western blotting is a technique that involves the separation of proteins by gel electrophoresis, their blotting or transfer to a membrane, and selective immunodetection of an immobilized antigen. This is an important and routine method for protein analysis that depends on the specificity of antibod...

  20. Variations in Western blot banding patterns of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus.

    OpenAIRE

    Burke, D S; Redfield, R R; Putman, P; Alexander, S S

    1987-01-01

    Serum samples from 27 patients infected with human T-cell lymphotropic virus type III (14 with acquired immune deficiency syndrome [AIDS] and 13 with AIDS-related complex) were examined for antibodies to viral proteins by the Western blot method and with four different commercial solid-phase enzyme-linked immunosorbent assays (ELISAs). Virus-specific bands on blots at molecular masses of 64, 55, 53, 41, 31, 24, and 17 kilodaltons were observed. Rank correlation matrices were calculated to rel...

  1. Comparison of western blot analysis and immunocytochemical detection of P-glycoprotein in multidrug resistant cells.

    OpenAIRE

    Friedlander, M L; Bell, D R; Leary, J; Davey, R A

    1989-01-01

    A sensitive immunocytochemical technique was developed to detect a 170,000 dalton cell membrane glycoprotein (P-gp) in cell lines resistant to vincristine and vinblastine with varying degrees of resistance. P-gp was shown very clearly using the C219 monoclonal antibody and immunocytochemical detection with either antialkaline phosphate or peroxidase-antiperoxidase with silver gold intensification. There was good correlation between the results obtained with immunocytochemical detection of P-g...

  2. The Dot Blot ELISA.

    Science.gov (United States)

    Gerbig, Donald G., Jr.; Fenk, Christopher J.; Goodhart, Amy S.

    2000-01-01

    Uses two laboratory techniques, Enzyme Linked Immunosorbent Assay (ELISA) and Western Blot, to demonstrate antibody-antigen binding concepts. Includes a list of required materials and directions for the procedure, and makes suggestions for classroom applications. (Contains 13 references.) (YDS)

  3. Cross-Reactions between Toxocara canis and Ascaris suum in the diagnosis of visceral larva migrans by western blotting technique

    Directory of Open Access Journals (Sweden)

    NUNES Cáris Maroni

    1997-01-01

    Full Text Available Visceral larva migrans (VLM is a clinical syndrome caused by infection of man by Toxocara spp, the common roundworm of dogs and cats. Tissue migration of larval stages causes illness specially in children. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. After the introduction of the enzyme-linked immunosorbent assay (ELISA using the larval excretory-secretory antigen of T. canis (TES, the diagnosis specificity was greatly improved although cross-reactivity with other helminths are still being reported. In Brazil, diagnosis is routinely made after absorption of serum samples with Ascaris suum antigens, a nematode antigenicaly related with Ascaris lumbricoides which is a common intestinal nematode of children. In order to identify T. canis antigens that cross react to A. suum antigens we analyzed TES antigen by SDS-PAGE and Western blotting techniques. When we used serum samples from patients suspected of VLM and positive result by ELISA as well as a reference serum sample numerous bands were seen (molecular weight of 210-200 kDa, 116-97 kDa, 55-50 kDa and 35-29 kDa. Among these there is at least one band with molecular weight around 55-66 kDa that seem to be responsible for the cross-reactivity between T. canis e A. suum once it disappears when previous absorption of serum samples with A. suum antigens is performed

  4. Serodiagnosis of grass carp reovirus infection in grass carp Ctenopharyngodon idella by a novel Western blot technique.

    Science.gov (United States)

    He, Yongxing; Jiang, Yousheng; Lu, Liqun

    2013-12-01

    Frequent outbreaks of grass carp hemorrhagic disease, caused by grass carp reovirus (GCRV) infection, pose as serious threats to the production of grass carp Ctenopharyngodon idella. Although various nucleic acids-based diagnostic methods have been shown effective, lack of commercial monoclonal antibody against grass carp IgM has impeded the development of any reliable immunoassays in detection of GCRV infection. The present study describes the preparation and screening of monoclonal antibodies against the constant region of grass carp IgM protein, and the development of a Western blot (WB) protocol for the specific detection of antibodies against outer capsid VP7 protein of GCRV that serves as antibody-capture antigen in the immunoassay. In comparison to a conventional RT-PCR method, validity of the WB is further demonstrated by testing on clinical fish serum samples collected from a grass carp farm in Jiangxi Province during disease pandemic in 2011. In conclusion, the WB technique established in this study could be employed for specific serodiagnosis of GCRV infection. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Modification of T-cell antigenic properties of tetanus toxoid by SDS-PAGE separation. Implications for T-cell blotting

    DEFF Research Database (Denmark)

    Christensen, C B; Theander, T G

    1997-01-01

    Using Tetanus Toxoid (TT) as a model antigen the T-cell Blotting method was evaluated. Peripheral blood mononuclear cell (PBMC) cultures were stimulated by blotted nitrocellulose-bound TT or soluble TT. SDS-Poly-Acrylamide-Gel-Electrophoresis separated TT only induced proliferation in 20...... that SDS-PAGE alters the ability of TT to induce T-cell proliferation, possibly due to unpolymerized acrylamide binding to proteins during SDS-PAGE. The use of SDS-PAGE T-cell blotting in the screening for T-cell antigens must therefore be reconsidered. We suggest the use of SDS-Agarose Gel Electrophoresis......% of the PBMC cultures whereas proliferation was induced in 79% of the same cultures offered similar treated TT (except for the PAGE separation). When T-cell blotting was performed with TT separated in a SDS-agarose matrix, proliferation was induced in 80% of donors responding to soluble TT. The results show...

  6. Microfluidic Western blotting.

    Science.gov (United States)

    Hughes, Alex J; Herr, Amy E

    2012-12-26

    Rapid, quantitative Western blotting is a long-sought bioanalytical goal in the life sciences. To this end, we describe a Western blotting assay conducted in a single glass microchannel under purely electronic control. The μWestern blot is comprised of multiple steps: sample enrichment, protein sizing, protein immobilization (blotting), and in situ antibody probing. To validate the microfluidic assay, we apply the μWestern blot to analyses of human sera (HIV immunoreactivity) and cell lysate (NFκB). Analytical performance advances are achieved, including: short durations of 10-60 min, multiplexed analyte detection, mass sensitivity at the femtogram level, high-sensitivity 50-pM detection limits, and quantitation capability over a 3.6-log dynamic range. Performance gains are attributed to favorable transport and reaction conditions on the microscale. The multistep assay design relies on a photopatternable (blue light) and photoreactive (UV light) polyacrylamide gel. This hydrophilic polymer constitutes both a separation matrix for protein sizing and, after brief UV exposure, a protein immobilization scaffold for subsequent antibody probing of immobilized protein bands. We observe protein capture efficiencies exceeding 75% under sizing conditions. This compact microfluidic design supports demonstration of a 48-plex μWestern blot in a standard microscope slide form factor. Taken together, the μWestern blot establishes a foundation for rapid, targeted proteomics by merging exceptional specificity with the throughput advantages of multiplexing, as is relevant to a broad range of biological inquiry.

  7. A study of the technique of western blot for diagnosis of lyme disease caused by Borrelia afzelii in China.

    Science.gov (United States)

    Liu, Zhi Yun; Hao, Qin; Hou, Xue Xia; Jiang, Yi; Geng, Zhen; Wu, Yi Mou; Wan, Kang Lin

    2013-03-01

    To study the technique of Western blot for the diagnosis of Lyme disease caused by Borrelia afzelii in China and to establish the standard criteria by operational procedure. FP1, which is the representative strain of B. afzelii in China, was analyzed by SDS-PAGE, electro transfer and immunoblotting assays. The molecular weights of the protein bands of FP1 were analyzed by Gel-Pro analysis software. In a study using 451 serum samples (159 patients with Lyme disease and 292 controls), all observed bands were recorded. The accuracy of the WB as a diagnostic test was established by using the ROC curve and Youden index. Criteria for a positive diagnosis of Lyme disease were established as at least one band of P83/100, P58, P39, OspB, OspA, P30, P28, OspC, P17, and P14 in the IgG test and at least one band of P83/100, P58, P39, OspA, P30, P28, OspC, P17, and P41 in the IgM test. For IgG criteria, the sensitivity, specificity and Youden index were 69.8%, 98.3%, and 0.681, respectively; for IgM criteria, the sensitivity, specificity and Youden index were 47%, 94.2%, and 0.412, respectively. Establishment of WB criteria for B. afzelii is important in validating the diagnostic assays for Lyme disease in China. Copyright © 2013 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  8. Modification of T-cell antigenic properties of tetanus toxoid by SDS-PAGE separation. Implications for T-cell blotting

    DEFF Research Database (Denmark)

    Christensen, C B; Theander, T G

    1997-01-01

    Using Tetanus Toxoid (TT) as a model antigen the T-cell Blotting method was evaluated. Peripheral blood mononuclear cell (PBMC) cultures were stimulated by blotted nitrocellulose-bound TT or soluble TT. SDS-Poly-Acrylamide-Gel-Electrophoresis separated TT only induced proliferation in 20% of the ......Using Tetanus Toxoid (TT) as a model antigen the T-cell Blotting method was evaluated. Peripheral blood mononuclear cell (PBMC) cultures were stimulated by blotted nitrocellulose-bound TT or soluble TT. SDS-Poly-Acrylamide-Gel-Electrophoresis separated TT only induced proliferation in 20......% of the PBMC cultures whereas proliferation was induced in 79% of the same cultures offered similar treated TT (except for the PAGE separation). When T-cell blotting was performed with TT separated in a SDS-agarose matrix, proliferation was induced in 80% of donors responding to soluble TT. The results show...... that SDS-PAGE alters the ability of TT to induce T-cell proliferation, possibly due to unpolymerized acrylamide binding to proteins during SDS-PAGE. The use of SDS-PAGE T-cell blotting in the screening for T-cell antigens must therefore be reconsidered. We suggest the use of SDS-Agarose Gel Electrophoresis...

  9. La técnica de Western Blot como criterio de identidad para la vacuna antimeningocócica Men B Western Blot technique as an identity criterion for Men B antimeningococcal vaccine

    Directory of Open Access Journals (Sweden)

    R. Rosario Diéguez Castro

    2009-12-01

    Full Text Available Se desarrolló y validó la técnica de Western Blot aplicada a la vacuna antimeningocócica Men B producida en el Instituto Finlay con el objetivo de demostrar un criterio de identidad. En el estudio de las proteínas antigénicas de la vacuna, P1.15 y P1.4 en vesícula de membrana externa,monograneles y producto final se emplearon en la identificación anticuerpos monoclonales específicos para estas proteínas. Los parámetros desarrollados en la validación de la técnica fueron: especificidad, límite de detección, repetibilidad, precisión intermedia, reproducibilidad y robustez. El método cumplió con los parámetros señalados, por lo que se consideró validado.Western Blot technique was developed and validated, applied to Men B meningococcal vaccine produced in "Carlos J, Finlay" Institute to demonstrate an identity criterion. In study of antigenic proteins of the vaccine, we used P1.15 y P1.4 in vesicle of external membrane, monogranels, and end product to identify the monoclonal antibodies specific of these proteins. Parameters developed in technique validation included: specificity, detection limit, repetition, average accuracy, reproduction, and strength. Method fulfilled with specified parameters, thus considering its validation.

  10. La técnica de Western Blot aplicada a la vacuna antimeningocócica VA MENGOC BC® Western Blot technique applied to VA MENCOG BC® antimeningococcal vaccine

    Directory of Open Access Journals (Sweden)

    R. Rosario Diéguez Castro

    2009-12-01

    Full Text Available Se desarrolló y validó la técnica de Western Blot aplicada a la vacuna antimeningocócica VA MENGOC BC® producida en el Instituto Finlay con el objetivo de demostrar criterio de identidad. Con el empleo de esta técnica se identificaron las proteínas antigénicas del tipo P1, P3, P5, 70 y 80 K presentes en la vesícula de membrana externa y vacuna final, por lo cual se utilizó como antisuero la gamma antimeningocócica. Los parámetros desarrollados en la validación de la técnica fueron: especificidad, límite de detección, repetibilidad, precisión intermedia, reproducibilidad y robustez. La técnica de identidad cumplió con los parámetros señalados anteriormente, por lo que se considera validada.ABSTRACT Western Blot technique applied to VA MENGOC BC® antimeningococcal vaccine was developed and validated and produced in "Carlos J. Finlay" Institute to demonstrate the identity criterion. Using this technique it was possible to identify antigenic proteins type P1, P3, P5 70 and 80 K present in the vesicle of external membrane and final vaccine, thus, we used the antimeningococcal gamma. Parameters developed in validation of this technique included: specificity, detection limit, repetition, average accuracy, reproduction, and strength, identity technique fulfilled with abovementioned parameters, considering like validated

  11. The Western Blot.

    Science.gov (United States)

    Hnasko, Thomas S; Hnasko, Robert M

    2015-01-01

    Western blotting is a technique that involves the separation of proteins by gel electrophoresis, their blotting or transfer to a membrane, and selective immunodetection of an immobilized antigen. This is an important and routine method for protein analysis that depends on the specificity of antibody-antigen interaction and is useful for the qualitative or semiquantitative identification of specific proteins and their molecular weight from a complex mixture. This chapter will outline the requisite steps including gel electrophoresis of a protein sample, transfer of protein from a gel to a membrane support, and immunodetection of a target antigen.

  12. Detection of Sleeping Beauty transposition in the genome of host cells by non-radioactive Southern blot analysis

    Energy Technology Data Exchange (ETDEWEB)

    Aravalli, Rajagopal N., E-mail: aravalli@umn.edu [Department of Radiology, University of Minnesota Medical School, MMC 292, 420 Delaware Street SE, Minneapolis, MN 55455 (United States); Park, Chang W. [Department of Medicine, University of Minnesota Medical School, MMC 36, 420 Delaware Street SE, Minneapolis, MN 55455 (United States); Steer, Clifford J., E-mail: steer001@umn.edu [Department of Medicine, University of Minnesota Medical School, MMC 36, 420 Delaware Street SE, Minneapolis, MN 55455 (United States); Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455 (United States)

    2016-08-26

    The Sleeping Beauty transposon (SB-Tn) system is being used widely as a DNA vector for the delivery of therapeutic transgenes, as well as a tool for the insertional mutagenesis in animal models. In order to accurately assess the insertional potential and properties related to the integration of SB it is essential to determine the copy number of SB-Tn in the host genome. Recently developed SB100X transposase has demonstrated an integration rate that was much higher than the original SB10 and that of other versions of hyperactive SB transposases, such as HSB3 or HSB17. In this study, we have constructed a series of SB vectors carrying either a DsRed or a human β-globin transgene that was encompassed by cHS4 insulator elements, and containing the SB100X transposase gene outside the SB-Tn unit within the same vector in cis configuration. These SB-Tn constructs were introduced into the K-562 erythroid cell line, and their presence in the genomes of host cells was analyzed by Southern blot analysis using non-radioactive probes. Many copies of SB-Tn insertions were detected in host cells regardless of transgene sequences or the presence of cHS4 insulator elements. Interestingly, the size difference of 2.4 kb between insulated SB and non-insulated controls did not reflect the proportional difference in copy numbers of inserted SB-Tns. We then attempted methylation-sensitive Southern blots to assess the potential influence of cHS4 insulator elements on the epigenetic modification of SB-Tn. Our results indicated that SB100X was able to integrate at multiple sites with the number of SB-Tn copies larger than 6 kb in size. In addition, the non-radioactive Southern blot protocols developed here will be useful to detect integrated SB-Tn copies in any mammalian cell type.

  13. Hydrogel Pore-Size Modulation for Enhanced Single-Cell Western Blotting.

    Science.gov (United States)

    Duncombe, Todd A; Kang, Chi-Chih; Maity, Santanu; Ward, Toby M; Pegram, Mark D; Murthy, Niren; Herr, Amy E

    2016-01-13

    Pore-gradient microgel arrays enable thousands of parallel high-resolution single-cell protein electrophoresis separations for targets accross a wide molecular mass (25-289 kDa), yet within 1 mm separation distances. Dual crosslinked hydrogels facilitate gel-pore expansion after electrophoresis for efficient and uniform immunoprobing. The photopatterned, light-activated, and acid-expandable hydrogel underpins single-cell protein analysis, here for oncoprotein-related signaling in human breast biopsy. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Modified western blot technique in fast detection of heme oxygenase (HO-1/HO-2) in various tissues and organs of experimental animals.

    Science.gov (United States)

    Andres, Marta M; Luszczki, Jarogniew J

    2004-01-01

    The present study was aimed at determining all indispensable conditions required to detect heme oxygenase (HO) with western blot technique. Our modified immunoblotting method allowed the detection of HO after 2 hours of electro-transferring onto nitrocellulose membranes that evidently shortened the time of research study without any loss of sensitivity and specificity to detect HO in various tissues and organs of experimental animals. HO was detected in the brain, heart, kidney, liver, lung, spleen, testis, and thymus of the examined mouse and rat, in a quantity for providing evidence that this modified immunoblotting technique is sensitive enough to elicit the existence of this enzyme in various animals' tissues and organs. In conclusion, our modified western blot method permits the fast detection of HO that may be useful in further more advanced quantitative studies.

  15. Western blot analysis of adhesive interactions under fluid shear conditions: the blot rolling assay.

    Science.gov (United States)

    Sackstein, Robert; Fuhlbrigge, Robert

    2015-01-01

    Western blotting has proven to be an important technique in the analysis of receptor-ligand interactions (i.e., by ligand blotting) and for identifying molecules mediating cell attachment (i.e., by cell blotting). Conventional ligand blotting and cell blotting methods employ non-dynamic (static) incubation conditions, whereby molecules or cells of interest are placed in suspension and overlaid on membranes. However, many cell-cell and cell-matrix adhesive interactions occur under fluid shear conditions, and shear stress itself mediates and/or facilitates the engagement of these physiologically appropriate receptors and ligands. Notably, shear forces critically influence the adhesion of circulating cells and platelets to vessel walls in physiologic cell migration and hemostasis, as well as in inflammatory and thrombotic disorders, cancer metastasis, and atherosclerosis. Use of non-dynamic blotting conditions to analyze such interactions can introduce bias, overtly missing relevant effectors and/or exaggerating the relative role(s) of non-physiologic adhesion molecules. To address this shortfall, we have developed a new technique for identifying binding interactions under fluid shear conditions, the "blot rolling assay." Using this method, molecules in a complex mixture are resolved by gel electrophoresis, transferred to a membrane that is rendered semitransparent, and the membrane is then incorporated into a parallel-plate flow chamber apparatus. Under controlled flow conditions, cells or particles bearing adhesion proteins of interest are then introduced into the chamber and interactions with individual immobilized molecules (bands) can be visualized in real time. The substrate molecule(s) supporting adhesion under fluid shear can then be identified by staining with specific antibodies or by excising the relevant band(s) and performing mass spectrometry or microsequencing of the isolated material. This method thus allows for the identification, within a complex

  16. Single cell–resolution western blotting

    Science.gov (United States)

    Kang, Chi-Chih; Yamauchi, Kevin A; Vlassakis, Julea; Sinkala, Elly; Duncombe, Todd A; Herr, Amy E

    2017-01-01

    This protocol describes how to perform western blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state. like conventional western blotting, single-cell western blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g., isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). the gel layer functions as both a molecular sieving matrix during PAGE and a blotting scaffold during immunoprobing. scWB involves five main stages: (i) gravity settling of cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. We also describe microdevice fabrication for both uniform and pore-gradient microgels. to extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. once the microdevice has been fabricated, the assay can be completed in 4–6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. the technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine. PMID:27466711

  17. Valid application of western blotting.

    Science.gov (United States)

    Wu, Liuji; Hu, Xiuli; Tang, Haitao; Han, Zanping; Chen, Yanhui

    2014-05-01

    Western blotting is a powerful and commonly used tool to identify and quantify a specific protein in a complex mixture. However, the systematic errors in the application of western blotting analysis are frequently to be found, which may compromise the interpretation of results. To make a valid application of western blotting, it is essential to begin with three independent biological replicates. Subsequently, a more reliable normalization method is in urgent need for western blotting analysis and using reference proteins is the currently preferred method of normalization. Additionally, identification of valid reference proteins is crucial for western blotting analysis and it should be examined carefully in relation to the cell or tissue types when using housekeeping proteins as internal standards.

  18. Evaluation of a Western blot technique (Helicoblot 2.1) for the diagnosis of Helicobacter pylori infection in children.

    Science.gov (United States)

    Oğünç, Dilara; Artan, Reha; Ongüt, Gözde; Gelen, Tekinalp; Colak, Dilek; Dönmez, Levent; Gültekin, Meral

    2003-04-01

    We evaluated the performance of Helicoblot 2.1 which differentiates the reactivity to each of the various Helicobacter pylori antigens, and compared the results with those obtained by standard techniques (rapid urease test and histological examination of gastric biopsy) in symptomatic children of different ages living in Antalya, Turkey. Eighty-eight children (mean age, 9.15 years) were divided into two groups. The first group included 66 children who were found to be infected with H. pylori. The second group included 22 children who were negative for H. pylori. Serum samples collected from all patients were tested for H. pylori IgG antibodies by immunoblot assay (Helicoblot 2.1). The sensitivity, specificity, positive and negative predictive values for detection of H. pylori infection were 80%, 100%, 100% and 85%, respectively. In children under 7 years of age, the sensitivity of the test was found to be lower than other age groups (P0.05). Helicoblot 2.1 is a useful non-invasive diagnostic tool for H. pylori infection in children over 6 years of age.

  19. Northern blotting analysis

    DEFF Research Database (Denmark)

    Josefsen, Knud; Nielsen, Henrik

    2011-01-01

    Northern blotting analysis is a classical method for analysis of the size and steady-state level of a specific RNA in a complex sample. In short, the RNA is size-fractionated by gel electrophoresis and transferred by blotting onto a membrane to which the RNA is covalently bound. Then, the membrane...... the gap to the more laborious nuclease protection experiments....

  20. Northern blotting analysis

    DEFF Research Database (Denmark)

    Josefsen, Knud; Nielsen, Henrik

    2011-01-01

    Northern blotting analysis is a classical method for analysis of the size and steady-state level of a specific RNA in a complex sample. In short, the RNA is size-fractionated by gel electrophoresis and transferred by blotting onto a membrane to which the RNA is covalently bound. Then, the membran...

  1. Western blotting using chemiluminescent substrates.

    Science.gov (United States)

    Alegria-Schaffer, Alice

    2014-01-01

    Western blotting is a powerful and commonly used tool to identify and quantify a specific protein in a complex mixture (Towbin et al., 1979). The technique enables indirect detection of protein samples immobilized on a nitrocellulose or polyvinylidene fluoride (PVDF) membrane. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Note sur la présence de lames aménagées par technique de Kostienki dans les couches gravettiennes du Blot (Cerzat,Haute-Loire).

    OpenAIRE

    Klaric , Laurent

    2000-01-01

    International audience; The unprecedented presence of Kostienki-technique prepared blades (also called Kostienki knives) in the Gravettian layers at Le Blot leads to a new analysis of these artefacts. Thorough technological study has pointed to the possible role of these items as cores, in association with or complementary to burin-forms, in particular context of backed-bladelet production. Le Blot is the second French site yielding such artefacts, the other being Corbiac (Dordogne). The aim ...

  3. Western blotting: an introduction.

    Science.gov (United States)

    Kurien, Biji T; Scofield, R Hal

    2015-01-01

    Western blotting is an important procedure for the immunodetection of proteins, particularly proteins that are of low abundance. This process involves the transfer of protein patterns from gel to microporous membrane. Electrophoretic as well as non-electrophoretic transfer of proteins to membranes was first described in 1979. Protein blotting has evolved greatly since the inception of this protocol, allowing protein transfer to be accomplished in a variety of ways.

  4. A comparison of the immune parameters of dogs infected with visceral leishmaniasis using Western blot and neutralization techniques Comparação dos parâmetros imunológicos de cães infectados com leishmaniose visceral usando as técnicas de Western blot e neutralização

    Directory of Open Access Journals (Sweden)

    Yeda L. Nogueira

    2007-12-01

    Full Text Available The Western blot technique was used to demonstrate the presence of antibodies in the blood of dogs that presented canine visceral leishmaniasis. This technique was used against some specific molecules present in the lysate of the promastigote form of Leshmania chagasi.Through the association of the results of the Western blot technique with the morphological alterations seen as a result of the serum neutralization technique performed in McCoy cells (which mimetizes the macrophage it was possible to observe the role of some molecules of great relevance in determining the disease in symptomatic dogs as well as that of some other molecules associated with asymptomatic infected dogs that may become transmitters as well as differentiating them as asymptomatic resistant dogs. In the sera analyses carried out during the immunobloting a variation of 9 to 27 immunoreacting bands was observed, which were then compared using Dice's similarity coefficient. In the dendrogram constructed on the basis of the coefficient, 50% similarity was observed among the total number of reagent bands with the promastigote lysate, thus creating five groups. The main difference observed related to the clinical condition of the dogs: symptomatic and asymptomatic dogs were found in separate groups. The asymptomatic group of dogs was distributed in two different places in the dendrogram because they presented two different behavior patterns regarding the cellular morphology in the serum neutralization reaction: the presence or absence of cellular lysis. According to this analysis it is possible to evaluate the immune status and associate it with specific markers observed in the reaction found in the Western blot strips.A técnica de Western blot foi utilizada para demonstrar a presença de anticorpos do soro de cães, que apresentavam leishmaniose visceral canina, contra algumas moléculas específicas no lisado da forma promastigota de Leshmania chagasi.Através da associa

  5. Multiplexed Western Blotting Using Microchip Electrophoresis.

    Science.gov (United States)

    Jin, Shi; Furtaw, Michael D; Chen, Huaxian; Lamb, Don T; Ferguson, Stephen A; Arvin, Natalie E; Dawod, Mohamed; Kennedy, Robert T

    2016-07-05

    Western blotting is a commonly used protein assay that combines the selectivity of electrophoretic separation and immunoassay. The technique is limited by long time, manual operation with mediocre reproducibility, and large sample consumption, typically 10-20 μg per assay. Western blots are also usually used to measure only one protein per assay with an additional housekeeping protein for normalization. Measurement of multiple proteins is possible; however, it requires stripping membranes of antibody and then reprobing with a second antibody. Miniaturized alternatives to Western blot based on microfluidic or capillary electrophoresis have been developed that enable higher-throughput, automation, and greater mass sensitivity. In one approach, proteins are separated by electrophoresis on a microchip that is dragged along a polyvinylidene fluoride membrane so that as proteins exit the chip they are captured on the membrane for immunoassay. In this work, we improve this method to allow multiplexed protein detection. Multiple injections made from the same sample can be deposited in separate tracks so that each is probed with a different antibody. To further enhance multiplexing capability, the electrophoresis channel dimensions were optimized for resolution while keeping separation and blotting times to less than 8 min. Using a 15 μm deep × 50 μm wide × 8.6 cm long channel, it is possible to achieve baseline resolution of proteins that differ by 5% in molecular weight, e.g., ERK1 (44 kDa) from ERK2 (42 kDa). This resolution allows similar proteins detected by cross-reactive antibodies in a single track. We demonstrate detection of 11 proteins from 9 injections from a single Jurkat cell lysate sample consisting of 400 ng of total protein using this procedure. Thus, multiplexed Western blots are possible without cumbersome stripping and reprobing steps.

  6. An improved method for western blotting when extracting proteins from mammalian cells cultured on a collagen gel under serum-free conditions.

    Science.gov (United States)

    Ishihara, Seiichiro; Mizutani, Takeomi; Kawabata, Kazushige; Haga, Hisashi

    2016-01-01

    Western blotting is a widely used method for detection and quantification of specific proteins extracted from mammalian cells. In the conventional method of protein extraction, we found that collagen-containing gels interfered with detection of the p65 protein (one of the subunits in the NF-κB family of proteins) in human lung adenocarcinoma A549 cells cultured on a collagen gel containing serum. In contrast, the collagen gels did not affect detection of the GAPDH protein. Then, we established an improved method for preparation of protein extracts (using trichloroacetic acid fixation and collagenase treatment) from the cells cultured on the collagen gel. Using the improved method, we were able to detect p65 proteins without loss in A549 cells cultured on a collagen gel under serum-free conditions, but we could not detect the proteins if serum was present in cell culture. Thus, using western blotting and serum-free culture conditions, we succeeded in comparing the p65 expression between the cells grown in a plastic dish and cells grown on a collagen gel.

  7. Northern blotting analysis

    DEFF Research Database (Denmark)

    Josefsen, Knud; Nielsen, Henrik

    2011-01-01

    is analysed by hybridization to one or more specific probes that are labelled for subsequent detection. Northern blotting is relatively simple to perform, inexpensive, and not plagued by artefacts. Recent developments of hybridization membranes and buffers have resulted in increased sensitivity closing...

  8. Recent Advances in Microscale Western Blotting.

    Science.gov (United States)

    Sanders, Brittany J; Kim, Daniel C; Dunn, Robert C

    2016-10-21

    Western blotting is a ubiquitous tool used extensively in the clinical and research settings to identify proteins and characterize their levels. It has rapidly become a mainstay in research laboratories due to its specificity, low cost, and ease of use. The specificity arises from the orthogonal processes used to identify proteins. Samples are first separated based on size and then probed with antibodies specific for the protein of interest. This confirmatory approach helps avoid pitfalls associated with antibody cross-reactivity and specificity issues. While the technique has evolved since its inception, the last decade has witnessed a paradigm shift in Western blotting technology. The introduction of capillary and microfluidic platforms has significantly decreased time and sample requirements while enabling high-throughput capabilities. These advances have enabled Western analysis down to the single cell level in highly parallel formats, opening vast new opportunities for studying cellular heterogeneity. Recent innovations in microscale Western blotting are surveyed, and the potential for enhancing detection using advances in label-free biosensing is briefly discussed.

  9. A Proteomic Evaluation of Sympathetic Activity Biomarkers of the Hypothalamus-Pituitary-Adrenal Axis by Western Blotting Technique Following Experimental Traumatic Brain Injury.

    Science.gov (United States)

    Toklu, Hale Zerrin; Sakarya, Yasemin; Tümer, Nihal

    2017-01-01

    Endocrine disorders and autonomic dysfunction are common paradigms following traumatic brain injury (TBI). Alterations in the hypothalamus-pituitary-adrenal (HPA) axis following TBI may result in impaired vasopressor response, energy imbalance, fatigue, depression, or neurological disorders. Autonomic dysfunction is a common disorder following TBI. The sympathetic activity markers on HPA axis can be measured by Western blot protein analysis. Tyrosine hydroxylase, dopamine beta hydroxylase are the key enzymes for the synthesis of norepinephrine; and neuropeptide Y (NPY) is the peptide that is co-stored and co-released with norepinephrine. Thus, the present chapter reviews the experimental protocols for Western blot protein analysis for the measurement of biomarkers that indicate sympathetic activity in brain regions (hypothalamus, pituitary, cerebral cortex, and cerebellum) following TBI.

  10. Cell Phone Detection Techniques

    Energy Technology Data Exchange (ETDEWEB)

    Pratt, Richard M.; Bunch, Kyle J.; Puzycki, David J.; Slaugh, Ryan W.; Good, Morris S.; McMakin, Douglas L.

    2007-10-01

    A team composed of Rick Pratt, Dave Puczyki, Kyle Bunch, Ryan Slaugh, Morris Good, and Doug McMakin teamed together to attempt to exploit cellular telephone features and detect if a person was carrying a cellular telephone into a Limited Area. The cell phone’s electromagnetic properties were measured, analyzed, and tested in over 10 different ways to determine if an exploitable signature exists. The method that appears to have the most potential for success without adding an external tag is to measure the RF spectrum, not in the cell phone band, but between 240 and 400MHz. Figures 1- 7 show the detected signal levels from cell phones from three different manufacturers.

  11. Studying protein-protein interactions via blot overlay/far western blot.

    Science.gov (United States)

    Hall, Randy A

    2015-01-01

    Blot overlay is a useful method for studying protein-protein interactions. This technique involves fractionating proteins on SDS-PAGE, blotting to nitrocellulose or PVDF membrane, and then incubating with a probe of interest. The probe is typically a protein that is radiolabeled, biotinylated, or simply visualized with a specific antibody. When the probe is visualized via antibody detection, this technique is often referred to as "Far Western blot." Many different kinds of protein-protein interactions can be studied via blot overlay, and the method is applicable to screens for unknown protein-protein interactions as well as to the detailed characterization of known interactions.

  12. Studying protein-protein interactions via blot overlay or Far Western blot.

    Science.gov (United States)

    Hall, Randy A

    2004-01-01

    Blot overlay is a useful method for studying protein-protein interactions. This technique involves fractionating proteins on SDS-PAGE, blotting to nitrocellulose or PVDF membrane, and then incubating with a probe of interest. The probe is typically a protein that is radiolabeled, biotinylated, or simply visualized with a specific antibody. When the probe is visualized via antibody detection, this technique is often referred to as "Far Western blot." Many different kinds of protein-protein interactions can be studied via blot overlay, and the method is applicable to screens for unknown protein-protein interactions as well as to the detailed characterization of known interactions.

  13. Lectin-Array Blotting.

    Science.gov (United States)

    Pazos, Raquel; Echevarria, Juan; Hernandez, Alvaro; Reichardt, Niels-Christian

    2017-09-01

    Aberrant protein glycosylation is a hallmark of cancer, infectious diseases, and autoimmune or neurodegenerative disorders. Unlocking the potential of glycans as disease markers will require rapid and unbiased glycoproteomics methods for glycan biomarker discovery. The present method is a facile and rapid protocol for qualitative analysis of protein glycosylation in complex biological mixtures. While traditional lectin arrays only provide an average signal for the glycans in the mixture, which is usually dominated by the most abundant proteins, our method provides individual lectin binding profiles for all proteins separated in the gel electrophoresis step. Proteins do not have to be excised from the gel for subsequent analysis via the lectin array but are transferred by contact diffusion from the gel to a glass slide presenting multiple copies of printed lectin arrays. Fluorescently marked glycoproteins are trapped by the printed lectins via specific carbohydrate-lectin interactions and after a washing step their binding profile with up to 20 lectin probes is analyzed with a fluorescent scanner. The method produces the equivalent of 20 lectin blots in a single experiment, giving detailed insight into the binding epitopes present in the fractionated proteins. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  14. TLC blot (far-eastern blot) and its applications.

    Science.gov (United States)

    Taki, Takao; Gonzalez, Tania Valdes; Goto-Inoue, Naoko; Hayasaka, Takahiro; Setou, Mitsutoshi

    2009-01-01

    A simple method for transfer of lipids including phospholipids, glycolipids, and neutral lipids from a high-performance thin-layer chromatography (HPTLC) plate to a polyvinylidene difluoride (PVDF) membrane, called TLC blot (far-eastern blot), is presented. Lipids separated on a HPTLC plate are blotted quantitatively. This procedure made it possible to purify individual lipids from a blotted membrane in a short time. Binding study, immunodetection, and mass spectrometric analysis are available for PVDF membrane. Furthermore, the world of molecular species imaging is opened by a scanning analysis with a combination of TLC blot and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometer (TLC-Blot/MALDI-TOF MS).

  15. Estandarización de la técnica de Western blot para el diagnóstico de la fasciolosis humana utilizando antígenos de excreción-secreción de Fasciola hepática Western blot technique standardization of the diagnosis of human fasciolosis using Fasciola hepatica excreted-secreted antigens

    Directory of Open Access Journals (Sweden)

    Hermes Escalante

    2011-09-01

    finding of parasite eggs in the stool microscopy. Antigens of 10, 12, 17, 23, 27, 30, 36, 43, 66 and 136 kDa were detected and used to develop the Western blot technique. The sensitivity was evaluated using sera from 67 fasciolosis patients, and the specificity using sera from 57 patients with other parasitic diseases, and 10 from healthy individuals. Results. Out of the 67 sera, 64 reacted with the 23 kDa band and 61 with the one of 17 kDa. These two bands were not detected in sera from patients with other parasitic diseases or in those from healthy volunteers and thus could be considered specific and diagnostic. Conclusions. The sensitivity of the test, using the bands of 17 and 23 kDa, was 95.5% for positive reactions to at least one of these two bands, being its specificity 100% with a positive predictive value of 100% and negative predictive value of 95.71%.

  16. Western Blotting of the Endocannabinoid System.

    Science.gov (United States)

    Wager-Miller, Jim; Mackie, Ken

    2016-01-01

    Measuring expression levels of G protein-coupled receptors (GPCRs) is an important step for understanding the distribution, function, and regulation of these receptors. A common approach for detecting proteins from complex biological systems is Western blotting. In this chapter, we describe a general approach to Western blotting protein components of the endocannabinoid system using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nitrocellulose membranes, with a focus on detecting type 1 cannabinoid (CB1) receptors. When this technique is carefully used, specifically with validation of the primary antibodies, it can provide quantitative information on protein expression levels. Additional information can also be inferred from Western blotting such as potential posttranslational modifications that can be further evaluated by specific analytical techniques.

  17. TLC-Blot (Far-Eastern Blot) and Its Application to Functional Lipidomics.

    Science.gov (United States)

    Taki, Takao

    2015-01-01

    A simple method for transfer of lipids-including phospholipids, glycolipids, and neutral lipids-from a high performance thin layer chromatography (HPTLC) plate to a polyvinylidene difluoride (PVDF) membrane, TLC-Blot (Far-Eastern Blot), and its biochemical applications are presented. This chapter presents the conventional procedures for separating lipid from tissue samples, cultured cells, and serum and the subsequent development of TLC. Individual lipids separated on an HPTLC plate can be transferred to the PVDF membrane quantitatively and also isolated from the lipid-blotted membrane by a one-step purification procedure. Immunodetection with monoclonal antibodies and treatment with lipid-metabolizing enzymes on the lipid-blotted membrane are possible. The method for identification of individual lipids transferred on the PVDF membrane using matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (TLC-Blot/MALDI-TOF MS) is shown as a functional lipidomics application.

  18. Western blotting using capillary electrophoresis.

    Science.gov (United States)

    Anderson, Gwendolyn J; M Cipolla, Cynthia; Kennedy, Robert T

    2011-02-15

    A microscale Western blotting system based on separating sodium-dodecyl sulfate protein complexes by capillary gel electrophoresis followed by deposition onto a blotting membrane for immunoassay is described. In the system, the separation capillary is grounded through a sheath capillary to a mobile X-Y translation stage which moves a blotting membrane past the capillary outlet for protein deposition. The blotting membrane is moistened with a methanol and buffer mixture to facilitate protein adsorption. Although discrete protein zones could be detected, bands were broadened by ∼1.7-fold by transfer to membrane. A complete Western blot for lysozyme was completed in about one hour with 50 pg mass detection limit from low microgram per milliliter samples. These results demonstrate substantial reduction in time requirements and improvement in mass sensitivity compared to conventional Western blots. Western blotting using capillary electrophoresis shows promise to analyze low volume samples with reduced reagents and time, while retaining the information content of a typical Western blot.

  19. Low Proviral Load is Associated with Indeterminate Western Blot Patterns in Human T-Cell Lymphotropic Virus Type 1 Infected Individuals: Could Punctual Mutations be Related?

    Directory of Open Access Journals (Sweden)

    Camila Cánepa

    2015-10-01

    Full Text Available Background: indeterminate Western blot (WB patterns are a major concern for diagnosis of human T-cell lymphotropic virus type 1 (HTLV-1 infection, even in non-endemic areas. Objectives: (a to define the prevalence of indeterminate WB among different populations from Argentina; (b to evaluate if low proviral load (PVL is associated with indeterminate WB profiles; and (c to describe mutations in LTR and tax sequence of these cases. Results: Among 2031 samples, 294 were reactive by screening. Of them, 48 (16.3% were WB indeterminate and of those 15 (31.3% were PCR+. Quantitative real-time PCR (qPCR was performed to 52 HTLV-1+ samples, classified as Group 1 (G1: 25 WB+ samples from individuals with pathologies; Group 2 (G2: 18 WB+ samples from asymptomatic carriers (AC; and Group 3 (G3: 9 seroindeterminate samples from AC. Median PVL was 4.78, 2.38, and 0.15 HTLV-1 copies/100 PBMCs, respectively; a significant difference (p=0.003 was observed. Age and sex were associated with PVL in G1 and G2, respectively. Mutations in the distal and central regions of Tax Responsive Elements (TRE 1 and 2 of G3 were observed, though not associated with PVL.The 8403A>G mutation of the distal region, previously related to high PVL, was absent in G3 but present in 50% of WB+ samples (p = 0.03. Conclusions: indeterminateWBresults confirmed later as HTLV-1 positive may be associated with low PVL levels. Mutations in LTR and tax are described; their functional relevance remains to be determined.

  20. Antibody Validation by Western Blotting.

    Science.gov (United States)

    Signore, Michele; Manganelli, Valeria; Hodge, Alex

    2017-01-01

    Validation of antibodies is an integral part of translational research, particularly for biomarker discovery. Assaying the specificity of the reagent (antibody) and confirming the identity of the protein biomarker is of critical importance prior to implementing any biomarker in clinical studies, and the lack of such quality control tests may result in unexpected and/or misleading results.Antibody validation is the procedure in which a single antibody is thoroughly assayed for sensitivity and specificity. Although a plethora of commercial antibodies exist, antibody specificity must be extensively demonstrated using diverse complex biological samples, rather than purified recombinant proteins, prior to use in clinical translational research. In the simplest iteration, antibody specificity is determined by the presence of a single band in a complex biological sample, at the expected molecular weight, on a Western blot.To date, numerous Western blotting procedures are available, based on either manual or automated systems and spanning the spectrum of single blots to multiplex blots. X-ray film is still employed in many research laboratories, but digital imaging has become a gold standard in immunoblotting. The basic principles of Western blotting are (a) separation of protein mixtures by gel electrophoresis, (b) transfer of the proteins to a blot, (c) probing the blot for a protein or proteins of interest, and (d) subsequent detection of the protein by chemiluminescent, fluorescent, or colorimetric methods. This chapter focuses on the chemiluminescent detection of proteins using a manual Western blotting system and a vacuum-enhanced detection system (SNAP i.d.™, Millipore).

  1. A rapid Western blotting protocol for the Xenopus oocyte.

    Science.gov (United States)

    Lin-Moshier, Yaping; Marchant, Jonathan S

    2013-03-01

    Often experimentalists require a quantitative assessment of the levels of heterologously expressed proteins to best interpret changed Ca(2+) signaling patterns. Here, we detail a rapid and convenient western blotting method for individual Xenopus oocytes. The method exploits recently introduced rapid blotting systems, commercially available from Invitrogen (iBlot) or Bio-Rad (Trans-Blot Turbo). The key advantage is speed: from live cell to transferred membrane in western blotting to assess relative expression levels, even after a long day of Ca(2+) imaging experiments.

  2. Multistrip Western blotting: a tool for comparative quantitative analysis of multiple proteins.

    Science.gov (United States)

    Aksamitiene, Edita; Hoek, Jan B; Kiyatkin, Anatoly

    2015-01-01

    The qualitative and quantitative measurements of protein abundance and modification states are essential in understanding their functions in diverse cellular processes. Typical Western blotting, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. Multistrip Western blotting is a modified immunoblotting procedure based on simultaneous electrophoretic transfer of proteins from multiple strips of polyacrylamide gels to a single membrane sheet. In comparison with the conventional technique, Multistrip Western blotting increases data output per single blotting cycle up to tenfold; allows concurrent measurement of up to nine different total and/or posttranslationally modified protein expression obtained from the same loading of the sample; and substantially improves the data accuracy by reducing immunoblotting-derived signal errors. This approach enables statistically reliable comparison of different or repeated sets of data and therefore is advantageous to apply in biomedical diagnostics, systems biology, and cell signaling research.

  3. Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS).

    Science.gov (United States)

    Gilda, Jennifer E; Ghosh, Rajeshwary; Cheah, Jenice X; West, Toni M; Bodine, Sue C; Gomes, Aldrin V

    2015-01-01

    Western blotting is a commonly used technique in biological research. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Investigation of some conditions that are commonly used and often modified in Western blotting, as well as some commercial antibodies, showed that published articles often fail to report critical parameters needed to reproduce the results. These parameters include the amount of protein loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized. In the present study, comparison of ubiquitinated proteins in rat heart and liver samples showed different results depending on the antibody utilized. Validation of five commercial ubiquitin antibodies using purified ubiquitinated proteins, ubiquitin chains and free ubiquitin showed that these antibodies differ in their ability to detect free ubiquitin or ubiquitinated proteins. Investigating proteins modified with interferon-stimulated gene 15 (ISG15) in young and old rat hearts using six commercially available antibodies showed that most antibodies gave different semi-quantitative results, suggesting large variability among antibodies. Evidence showing the importance of the Western blot buffer and the concentration of antibody used is presented. Hence there is a critical need for comprehensive reporting of experimental conditions to improve the accuracy and reproducibility of Western blot analysis. A Western blotting minimal reporting standard (WBMRS) is suggested to improve the reproducibility of Western blot analysis.

  4. Nebulin-like protein in the earthworm Eisenia foetida. Immunocytochemical electron microscopic study and western blot analysis of several muscle cell types.

    Science.gov (United States)

    Royuela, M; Fraile, B; Paniagua, R

    1997-07-01

    Nebulin is a giant protein (500-900 kDa), which has been reported only in the skeletal muscle (not in cardiac muscle) of vertebrates. The possible presence and distribution of nebulin-like proteins in obliquely striated muscles (body wall and inner muscular layer of the pseudoheart) and smooth muscle (outer muscular layer of the pseudoheart) from the earthworm Eisenia foetida have been examined by means of Western blotting analysis and immunoelectron microscopy, using antibodies against mouse nebulin. The results were compared with those obtained in skeletal, cardiac and smooth muscles of the mouse. In the mouse, immunoreaction to nebulin was observed only in the skeletal muscle and extended along the length of the thin filament. In the earthworm, immunoreaction to a nebulin-like protein was found in the muscle of the body wall and the inner muscular layer of the pseudoheart, but not in the outer muscular layer of the pseudoheart. By electron microscopy, immunolabeling to this protein was observed along the whole length of the thin filament. Western blotting analysis of this nebulin-like protein showed a single band at an estimated molecular mass between 350 and 450 kDa that is slightly lower than that of mouse skeletal muscle nebulin.

  5. Quantitative computerized western blotting in detail.

    Science.gov (United States)

    Talmi-Frank, Dalit; Jaffe, Charles L; Baneth, Gad

    2015-01-01

    The analysis of antibody reactivity against multiple antigens separated according to their molecular weights is facilitated by western blotting. The distinction between immune dominant and recessive antigens is often difficult and carried out by qualitative or empirical means. Quantitative computerized western blotting (QCWB) analyzes reactivity to specific antigens by providing a statistically measurable value for each band allowing differentiation between immunodominant and immunorecessive determinants. QCWB is useful for both single time point analysis and longitudinal studies where multiple time points are evaluated and the relativities against individual bands compared. This technique can be employed to study humoral responses to complex antigenic mixtures such as allergens and infectious agents, or identify serologic markers for early diagnosis of cancer, autoimmune or infectious diseases, or to monitor patient's clinical status.

  6. Methodological considerations for improving Western blot analysis.

    Science.gov (United States)

    MacPhee, Daniel J

    2010-01-01

    The need for a technique that could allow the determination of antigen specificity of antisera led to the development of a method that allowed the production of a replica of proteins, which had been separated electrophoretically on polyacrylamide gels, on to a nitrocellulose membrane. This method was coined Western blotting and is very useful to study the presence, relative abundance, relative molecular mass, post-translational modification, and interaction of specific proteins. As a result it is utilized routinely in many fields of scientific research such as chemistry, biology and biomedical sciences. This review serves to touch on some of the methodological conditions that should be considered to improve Western blot analysis, particularly as a guide for graduate students but also scientists who wish to continue adapting this now fundamental research tool. Copyright 2009 Elsevier Inc. All rights reserved.

  7. Dealing with large sample sizes: comparison of a new one spot dot blot method to western blot.

    Science.gov (United States)

    Putra, Sulistyo Emantoko Dwi; Tsuprykov, Oleg; Von Websky, Karoline; Ritter, Teresa; Reichetzeder, Christoph; Hocher, Berthold

    2014-01-01

    Western blot is the gold standard method to determine individual protein expression levels. However, western blot is technically difficult to perform in large sample sizes because it is a time consuming and labor intensive process. Dot blot is often used instead when dealing with large sample sizes, but the main disadvantage of the existing dot blot techniques, is the absence of signal normalization to a housekeeping protein. In this study we established a one dot two development signals (ODTDS) dot blot method employing two different signal development systems. The first signal from the protein of interest was detected by horseradish peroxidase (HRP). The second signal, detecting the housekeeping protein, was obtained by using alkaline phosphatase (AP). Inter-assay results variations within ODTDS dot blot and western blot and intra-assay variations between both methods were low (1.04-5.71%) as assessed by coefficient of variation. ODTDS dot blot technique can be used instead of western blot when dealing with large sample sizes without a reduction in results accuracy.

  8. Basic Techniques in Mammalian Cell Tissue Culture.

    Science.gov (United States)

    Phelan, Katy; May, Kristin M

    2016-11-01

    Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  9. The Design of a Quantitative Western Blot Experiment

    OpenAIRE

    Taylor, Sean C.; Posch, Anton

    2014-01-01

    Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in...

  10. New Approaches to Quantitative Western Blotting

    OpenAIRE

    Hagner-McWhirter, A.; Soderquist, K.; Grimsby, S.; Winkvist, M.

    2011-01-01

    Fluorescent detection in Western blotting offers high sensitivity, broad dynamic range and stability of signals. This makes it highly suitable for quantitative Western blotting. Here we show how fluorescent Western blotting can be used for simultaneously detection of up to three different proteins on the same blot at the same time and for detection of proteins of the same molecular weight without stripping and reprobing. We also demonstrate how fluorescent Western blotting with 3 layer probin...

  11. Western blot profile in HIV infection

    Directory of Open Access Journals (Sweden)

    Sudha T

    2006-01-01

    Full Text Available Background: Although the overall sensitivity and specificity of the western blot (WB test for detection of antibodies to various viral proteins is high, there has been a substantial difference in the timing of the appearance of antibody bands and their intensities during different stages of HIV infection. Aims: Mapping different band patterns of Western blot results and correlating them with stages of HIV infection. Methods: We performed a retrospective study with 1,467 HIV-1 infected cases confirmed by WB test between January 2002 to July 2005, with the objective of mapping different band patterns of western blot results and determining whether the presence or absence of certain bands was associated with any specific stage of HIV infection. For the interpretation of the WB results in this study, the guidelines recommended by NACO, India were followed. Results: Reactivity with all the bands was the most commonly observed WB pattern, occurring in 92.91% (1363/1467 of cases, whereas the other 7.09% showed uncommon band patterns. Of all individual bands, p31 band was the most frequently missing one, absent in 7.09% cases. On classifying the WB reactive cases by the WHO clinical staging system, 38.45% (564/1467 were in Stage 1, 47.99% (704/1467 in stages 2 and 3 and 13.56% in stage 4. Correlation of CD4 cell counts with the various uncommon band patterns showed that only 5.56% (4/72 had counts in the 200-500 cells/µl range, whereas 45.83% and 48.61% had counts of < 200 and> 500 cells/µl respectively. Conclusion: Interpretation of the WB band pattern in combination with clinical features may be occasionally useful in predicting the stage of HIV infection.

  12. Cost-effective and rapid lysis of Saccharomyces cerevisiae cells for quantitative western blot analysis of proteins, including phosphorylated eIF2α.

    Science.gov (United States)

    Lee, Su Jung; Ramesh, Rashmi; de Boor, Valerie; Gebler, Jan M; Silva, Richard C; Sattlegger, Evelyn

    2017-09-01

    The common method for liberating proteins from Saccharomyces cerevisiae cells involves mechanical cell disruption using glass beads and buffer containing inhibitors (protease, phosphatase and/or kinase inhibitors), followed by centrifugation to remove cell debris. This procedure requires the use of costly inhibitors and is laborious, in particular when many samples need to be processed. Also, enzymatic reactions can still occur during harvesting and cell breakage. As a result low-abundance and labile proteins may be degraded, and enzymes such as kinases and phosphatases may still modify proteins during and after cell lysis. We believe that our rapid sample preparation method helps overcome the above issues and offers the following advantages: (a) it is cost-effective, as no inhibitors and breaking buffer are needed; (b) cell breakage is fast (about 15 min) since it only involves a few steps; (c) the use of formaldehyde inactivates endogenous proteases prior to cell lysis, dramatically reducing the risk of protein degradation; (d) centrifugation steps only occur prior to cell lysis, circumventing the problem of losing protein complexes, in particular if cells were treated with formaldehyde intended to stabilize and capture large protein complexes; and (e) since formaldehyde has the potential to instantly terminate protein activity, this method also allows the study of enzymes in live cells, i.e. in their true physiological environment, such as the short-term effect of a drug on enzyme activity. Taken together, the rapid sample preparation procedure provides a more accurate snapshot of the cell's protein content at the time of harvesting. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  13. A Western Blot-based Investigation of the Yeast Secretory Pathway Designed for an Intermediate-Level Undergraduate Cell Biology Laboratory

    Science.gov (United States)

    Hood-DeGrenier, Jennifer K.

    2008-01-01

    The movement of newly synthesized proteins through the endomembrane system of eukaryotic cells, often referred to generally as the secretory pathway, is a topic covered in most intermediate-level undergraduate cell biology courses. An article previously published in this journal described a laboratory exercise in which yeast mutants defective in…

  14. Application of Monoclonal Antibodies against Bioactive Natural Products: Eastern Blotting and Preparation of Knockout Extract

    Directory of Open Access Journals (Sweden)

    Hiroyuki Tanaka

    2012-01-01

    Full Text Available Matrix-assisted laser desorption/ionization (MALDI tof mass spectrometry was used for the confirmation of hapten number in synthesized antigen. As application of MAb, the MAbs against ginsenosides and glycyrrhizin have been prepared resulting in the development of two new techniques that we named the eastern blotting method and the knockout extract preparation. In eastern blotting technique, glycosides like ginsenosides and glycyrrhizin separated by silica gel TLC were blotted to PVDF membrane that was treated with a NaIO4 solution followed by BSA resulted in glycoside-BSA conjugate on a PVDF membrane. The blotted spots were stained by MAb. Double staining of eastern blotting for ginsenosides using antiginsenoside Rb1 and Rg1 MAbs promoted complete identification of ginsenosides in Panax species. The immunoaffinity concentration of glycyrrhizin was determined by immunoaffinity column conjugated with antiglycyrrhizin MAb resulting in the glycyrrhizin-knockout extract, which was determined by the synergic effect with glycyrrhizin on NO production using the cell line.

  15. BIOMED-2 multiplex immunoglobulin/T-cell receptor polymerase chain reaction protocols can reliably replace Southern blot analysis in routine clonality diagnostics

    NARCIS (Netherlands)

    Y. Sandberg (Yorick); E.J. van Gastel-Mol (Ellen); B. Verhaaf (Brenda); K.H. Lam (King); J.J.M. van Dongen (Jacques); A.W. Langerak (Anton)

    2005-01-01

    textabstractTo establish the most sensitive and efficient strategy of clonality diagnostics via immunoglobulin and T-cell receptor gene rearrangement studies in suspected lymphoproliferative disorders, we evaluated 300 samples (from 218 patients) submitted consecutively for

  16. The Design of a Quantitative Western Blot Experiment

    Directory of Open Access Journals (Sweden)

    Sean C. Taylor

    2014-01-01

    Full Text Available Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013 and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting.

  17. The design of a quantitative western blot experiment.

    Science.gov (United States)

    Taylor, Sean C; Posch, Anton

    2014-01-01

    Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013) and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting.

  18. A review of photovoltaic cells cooling techniques

    Science.gov (United States)

    Zubeer, Swar A.; Mohammed, H. A.; Ilkan, Mustafa

    2017-11-01

    This paper highlights different cooling techniques to reduce the operating temperature of the PV cells. This review paper focuses on the improvement of the performance of the small domestic use PV systems by keeping the temperature of the cells as low as possible and uniform. Different cooling techniques have been investigated experimentally and numerically the impact of the operating temperature of the cells on the electrical and thermal performance of the PV systems. The advantages and disadvantages of ribbed wall heat sink cooling, array air duct cooling installed beneath the PV panel, water spray cooling technique and back surface water cooling are examined in this paper to identify their effective impact on the PV panel performance. It was identified that the water spray cooling system has a proper impact on the PV panel performance. So the water cooling is one way to enhance the electrical efficiency of the PV panel.

  19. A review of photovoltaic cells cooling techniques

    Directory of Open Access Journals (Sweden)

    Zubeer Swar A.

    2017-01-01

    Full Text Available This paper highlights different cooling techniques to reduce the operating temperature of the PV cells. This review paper focuses on the improvement of the performance of the small domestic use PV systems by keeping the temperature of the cells as low as possible and uniform. Different cooling techniques have been investigated experimentally and numerically the impact of the operating temperature of the cells on the electrical and thermal performance of the PV systems. The advantages and disadvantages of ribbed wall heat sink cooling, array air duct cooling installed beneath the PV panel, water spray cooling technique and back surface water cooling are examined in this paper to identify their effective impact on the PV panel performance. It was identified that the water spray cooling system has a proper impact on the PV panel performance. So the water cooling is one way to enhance the electrical efficiency of the PV panel.

  20. The line blot assay: problems with titrating first and second antibodies for Western blot and immunohistochemistry assays?

    Science.gov (United States)

    Rojas-Espinosa, O; Silva-Miranda, M; Wek-Rodriguez, K; Arce-Paredes, P

    2006-01-01

    We describe a technique designed to assess the optimal dilution of primary and secondary antibodies, to be used in Western blot, dot blot, the multi-antigen print immunoassay (MAPIA) and immunohistochemistry assays. The method that we call "line blot" is not an alternative but a practical, complementary tool for the above techniques that assures definitive results are obtained from single assays, so there is no need to repeat the assay. As with most immunoenzymatic assays, the line blot assay is very sensitive, allowing the detection of absolute amounts of antigen as low as 2.5 ng in the 0.5 cm-long segment line (see Results), depending on the strength of the secondary, enzyme-labelled antibody.

  1. Analysis of changes during subclone development and ageing of human antibody-producing heterohybridoma cells by northern blot and flow cytometry.

    Science.gov (United States)

    Borth, N; Strutzenberger, K; Kunert, R; Steinfellner, W; Katinger, H

    1999-01-08

    The economic importance of obtaining high-producing subclones for large scale production of pharmaceutical proteins is self-evident. However, few papers have studied the changes that occur during subclone development. This information would be important for further improvement of screening and subcloning protocols. We have therefore compared subclones of a human-mouse heterohybridoma cell line producing a human antibody againt HIV-1. Three subclones with low, medium and high specific production rates were selected for this study and their light and heavy chain mRNA content, the intracellular content of light and heavy chain and the specific secretion rates compared. In addition the long time stability of antibody expression in the highest producing subclone was analysed for one year. For the three subclones a correlation between the intracellular content in light chain and the secretion rate was found, while the intracellular content in heavy chain was the same for all three subclones. These results indicate that the assembly in the endoplasmic reticulum (ER) is one of the major rate limiting factors in antibody production. During long time cultivation of the heterohybridoma cell line a continuous decrease in light and heavy chain production was seen without the appearance of a non producing sub-population.

  2. Prolonged incubation and stacked film exposure improve sensitivity in western blotting.

    Science.gov (United States)

    Luo, Haitao; Rankin, Gary O; Straley, Shannon; Chen, Yi Charlie

    2011-01-01

    Western blotting is a basic technique for protein detection. For proteins of less abundance or antibodies of poorer quality, an increased sensitivity is often desired. Although it is commonly known that higher concentrations of antibodies and prolonged film exposure times will help improve sensitivity in western blots, both measures come with their own risks, and it is often unclear to which extent these measures should be applied. We conducted time-course studies to investigate protein-antibody interactions and primary antibody-secondary antibody interactions in western blotting. We also propose a protocol of stacked film exposure and have tested it in standard curves and cancer cell samples. Our study found that protein-primary antibody interactions and primary antibody-secondary antibody interactions could take a longer time than commonly used "one hour" or "overnight", and in some cases longer than 48h, to reach its maximum binding. We also show that the modified protocol of stacked film exposure works well for both standard curves and biological samples, reaching a maximum sensitivity in western blots without blurring target signals or increasing backgrounds. In addition to regular optimization of antibody concentrations and film exposure time, a prolonged incubation with antibodies and stacked film exposure will also help improve sensitivity and reduce background in western blotting. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Western Blotting Analysis of CCN Proteins in Calcified Tissues.

    Science.gov (United States)

    Kawaki, Harumi; Kubota, Satoshi; Takigawa, Masaharu

    2017-01-01

    Western blotting is widely used for protein analysis. We routinely perform such analysis for evaluating the production levels of CCN family proteins in a variety of cells under various conditions. In this chapter, we describe our Western blotting protocol to estimate protein production profiles of CCN family members after having assessed the specificity of the antibodies against each CCN member protein to ensure no cross-reaction with other CCN member proteins.

  4. Western blotting by thin-film direct coating.

    Science.gov (United States)

    Yen, Yi-Kuang; Jiang, Yi-Wei; Chang, Shih-Chung; Wang, An-Bang

    2014-05-20

    A novel thin-film direct coating (TDC) technique was developed to markedly reduce the amount of antibody required for Western blotting (WB). Automatic application of the technique for a few seconds easily and homogeneously coats the specific primary antibody on the polyvinylidene fluoride (PVDF) membrane. While conventional WB requires 0.4 μg of the primary antibody, the proposed technique only uses 4 × 10(-2) μg, which can be reduced further to 4 × 10(-5) μg by reducing the coater width. Moreover, the proposed process reduces antibody probing times from 60 to 10 min. The quantification capability of TDC WB showed high linearity within a 4-log2 dynamic range for detecting target antigen glutathione-S-transferase. Furthermore, TDC WB can specifically detect the extrinsic glutathione-S-transferase added in the Escherichia coli or 293T cell lysate with better staining sensitivity than conventional WB. TDC WB can also clearly probe the intrinsic β-actin, α-tubulin, and glyceraldehyde 3-phosphate dehydrogenase, which are usually used as control proteins in biological experiments. This novel technique has been shown to not only have valuable potential for increasing WB efficiency but also for providing significant material savings for future biomedical applications.

  5. Immunocytochemical electron microscopic study and Western blot analysis of caldesmon and calponin in striated muscle of the fruit fly Drosophila melanogaster and in several muscle cell types of the earthworm Eisenia foetida.

    Science.gov (United States)

    Royuela, M; Fraile, B; Picazo, M L; Paniagua, R

    1997-01-01

    Caldesmon and calponin are two proteins that are characteristic of vertebrate smooth muscle. In invertebrates, caldesmon has only been studied in some molluscan muscles, and no previous references to calponin have been found. The aim of this paper was to investigate the presence and distribution of caldesmon and calponin in several invertebrate muscle cell types, classified according to their ultrastructural pattern: transversely striated muscle (flight muscle from Drosophila melanogaster), obliquely striated muscle (muscular body wall and inner muscular layer of the pseudoheart from the earthworm Eisenia foetida), and a muscle of doubtful classification which seems to be intermediate between smooth muscle and obliquely striated muscle (outer muscular layer of the pseudoheart, from E. foetida), using electron microscopy immunocytochemistry and Western blot analysis. Immunoreactions to both caldesmon and calponin were observed in the outer muscular layer cells from the earthworm pseudoheart but neither in the transversely striated muscle of D. melanogaster nor in the obliquely striated muscle from the earthworm. Present findings suggest that caldesmon- and calponin-like proteins are also present in invertebrate muscle cells, but only in those that are ultrastructurally similar to the vertebrate smooth muscle cells. Since discrepancies in the classification of some invertebrate muscles are common in the literature, the use of distinctive markers, such as troponin, caldesmon and calponin may improve our understanding of the nature and properties of many invertebrate muscles showing an ultrastructural pattern that does not resemble any of the classic muscle types.

  6. Uso da técnica de Southern Blot/Hibridização associada à reação em cadeia da polimerase para aumentar a sensibilidade no diagnóstico das infecções por hemoplasmas em gatos domésticos: Use of Southern Blot/Hybridization technique associated to polymerase chain reaction to improve the sensitivity in the diagnosis of hemoplasma infections in domestic cats

    Directory of Open Access Journals (Sweden)

    Daniel B. Macieira

    2009-12-01

    Full Text Available O objetivo deste trabalho foi verificar se a técnica de Southern Blot/Hibridização (SB em associação à reação de polimerização em cadeia (PCR aumenta a sensibilidade na detecção de DNA de hemoplasmas em gatos domésticos (Felis catus. O sangue total foi coletado em tubos contendo o anticoagulante ácido etilenodiamino tetra-acético, o DNA extraído a partir de 149 animais e a PCR realizada com o uso de sequências iniciadoras espécie-específicas, para amplificar subunidade 16S do RNA ribossomal de Mycoplasma haemofelis e 'Candidatus M. haemominutum' dessas amostras. Para a hibridização, foram utilizadas sondas específicas quimicamente marcadas, e os resultados visualizados por meio da adição de substrato quimiluminescente seguida de autoradiografia. Dezoito (12,1% das 149 amostras testadas apresentaram resultado PCR-positivo para o DNA de hemoplasmas. A técnica de SB mostrou que 24/149 (16,1% amostras apresentaram resultado positivo para hemoplasmas, confirmando os 18 resultados PCR-positivos, além de revelar seis outros adicionais (p The aim of this study was to determine whether Southern Blot/Hybridization (SB associated to Polymerase Chain Reaction (PCR improves the sensitivity in the detection of hemoplasma DNA in domestic cats (Felis catus. Whole blood was collected in tubes containing the anticoagulant ethylenediamine tetra-acetic acid and DNA extracted from 149 animals. PCR was performed using species specific primers to amplify the 16S ribosomal RNA subunit of Mycoplasma haemofelis and 'Candidatus M. haemominutum' from these samples. Hybridization was performed using a 16S rDNA probes chemically labeled and the results were visualized using a chemiluminescent substrate addition followed by autoradiography. Eighteen (12.1% of the 149 tested samples had a positive PCR result for hemoplasma species DNA. SB/hybridization technique showed that 24/149 (16.1% samples were positive for hemoplasmas, confirming the 18 PCR

  7. Western Blotting Using PVDF Membranes and Its Downstream Applications.

    Science.gov (United States)

    Komatsu, Setsuko

    2015-01-01

    Western blotting using polyvinylidene difluoride (PVDF) membranes is one of the most popular techniques for detection and characterization of proteins. If this technique is combined with immunodetection, the behavior of a particular protein can be clarified. On the other hand, if it is combined with Edman sequencing, the primary structure of the protein can be determined. A protein sample is transferred from an SDS-polyacrylamide gel electrophoresis (PAGE) gel onto a PVDF membrane by electroblotting. The membrane carrying the protein is either used for immunodetection or protein sequencing. SDS-PAGE followed by Western blotting combined with immunodetection using antibodies can easily detect protein behavior in crude protein mixtures. Furthermore, two-dimensional PAGE followed by Western blotting and Edman sequencing allows effective sequence determination of crude protein mixtures that may not be easily purified by conventional column chromatography.

  8. Routine Western blot to check autophagic flux: cautions and recommendations.

    Science.gov (United States)

    Gómez-Sánchez, Rubén; Pizarro-Estrella, Elisa; Yakhine-Diop, Sokhna M S; Rodríguez-Arribas, Mario; Bravo-San Pedro, José M; Fuentes, José M; González-Polo, Rosa A

    2015-05-15

    At present, the analysis of autophagic flux by Western blotting (WB), which measures two of the most important markers of autophagy, i.e., microtubule-associated protein 1 light chain 3 (LC3) and p62, is widely accepted in the scientific community. In this study, we addressed the possible disadvantages and limitations that this method presents for a correct interpretation of the results according to the lysis buffer used for extracting proteins. Here, we tested the LC3 and p62 protein levels by WB in four cell models (mouse embryonic and human fibroblasts (MEFs and HFs, respectively), N27 rat mesencephalic dopaminergic neurons and SH-SY5Y human neuroblastoma cells). The cells were exposed to the autophagy inhibitor bafilomycin A1 (Baf. A1) in combination (or not) with nutrient deprivation to induce autophagy, and they were lysed by using four different buffers (nonyl phenoxypolyethoxylethanol (NP-40), radioimmunoprecipitation assay (RIPA), Triton X-100, and sample buffer (SB) 1×). Based on our observations, we want to highlight that this technique is not always appropriate for analyzing and monitoring autophagy. In this report, we show conflicting data that hinder the correct interpretation of the results, especially in relation to p62 protein levels, at least in the models studied in this work. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Detection of Diverse and High Molecular Weight Nesprin-1 and Nesprin-2 Isoforms Using Western Blotting.

    Science.gov (United States)

    Carthew, James; Karakesisoglou, Iakowos

    2016-01-01

    Heavily utilized in cell and molecular biology, western blotting is considered a crucial technique for the detection and quantification of proteins within complex mixtures. In particular, the detection of members of the nesprin (nuclear envelope spectrin repeat protein) family has proven difficult to analyze due to their substantial isoform diversity, molecular weight variation, and the sheer size of both nesprin-1 and nesprin-2 giant protein variants (>800 kDa). Nesprin isoforms contain distinct domain signatures, perform differential cytoskeletal associations, occupy different subcellular compartments, and vary in their tissue expression profiles. This structural and functional variance highlights the need to distinguish between the full range of proteins within the nesprin protein family, allowing for greater understanding of their specific roles in cell biology and disease. Herein, we describe a western blotting protocol modified for the detection of low to high molecular weight (50-1000 kDa) nesprin proteins.

  10. Comparison of chromosome analysis using cell culture by coverslip technique with flask technique.

    Science.gov (United States)

    Sajapala, Suraphan; Buranawut, Kitti; NiwatArunyakasemsuk, Md

    2014-02-01

    To determine accuracy rate ofchromosome study from amniotic cellculture by coverslip technique compared with flask technique and to compared timing ofamniotic cell culture, amount ofamniotic cell culture media and cost ofamniotic cell culture. Cross sectional study. Department of Obstetrics and Gynecology, Phramongkutklao Hospital. Subjects: 70 pregnant women who underwent amniocentesis at Phramongkutklao Hospital during November 1, 2007 to February 29, 2008. Amniotic cell culture by flask technique and coverslip technique. Accuracy of amniotic cell culture for chromosome study by coverslip technique compared with flask technique. Totally 70 pregnant women who underwent to amniocentesis and dividedamniotic fluid to cell culture by flask technique and coverslip technique. 69 samples had similar resultfrom both techniques. The only one sample had cell culture failure inboth methods due to blood contamination. Accuracy in coverslip technique was 100% compared with flask technique. In timing of amniotic cell culture, amount ofamniotic cell culture media and cost of amniotic cell culture between 2 methods that coverslip technique was lesser than flask technique. There is statistically significant of accuracy in chromosome result between coverslip technique and flask technique. Coverslip technique was lesser than flask technique in timing, amniotic cell culture media and costs ofamniotic cell culture.

  11. β-actin as a loading control for plasma-based Western blot analysis of major depressive disorder patients.

    Science.gov (United States)

    Zhang, Rufang; Yang, Deyu; Zhou, Chanjuan; Cheng, Ke; Liu, Zhao; Chen, Liang; Fang, Liang; Xie, Peng

    2012-08-15

    Western blot analysis is a commonly used technique for determining specific protein levels in clinical samples. For normalization of protein levels in Western blot, a suitable loading control is required. On account of its relatively high and constant expression, β-actin has been widely employed in Western blot of cell cultures and tissue extracts. However, β-actin's presence in human plasma and this protein's putative role as a plasma-based loading control for Western blot analysis remain unknown. In this study, an enzyme-linked immunosorbent assay was used to determine the concentration of β-actin in human plasma, which is 6.29±0.54 ng/ml. In addition, the linearity of β-actin immunostaining and loaded protein amount was evaluated by Western blot, and a fine linearity (R²=0.974±0.012) was observed. Furthermore, the expression of plasma β-actin in major depressive disorder subjects and healthy controls was compared. The data revealed no statistically significant difference between these two groups. Moreover, the total coefficient of variation for β-actin expression in the two groups was 9.2±1.2%. These findings demonstrate that β-actin is present in human plasma and may possibly be used as a suitable loading control for plasma-based Western blot analysis in major depressive disorder. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Multianalyte on-chip native Western blotting.

    Science.gov (United States)

    Tia, Samuel Q; He, Mei; Kim, Dohyun; Herr, Amy E

    2011-05-01

    We introduce and characterize multiplexed native Western blotting in an automated and unified microfluidic format. While slab gel Western blotting is slow and laborious, conventional multiplexed blotting ("reblotting": probing one sample with multiple antibodies) requires even more resources. Here we detail three key advances that enable an automated and rapid microfluidic alternative to slab gel reblotting. First, we introduce both assay and microdevice designs that integrate protein blotting against multiple antibody blotting regions with native polyacrylamide gel electrophoresis. This microfluidic integration strategy overcomes nonspecific material losses inherent to harsh antibody stripping steps typically needed for conventional reblotting; said conditions can severely limit analyte quantitation. Second, to inform rational design of the multiplexed microfluidic device we develop an analytical model for analyte capture on the blotting regions. Comparison to empirical observations is reported, with capture efficiencies of >85%. Third, we introduce label free detection that makes simultaneous and quantitative multiplexed measurements possible without the need for prelabeling of sample. Assay linear dynamic range spans 8-800 nM with assay completion in 5 min. Owing to the speed, automation, enhanced quantitation capability, and the difficulty of conventional slab gel Western reblotting, microfluidic multiplexed native Western blotting should find use in systems biology, in particular in analyses of protein isoforms and multimeric protein complexes.

  13. Bioconjugation of quantum dot luminescent probes for Western blot analysis.

    Science.gov (United States)

    Makrides, Savvas C; Gasbarro, Christina; Bello, Job M

    2005-10-01

    Western blot analysis is a widely used technique for protein immunodetection. Its current format, however is unsuitable for multiplex detection of proteins, primarily due to intrinsic limitations of standard organic dyes employed as probes. Quantum dot (QD) semiconductor nanoparticles exhibit significant advantages over organic dyes, including their broad absorption bands, narrow, tunable, and symmetric emission spectra, large Stokes shifts, and excellent photostability. Here we describe a novel method for the functionalization of streptavidin-coated QDs with an in vivo biotinylated peptide (head-to-tail dimerized Z domain derived from protein A) that permits the facile conjugation of antibodies to QDs. In this study, we demonstrate the simultaneous detection of two different types of protein in a Western blot. The bioconjugation of QDs described here makes it possible to achieve multiplex detection of proteins in Western blot analysis in a more straightforward manner.

  14. Blame it on Southern, but it's a western blot.

    Science.gov (United States)

    Klionsky, Daniel J

    2017-01-02

    Edwin M. Southern is a professor emeritus at the University of Oxford. He is perhaps best known for development of the "Southern blot" (Dr. Southern was at the University of Edinburgh when he wrote his landmark paper). The Southern blot provided a scientific breakthrough by allowing scientists to detect a particular DNA sequence without first purifying it from the rest of the genome; the basic method involves the transfer of the DNA to a membrane, followed by detection with a specific probe. Although few people perform Southern blots as originally carried out by Southern, due in part to the more recent technique of the polymerase chain reaction, the basic concept continues to play an important role in molecular biology.

  15. Immunocytochemical electron microscopic study and western blot analysis of paramyosin in different invertebrate muscle cell types of the fruit fly Drosophila melanogaster, the earthworm Eisenia foetida, and the snail Helix aspersa.

    Science.gov (United States)

    Royuela, M; García-Anchuelo, R; Arenas, M I; Cervera, M; Fraile, B; Paniagua, R

    1996-04-01

    The presence and distribution pattern of paramyosin have been examined in different invertebrate muscle cell types by means of Western blot analysis and electron microscopy immunogold labelling. The muscles studied were: transversely striated muscle with continuous Z lines (flight muscle from Drosophila melanogaster), transversely striated muscle with discontinuous Z lines (heart muscle from the snail Helix aspersa), obliquely striated body wall muscle from the earthworm Eisenia foetida, and smooth muscles (retractor muscle from the snail and pseudoheart outer muscular layer from the earthworm). Paramyosin-like immunoreactivity was localized in thick filaments of all muscles studied. Immunogold particle density was similar along the whole thick filament length in insect flight muscle but it predominated in filament tips of fusiform thick filaments in both snail heart and earthworm body wall musculature when these filaments were observed in longitudinal sections. In obliquely sectioned thick filaments, immunolabelling was more abundant at the sites where filaments disappeared from the section. These results agree with the notion that paramyosin extended along the whole filament length, but that it can only be immunolabelled when it is not covered by myosin. In all muscles examined, immunolabelling density was lower in cross-sectioned myofilaments than in longitudinally sectioned myofilaments. This suggests that paramyosin does not form a continuous filament. The results of a semiquantitative analysis of paramyosin-like immunoreactivity indicated that it was more abundant in striated than in smooth muscles, and that, within striated muscles, transversely striated muscles contain more paramyosin than obliquely striated muscles.

  16. [Conservation of Malassezia strains in blotting paper].

    Science.gov (United States)

    Ramos, Laura; Ramadán, Silvana; López, Clara; Bulacio, Lucía; Mellado, Soledad

    2006-06-01

    Reference strains belonging to the genus Malassezia were analyzed to evaluate, by comparison, different preservation systems such us subculture, freezing at -80 degrees C in glycerol, and blotting paper-disc conservation. The viability, phenotypic and genotypic characteristics of the strains used in this study was evaluated. The blotting paper method was found to be advantageous to preserve Malassezia spp strains due to both, its simple implementation in the laboratory and its efficiency.

  17. Extracellular Vesicle Isolation and Analysis by Western Blotting.

    Science.gov (United States)

    Kowal, Emma J K; Ter-Ovanesyan, Dmitry; Regev, Aviv; Church, George M

    2017-01-01

    Extracellular vesicles (EVs) are released by mammalian cells and are thought to be important mediators of intercellular communication. There are many methods for isolating EVs from cell culture media, but the most popular methods involve purification based on ultracentrifugation . Here, we provide a detailed protocol for isolating EVs by differential ultracentrifugation and analyzing EV proteins (such as the tetraspanins CD9 , CD63 and CD81 ) by western blotting.

  18. Nonstripping "Rainbow" and Multiple Antigen Detection (MAD) Western Blotting.

    Science.gov (United States)

    Krajewski, Stan Stanislaw; Tsukamoto, Michelle M; Huang, Xianshu; Krajewski, Sebastian B

    2015-01-01

    A variation of immunoblotting method (the "Rainbow Western"), permits sequential detection of multiple antigens (MAD) on a single protein blot without stripping off prior antibodies. Because no stripping is involved, immobilized proteins are not lost from the membrane, thus allowing for multiple reprobings of the same membrane with different primary antibodies (≥12), retaining strong signal intensities for all sequential antibody probings. The procedure utilizes horseradish peroxidase (HRPase)-based detection with both a chemiluminescent and colorimetric substrate. Initial incubation of the blot with secondary antibody followed by colorimetric development prior to probing the blot with primary antibodies markedly reduces background in ECL-based detection procedures and permits sequential use of antibodies derived from a single species. In the "Rainbow Western," four different HRPase-colorimetric substrates that produce black, brown, red, and green colors are employed sequentially for detection and simultaneous display of four different antigens on the same membrane. By allowing large amounts of data to be obtained from a single blot, the MAD-immunoblotting and Rainbow Western methods have the potential for researchers to compare the expression of several proteins within a single biological sample. Both techniques could be particularly valuable for analysis of cellular populations that are difficult to isolate in large numbers or of clinical specimens where the amounts of protein samples is minute or only available on a one-time basis.

  19. The limits for detection of activated caspases of spermatozoa by western blot in human semen.

    Science.gov (United States)

    Brugnon, F; Pons-Rejraji, H; Artonne, C; Janny, L; Grizard, G

    2012-08-01

    Detection of activated caspases of spermatozoa could be helpful to evaluate male infertility. Although western blot is validated as a highly specific method to detect the proteins extracted from cells, the ability of this technique to detect activated sperm caspases in human semen may be limited. Indeed, round cells, which potentially contain some activated caspases, may be present in semen and interfere with the detection of activated sperm caspases. Moreover, it is necessary to evaluate the minimum amount of spermatozoa necessary to optimise the detection of activated caspases in semen samples. Our results showed that interference due to round cells contained in semen with activated caspase-3 requires separation of spermatozoa by density migration. This sperm preparation selects a mature sperm population that does not reflect the whole sperm population, and in infertile men with oligoasthenoteratozoospermia, the amount of spermatozoa thus selected is usually low. Moreover, the western blot technique's low detection sensitivity and the low level of caspase enzyme activity in human spermatozoa for activated caspase-3, -8 and -9 mean that large quantities of spermatozoa are needed to detect the expression of the activated caspases. These limitations prevent this method being used for routine analysis in clinical practice. © 2012 Blackwell Verlag GmbH.

  20. An Introductory Undergraduate Course Covering Animal Cell Culture Techniques

    Science.gov (United States)

    Mozdziak, Paul E.; Petitte, James N.; Carson, Susan D.

    2004-01-01

    Animal cell culture is a core laboratory technique in many molecular biology, developmental biology, and biotechnology laboratories. Cell culture is a relatively old technique that has been sparingly taught at the undergraduate level. The traditional methodology for acquiring cell culture training has been through trial and error, instruction when…

  1. Subcellular dissemination of prothymosin alpha at normal physiology: immunohistochemical vis-a-vis western blotting perspective.

    Science.gov (United States)

    Kijogi, Caroline Mwendwa; Khayeka-Wandabwa, Christopher; Sasaki, Keita; Tanaka, Yoshimasa; Kurosu, Hiroshi; Matsunaga, Hayato; Ueda, Hiroshi

    2016-03-01

    The cell type, cell status and specific localization of Prothymosin α (PTMA) within cells seemingly determine its function. PTMA undergoes 2 types of protease proteolytic modifications that are useful in elucidating its interactions with other molecules; a factor that typifies its roles. Preferably a nuclear protein, PTMA has been shown to function in the cytoplasm and extracellularly with much evidence leaning on pathognomonic status. As such, determination of its cellular distribution under normal physiological context while utilizing varied techniques is key to illuminating prospective validation of its distinct functions in different tissues. Differential distribution insights at normal physiology would also portent better basis for further clarification of its interactions and proteolytic modifications under pathological conditions like numerous cancer, ischemic stroke and immunomodulation. We therefore raised an antibody against the C terminal of PTMA to use in tandem with available antibody against the N terminal in a murine model to explicate the differences in its distribution in brain cell types and major peripheral organs through western blotting and immunohistochemical approaches. The newly generated antibody was applied against the N-terminal antibody to distinguish truncated versions of PTMA or deduce possible masking of the protein by other interacting molecules. Western blot analysis indicated presence of a truncated form of the protein only in the thymus, while immunohistochemical analysis showed that in brain hippocampus the full-length PTMA was stained prominently in the nucleus whereas in the stomach full-length PTMA staining was not observed in the nucleus but in the cytoplasm. Truncated PTMA could not be detected by western blotting when both antibodies were applied in all tissues examined except the thymus. However, immunohistochemistry revealed differential staining by these antibodies suggesting possible masking of epitopes by interacting

  2. High-Resolution Northern Blot for a Reliable Analysis of MicroRNAs and Their Precursors

    Directory of Open Access Journals (Sweden)

    Edyta Koscianska

    2011-01-01

    Full Text Available This protocol describes how to perform northern blot analyses to detect microRNAs and their precursors with single-nucleotide resolution, which is crucial for analyzing individual length variants and for evaluating relative quantities of unique microRNAs in cells. Northern blot analysis consists of resolving RNAs by gel electrophoresis, followed by transferring and fixing to nylon membranes as well as detecting by hybridization with radioactive probes. Earlier efforts to improve this method focused mainly on altering the sensitivity of short RNA detection. We have enhanced the resolution of the northern blot technique by optimizing the electrophoresis step. We have also investigated other steps of the procedure with the goal of enhancing the resolution of RNAs; herein, we present several recommendations to do so. Our protocol is applicable to analyses of all kinds of endogenous and exogenous RNAs, falling within length ranges of 20–30 and 50–70 nt, corresponding to microRNA and pre-microRNA lengths, respectively.

  3. Endothelial cell loss secondary to two different phacoemulsification techniques.

    Science.gov (United States)

    Kohlhaas, M; Klemm, M; Kammann, J; Richard, G

    1998-11-01

    The endothelial cell count after phacoemulsification serves as a sensitive indicator for the level of corneal damage caused by different phacoemulsification techniques. In a prospective and randomized study, the "Reversed Tip and Snip" technique and the "Divide and Conquer" technique were performed in groups of 30 patients each. The corneal endothelial cell count was measured preoperatively as well as 4 weeks and 3 months postoperatively. The endothelial cell count showed significant (P < .001) reduction by approximately 10% after the "Reversed Tip and Snip" technique and by approximately 15% (P < .001) after the "Divide and Conquer" technique. The latter produced a significantly (P < .001) greater cell loss. The "Reversed Tip and Snip" phacoemulsification technique produces less endothelial cell loss than the "Divide and Conquer" technique.

  4. Immunochemische detectiemethoden na western blotting van cytochroom P-450 iso-enzymen

    NARCIS (Netherlands)

    Laan CA; Jansen EHJM

    1992-01-01

    In this report a number of staining techniques on Western blots have been compared with respect to sensitivity, background staining, practical applicability and cost aspects. After electrophoresis of a rat microsomal liver sample followed by blotting, an incubation was performed of a primary

  5. Sensitive detection of fluorescence in western blotting by merging images.

    Science.gov (United States)

    Kondo, Yukari; Higa, Shinichiro; Iwasaki, Takeshi; Matsumoto, Tomoya; Maehara, Kazumitsu; Harada, Akihito; Baba, Yoshihiro; Fujita, Masatoshi; Ohkawa, Yasuyuki

    2018-01-01

    The western blotting technique is widely used to analyze protein expression levels and protein molecular weight. The chemiluminescence method is mainly used for detection due to its high sensitivity and ease of manipulation, but it is unsuitable for detailed analyses because it cannot be used to detect multiple proteins simultaneously. Recently, more attention has been paid to the fluorescence detection method because it is more quantitative and is suitable for the detection of multiple proteins simultaneously. However, fluorescence detection can be limited by poor image resolution and low detection sensitivity. Here, we describe a method to detect fluorescence in western blots using fluorescence microscopy to obtain high-resolution images. In this method, filters and fluorescent dyes are optimized to enhance detection sensitivity to a level similar to that of the chemiluminescence method.

  6. Glycosaminoglycan blotting and detection after electrophoresis separation.

    Science.gov (United States)

    Volpi, Nicola; Maccari, Francesca

    2015-01-01

    Separation of glycosaminoglycans (GAGs) by electrophoresis and their characterization to the microgram level are integral parts of biochemical research. Their blotting on membranes after electrophoresis offers the advantage to perform further analysis on single separated species such as identification with antibodies and/or recovery of single band. A method for the blotting and immobilizing of several nonsulfated and sulfated complex GAGs on membranes made hydrophilic and positively charged by cationic detergent after their separation by conventional agarose-gel electrophoresis is illustrated. This approach to the study of these complex macromolecules utilizes the capacity of agarose-gel electrophoresis to separate single species of polysaccharides from mixtures and the membrane technology for further preparative and analytical uses. Nitrocellulose membranes are derivatized with the cationic detergent cetylpyridinium chloride (CPC) and mixtures of GAGs are capillary blotted after their separation in agarose-gel electrophoresis. Single purified species of variously sulfated polysaccharides are transferred on derivatized membranes with an efficiency of 100 % and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining). This enables a lower amount limit of detection of 0.1 μg. Nonsulfated polyanions, for example hyaluronic acid (HA), may also be transferred to membranes with a limit of detection of approximately 0.1-0.5 μg after irreversible or reversible staining. The membranes may be stained with reversible staining and the same lanes used for immunological detection or other applications.

  7. Confocal laser scanning microscope, Raman microscopy and Western blotting to evaluate inflammatory response after myocardial infarction.

    Science.gov (United States)

    Riezzo, Irene; Cantatore, Santina; DeCarlo, Dania; Fiore, Carmela; Neri, Margherita; Turillazzi, Emanuela; Fineschi, Vittorio

    2015-01-01

    Cardiac muscle necrosis is associated with inflammatory cascade that clears the infarct from dead cells and matrix debris, and then replaces the damaged tissue with scar, through three overlapping phases: the inflammatory phase, the proliferative phase and the maturation phase. Western blotting, laser confocal microscopy, Raman microscopy are valuable tools for studying the inflammatory response following myocardial infarction both humoral and cellular phase, allowing the identification and semiquantitative analysis of proteins produced during the inflammatory cascade activation and the topographical distribution and expression of proteins and cells involved in myocardial inflammation. Confocal laser scanning microscopy (CLSM) is a relatively new technique for microscopic imaging, that allows greater resolution, optical sectioning of the sample and three-dimensional reconstruction of the same sample. Western blotting used to detect the presence of a specific protein with antibody-antigen interaction in the midst of a complex protein mixture extracted from cells, produced semi-quantitative data quite easy to interpret. Confocal Raman microscopy combines the three-dimensional optical resolution of confocal microscopy and the sensitivity to molecular vibrations, which characterizes Raman spectroscopy. The combined use of western blotting and confocal microscope allows detecting the presence of proteins in the sample and trying to observe the exact location within the tissue, or the topographical distribution of the same. Once demonstrated the presence of proteins (cytokines, chemokines, etc.) is important to know the topographical distribution, obtaining in this way additional information regarding the extension of the inflammatory process in function of the time stayed from the time of myocardial infarction. These methods may be useful to study and define the expression of a wide range of inflammatory mediators at several different timepoints providing a more

  8. A duplex approach for immunochemical staining and typing of protein in western blots

    NARCIS (Netherlands)

    Kuczius, T.; Brandstädter, L.; Karch, H.; Langeveld, J.P.M.

    2011-01-01

    The qualitative and semiquantitative Western blotting technique enables the detection of separate proteins and the determination of subtypes and fragments by specific immunological reactions. Protein typing on immunoblots is restricted to antibody-specific determination, with the result of a

  9. Investigation progress of imaging techniques monitoring stem cell therapy

    International Nuclear Information System (INIS)

    Wu Jun; An Rui

    2006-01-01

    Recently stem cell therapy has showed potential clinical application in diabetes mellitus, cardiovascular diseases, malignant tumor and trauma. Efficient techniques of non-invasively monitoring stem cell transplants will accelerate the development of stem cell therapies. This paper briefly reviews the clinical practice of stem cell, in addition, makes a review of monitoring methods including magnetic resonance and radionuclide imaging which have been used in stem cell therapy. (authors)

  10. AC impedance technique in PEM fuel cell diagnosis - A review

    Energy Technology Data Exchange (ETDEWEB)

    Yuan, Xiaozi; Wang, Haijiang; Colin Sun, Jian; Zhang, Jiujun [Institute for Fuel Cell Innovation, National Research Council (Canada)

    2007-12-15

    Because the AC impedance technique, also known as electrochemical impedance spectroscopy (EIS), is being utilized by more and more researchers in proton exchange membrane (PEM) fuel cell studies, the technique has developed into a primary tool in such research. In this paper the recent work on PEM fuel cells using the AC impedance technique is reviewed. Both in situ and ex situ impedance measurements are discussed, with primary focus on the in situ measurements. Within the domain of in situ studies, various methods for measuring the impedance of a PEM fuel cell are examined, and typical impedance spectra in several common scenarios are presented. Representative applications of the AC impedance technique in PEM fuel cell research are also discussed. Finally, the necessity of a time domain rapid AC impedance technique is briefly discussed. (author)

  11. Recombinant antigen-based immuno-slot blot method for serodiagnosis of syphilis

    Directory of Open Access Journals (Sweden)

    N.S. Sato

    2004-07-01

    Full Text Available Three recombinant antigens of Treponema pallidum Nichols strain were fused with GST, cloned and expressed in Escherichia coli, resulting in high levels of GST-rTp47 and GST-rTp17 expression, and supplementation with arginine tRNA for the AGR codon was needed to obtain GST-rTp15 overexpression. Purified fusion protein yields were 1.9, 1.7 and 5.3 mg/l of cell culture for GST-rTp47, GST-rTp17 and GST-rTp15, respectively. The identities of the antigens obtained were confirmed by automated DNA sequencing using ABI Prism 310 and peptide mapping by Finningan LC/MS. These recombinant antigens were evaluated by immuno-slot blot techniques applied to 137 serum samples from patients with a clinical and laboratory diagnosis of syphilis (61 samples, from healthy blood donors (50 samples, individuals with sexually transmitted disease other than syphilis (3 samples, and from individuals with other spirochetal diseases such as Lyme disease (20 samples and leptospirosis (3 samples. The assay had sensitivity of 95.1% (95% CI, 86.1 to 98.7% and a specificity of 94.7% (95% CI, 87.0 to 98.7%; a stronger reactivity was observed with fraction rTp17. The immunoreactivity results showed that fusion recombinant antigens based-immuno-slot blot techniques are suitable for use in diagnostic assays for syphilis.

  12. Analysis of sperm antigens by sodium dodecyl sulfate gel/protein blot radioimmunobinding method

    Energy Technology Data Exchange (ETDEWEB)

    Lee, C.Y.G. (Univ. of British Columbia, Vancouver, Canada); Huang, Y.S.; Hu, P.C.; Gomel, V.; Menge, A.C.

    1982-06-01

    A radioimmunobinding method based on the blotting of renatured proteins from sodium dodecyl sulfate gels on to nitrocellulose filter papers was developed to analyze the sperm antigens that elicit serum anti-sperm antibodies. In rabbits, serum anti-sperm antibodies were raised by immunization with homologous epididymal spermatozoa mixed with complete Freund's adjuvant. The raised antisera from either male or female rabbits were shown to react with three major sperm protein bands on sodium dodecyl sulfate gels with the corresponding molecular weights of about 70,000 +/- 5000, 14,000, and 13,000, respectively. In humans, the monoclonal antibodies against human sperm were raised by a hybridoma technique. Out of six independent hybrid cell lines that were generated, three of them were shown to secrete immunoglobulins that react with the same two protein bands on sodium dodecyl sulfate gels, which have the approximate molecular weight of 10,000. The same procedure was also used to analyze human serum samples that were shown to contain anti-sperm antibodies by the known techniques. Unique sperm antigens that elicit anti-sperm antibodies in humans were identified and correlated. The results of this study suggest that sodium dodecyl sulfate gel/protein blot radioimmunobinding method may be a sensitive and useful tool for the study of sperm antigens that elicit autoimmune responses and their association with human infertility.

  13. Western blot evaluation of siRNA delivery by pH-responsive peptides.

    Science.gov (United States)

    Liang, Wanling; Mason, A James; Lam, Jenny K W

    2013-01-01

    Gene silencing, via RNA interference (RNAi) technologies using small interfering RNA (siRNA), has been developed as an important tool for target identification and validation in drug discovery and has huge therapeutic potential. However, effective delivery into cells presents a major challenge to the use of siRNA. pH-responsive cell-penetrating peptides have attracted considerable attention in recent years as delivery vectors due to their ability to transport their cargos across the biological membrane and/or to promote endosomal escape and prevent lysosomal degradation. To evaluate the in vitro transfection efficiency of the pH-responsive peptide-based siRNA delivery system, the western blotting technique is commonly employed. This method offers a simple, efficient and economical way to study the gene silencing effect of the siRNA by analysing the protein of interest in a sample with minimum equipment requirement. This chapter provides a description of siRNA delivery and analysis by western blotting protocols for qualitatively and quantitatively assessing gene silencing efficiency and selectivity.

  14. Cell-Detection Technique for Automated Patch Clamping

    Science.gov (United States)

    McDowell, Mark; Gray, Elizabeth

    2008-01-01

    A unique and customizable machinevision and image-data-processing technique has been developed for use in automated identification of cells that are optimal for patch clamping. [Patch clamping (in which patch electrodes are pressed against cell membranes) is an electrophysiological technique widely applied for the study of ion channels, and of membrane proteins that regulate the flow of ions across the membranes. Patch clamping is used in many biological research fields such as neurobiology, pharmacology, and molecular biology.] While there exist several hardware techniques for automated patch clamping of cells, very few of those techniques incorporate machine vision for locating cells that are ideal subjects for patch clamping. In contrast, the present technique is embodied in a machine-vision algorithm that, in practical application, enables the user to identify good and bad cells for patch clamping in an image captured by a charge-coupled-device (CCD) camera attached to a microscope, within a processing time of one second. Hence, the present technique can save time, thereby increasing efficiency and reducing cost. The present technique involves the utilization of cell-feature metrics to accurately make decisions on the degree to which individual cells are "good" or "bad" candidates for patch clamping. These metrics include position coordinates (x,y) in the image plane, major-axis length, minor-axis length, area, elongation, roundness, smoothness, angle of orientation, and degree of inclusion in the field of view. The present technique does not require any special hardware beyond commercially available, off-the-shelf patch-clamping hardware: A standard patchclamping microscope system with an attached CCD camera, a personal computer with an imagedata- processing board, and some experience in utilizing imagedata- processing software are all that are needed. A cell image is first captured by the microscope CCD camera and image-data-processing board, then the image

  15. A flow cytometry technique to study intracellular signals NF-κB and STAT3 in peripheral blood mononuclear cells

    Directory of Open Access Journals (Sweden)

    Chavarin Patricia

    2007-07-01

    Full Text Available Abstract Background Cytokines have essential roles on intercellular communications and are effective in using a variety of intracellular pathways. Among this multitude of signalling pathways, the NF-κB (nuclear factor kappaB and STAT (signal transducer and activator of transcription families are among the most frequently investigated because of their importance. Indeed, they have important role in innate and adaptive immunity. Current techniques to study NF-κB and STAT rely on specific ELISAs, Western Blots and – most recently described – flow cytometry; so far, investigation of such signalling pathways are most commonly performed on homogeneous cells after purification. Results The present investigation aimed at developing a flow cytometry technique to study transcription factors in various cellular types such as mixtures of B-cells, T-lymphocytes and monocytes/macrophages stimulated in steady state conditions (in other words, as peripheral blood mononuclear cells. To achieve this goal, a two step procedure was carried out; the first one consisted of stimulating PBMCs with IL1β, sCD40L and/or IL10 in such a manner that optimal stimulus was found for each cell subset (and subsequent signal transduction, therefore screened by specific ELISA; the second step consisted of assessing confirmation and fine delineation of technical conditions by specific Western-Blotting for either NF-κB or STAT products. We then went on to sensitize the detection technique for mixed cells using 4 color flow cytometry. Conclusion In response to IL1β, or IL10, the levels of phosphorylated NF-κB and STAT3 – respectively – increased significantly for all the studied cell types. In contrast, B-cells and monocytes/macrophages – but, interestingly, not T-lymphocytes (in the context of PBMCs – responded significantly to sCD40L by increasing phosphorylated NF-κB.

  16. A Proteomics Approach to the Protein Normalization Problem: Selection of Unvarying Proteins for MS-Based Proteomics and Western Blotting.

    Science.gov (United States)

    Wiśniewski, Jacek R; Mann, Matthias

    2016-07-01

    Proteomics and other protein-based analysis methods such as Western blotting all face the challenge of discriminating changes in the levels of proteins of interest from inadvertent changes in the amount loaded for analysis. Mass-spectrometry-based proteomics can now estimate the relative and absolute amounts of thousands of proteins across diverse biological systems. We reasoned that this new technology could prove useful for selection of very stably expressed proteins that could serve as better loading controls than those traditionally employed. Large-scale proteomic analyses of SDS lysates of cultured cells and tissues revealed deglycase DJ-1 as the protein with the lowest variability in abundance among different cell types in human, mouse, and amphibian cells. The protein constitutes 0.069 ± 0.017% of total cellular protein and occurs at a specific concentration of 34.6 ± 8.7 pmol/mg of total protein. Since DJ-1 is ubiquitous and therefore easily detectable with several peptides, it can be helpful in normalization of proteomic data sets. In addition, DJ-1 appears to be an advantageous loading control for Western blot that is superior to those used commonly used, allowing comparisons between tissues and cells originating from evolutionarily distant vertebrate species. Notably, this is not possible by the detection and quantitation of housekeeping proteins, which are often used in the Western blot technique. The approach introduced here can be applied to select the most appropriate loading controls for MS-based proteomics or Western blotting in any biological system.

  17. The fastest Western in town: a contemporary twist on the classic Western blot analysis.

    Science.gov (United States)

    Silva, Jillian M; McMahon, Martin

    2014-02-05

    The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

  18. [Application of cell co-culture techniques in medical studies].

    Science.gov (United States)

    Luo, Yun; Sun, Gui-Bo; Qin, Meng; Yao, Fan; Sun, Xiao-Bo

    2012-11-01

    As the cell co-culture techniques can better imitate an in vivo environment, it is helpful in observing the interactions among cells and between cells and the culture environment, exploring the effect mechanisms of drugs and their possible targets and filling the gaps between the mono-layer cell culture and the whole animal experiments. In recently years, they has attracted much more attention from the medical sector, and thus becoming one of research hotspots in drug research and development and bio-pharmaceutical fields. The cell co-culture techniques, including direct and indirect methods, are mainly used for studying pathological basis, new-type treatment methods and drug activity screening. Existing cell co-culture techniques are used for more pharmacological studies on single drug and less studies on interaction of combined drugs, such as collaborative compatibility and attenuation and synergistic effect among traditional Chinese medicines (TCMs). In line with the action characteristics of multi-component and multi-target, the cell co-culture techniques provide certain reference value for future studies on the effect and mechanism of combined TCMs on organisms as well as new methods for studies on TCMs and their compounds. This essay summarizes cell co-culture methods and their application and look into the future of their application in studies on TCMs and compounds.

  19. A photoacoustic technique to measure the properties of single cells

    Science.gov (United States)

    Strohm, Eric M.; Berndl, Elizabeth S. L.; Kolios, Michael C.

    2013-03-01

    We demonstrate a new technique to non-invasively determine the diameter and sound speed of single cells using a combined ultrasonic and photoacoustic technique. Two cell lines, B16-F1 melanoma cells and MCF7 breast cancer cells were examined using this technique. Using a 200 MHz transducer, the ultrasound backscatter from a single cell in suspension was recorded. Immediately following, the cell was irradiated with a 532 nm laser and the resulting photoacoustic wave recorded by the same transducer. The melanoma cells contain optically absorbing melanin particles, which facilitated photoacoustic wave generation. MCF7 cells have negligible optical absorption at 532 nm; the cells were permeabilized and stained with trypan blue prior to measurements. The measured ultrasound and photoacoustic power spectra were compared to theoretical equations with the cell diameter and sound speed as variables (Anderson scattering model for ultrasound, and a thermoelastic expansion model for photoacoustics). The diameter and sound speed were extracted from the models where the spectral shape matched the measured signals. However the photoacoustic spectrum for the melanoma cell did not match theory, which is likely because melanin particles are located around the cytoplasm, and not within the nucleus. Therefore a photoacoustic finite element model of a cell was developed where the central region was not used to generate a photoacoustic wave. The resulting power spectrum was in better agreement with the measured signal than the thermoelastic expansion model. The MCF7 cell diameter obtained using the spectral matching method was 17.5 μm, similar to the optical measurement of 16 μm, while the melanoma cell diameter obtained was 22 μm, similar to the optical measurement of 21 μm. The sound speed measured from the MCF7 and melanoma cell was 1573 and 1560 m/s, respectively, which is within acceptable values that have been published in literature.

  20. Automated capillary Western dot blot method for the identity of a 15-valent pneumococcal conjugate vaccine.

    Science.gov (United States)

    Hamm, Melissa; Ha, Sha; Rustandi, Richard R

    2015-06-01

    Simple Western is a new technology that allows for the separation, blotting, and detection of proteins similar to a traditional Western except in a capillary format. Traditionally, identity assays for biological products are performed using either an enzyme-linked immunosorbent assay (ELISA) or a manual dot blot Western. Both techniques are usually very tedious, labor-intensive, and complicated for multivalent vaccines, and they can be difficult to transfer to other laboratories. An advantage this capillary Western technique has over the traditional manual dot blot Western method is the speed and the automation of electrophoresis separation, blotting, and detection steps performed in 96 capillaries. This article describes details of the development of an automated identity assay for a 15-valent pneumococcal conjugate vaccine, PCV15-CRM197, using capillary Western technology. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Single-cell mechanics: the parallel plates technique.

    Science.gov (United States)

    Bufi, Nathalie; Durand-Smet, Pauline; Asnacios, Atef

    2015-01-01

    We describe here the parallel plates technique which enables quantifying single-cell mechanics, either passive (cell deformability) or active (whole-cell traction forces). Based on the bending of glass microplates of calibrated stiffness, it is easy to implement on any microscope, and benefits from protocols and equipment already used in biology labs (coating of glass slides, pipette pullers, micromanipulators, etc.). We first present the principle of the technique, the design and calibration of the microplates, and various surface coatings corresponding to different cell-substrate interactions. Then we detail the specific cell preparation for the assays, and the different mechanical assays that can be carried out. Finally, we discuss the possible technical simplifications and the specificities of each mechanical protocol, as well as the possibility of extending the use of the parallel plates to investigate the mechanics of cell aggregates or tissues. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Induced Pluripotent Stem Cells: Emerging Techniques for Nuclear Reprogramming

    Science.gov (United States)

    Han, Ji Woong

    2011-01-01

    Abstract Introduction of four transcription factors, Oct3/4, Sox2, Klf4, and c-Myc, can successfully reprogram somatic cells into embryonic stem (ES)-like cells. These cells, which are referred to as induced pluripotent stem (iPS) cells, closely resemble embryonic stem cells in genomic, cell biologic, and phenotypic characteristics, and the creation of these special cells was a major triumph in cell biology. In contrast to pluripotent stem cells generated by somatic cell nuclear-transfer (SCNT) or ES cells derived from the inner cell mass (ICM) of the blastocyst, direct reprogramming provides a convenient and reliable means of generating pluripotent stem cells. iPS cells have already shown incredible potential for research and for therapeutic applications in regenerative medicine within just a few years of their discovery. In this review, current techniques of generating iPS cells and mechanisms of nuclear reprogramming are reviewed, and the potential for therapeutic applications is discussed. Antioxid. Redox Signal. 15, 1799–1820. PMID:21194386

  3. Development of a cell sheet transportation technique for regenerative medicine.

    Science.gov (United States)

    Oie, Yoshinori; Nozaki, Takayuki; Takayanagi, Hiroshi; Hara, Susumu; Hayashi, Ryuhei; Takeda, Shizu; Mori, Keisuke; Moriya, Noboru; Soma, Takeshi; Tsujikawa, Motokazu; Saito, Kazuo; Nishida, Kohji

    2014-05-01

    A transportation technique for cell sheets is necessary to standardize regenerative medicine. The aim of this article is to develop and evaluate a new transportation technique for cell sheets. We developed a transportation container with three basic functions: the maintenance of interior temperature, air pressure, and sterility. The interior temperature and air pressure were monitored by a recorder. Human oral mucosal epithelial cells obtained from two healthy volunteers were cultured on temperature-responsive culture dishes. The epithelial cell sheets were transported via an airplane between the Osaka University and Tohoku University using the developed cell transportation container. Histological and immunohistochemical analyses and flow cytometric analyses for cell viability and cell purity were performed for the cell sheets before and 12 h after transportation to assess the influence of transportation on the cell sheets. Sterility tests and screening for endotoxin and mycoplasma in the cell sheets were performed before and after transportation. During transportation via an airplane, the temperature inside the container was maintained above 32°C, and the changes in air pressure remained within 10 hPa. The cell sheets were well stratified and successfully harvested before and after transportation. The expression patterns of keratin 3/76, p63, and MUC16 were equivalent before and after transportation. However, the expression of ZO-1 in the cell sheet after transportation was slightly weaker than that before transportation. The cell viability was 72.0% before transportation and 77.3% after transportation. The epithelial purity was 94.6% before transportation and 87.9% after transportation. Sterility tests and screening for endotoxin and mycoplasma were negative for all cell sheets. The newly developed transportation technique for air travel is essential technology for regenerative medicine and promotes the standardization and spread of regenerative therapies.

  4. Red blood cell image enhancement techniques for cells with ...

    African Journals Online (AJOL)

    quality or challenging conditions of the images such as poor illumination of blood smear and most importantly overlapping RBC. The algorithm comprises of two RBC segmentation that can be selected based on the image quality, circle mask technique and grayscale blood smear image processing. Detail explanations ...

  5. Quantum dot-based western blot for sensitive detection of pig serum antibody to actinobacillus pleuropneumoniae

    Science.gov (United States)

    Cişmileanu, Ana; Sima, Cornelia; Grigoriu, Constantin

    2007-08-01

    A quantum dot - immunoglobulin conjugate specific for pig IgG, was obtained by carbodiimide chemistry. We used a Western blot technique for detecting specific antibodies against Actinobacillus pleuropneumoniae (A. pp), which cause porcine pleuropneumonia. The antigen used in this technique was Apx haemolysin which is an important virulence factor of A. pp and it induces protective immunity in vaccined pigs. The detection on Western blot membrane was possible at 1/50 dilution of quantum dot conjugate at a dilution of pig serum till 1/6400. The results for pig serum demonstrated a higher sensitivity of QD-based Western blot technique for the presence of antibodies specific for Apx haemolysin in comparison with similar classical techniques (with coloured substrate for enzyme present in secondary antibody conjugate).

  6. V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

    OpenAIRE

    Posch, Anton; Kohn, Jonathan; Oh, Kenneth; Hammond, Matt; Liu, Ning

    2013-01-01

    The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a "black box" because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-fr...

  7. A Streamlined Western Blot Exercise: An Efficient and Greener Approach in the Laboratory Classroom

    Science.gov (United States)

    Ness, Traci L.; Robinson, Rebekah L.; Mojadedi, Wais; Peavy, Lydia; Weiland, Mitch H.

    2015-01-01

    SDS-PAGE and western blotting are two commonly taught protein detection techniques in biochemistry and molecular biology laboratory classrooms. A pitfall associated with incorporating these techniques into the laboratory is the significant wait times that do not allow students to obtain timely results. The waiting associated with SDS-PAGE comes…

  8. Using a large area CMOS APS for direct chemiluminescence detection in Western blotting electrophoresis

    Science.gov (United States)

    Esposito, Michela; Newcombe, Jane; Anaxagoras, Thalis; Allinson, Nigel M.; Wells, Kevin

    2012-03-01

    Western blotting electrophoretic sequencing is an analytical technique widely used in Functional Proteomics to detect, recognize and quantify specific labelled proteins in biological samples. A commonly used label for western blotting is Enhanced ChemiLuminescence (ECL) reagents based on fluorescent light emission of Luminol at 425nm. Film emulsion is the conventional detection medium, but is characterized by non-linear response and limited dynamic range. Several western blotting digital imaging systems have being developed, mainly based on the use of cooled Charge Coupled Devices (CCDs) and single avalanche diodes that address these issues. Even so these systems present key drawbacks, such as a low frame rate and require operation at low temperature. Direct optical detection using Complementary Metal Oxide Semiconductor (CMOS) Active Pixel Sensors (APS)could represent a suitable digital alternative for this application. In this paper the authors demonstrate the viability of direct chemiluminescent light detection in western blotting electrophoresis using a CMOS APS at room temperature. Furthermore, in recent years, improvements in fabrication techniques have made available reliable processes for very large imagers, which can be now scaled up to wafer size, allowing direct contact imaging of full size western blotting samples. We propose using a novel wafer scale APS (12.8 cm×13.2 cm), with an array architecture using two different pixel geometries that can deliver an inherently low noise and high dynamic range image at the same time representing a dramatic improvement with respect to the current western blotting imaging systems.

  9. Investigation of Yeast Mitophagy with Fluorescence Microscopy and Western Blotting.

    Science.gov (United States)

    Nagumo, Sachiyo; Okamoto, Koji

    2017-03-24

    Selective clearance of superfluous or dysfunctional mitochondria is a fundamental process that depends on the autophagic membrane trafficking pathways found in many cell types. This catabolic event, called mitophagy, is conserved from yeast to humans and serves to control mitochondrial quality and quantity. In budding yeast, degradation of mitochondria occurs under various physiological conditions, such as respiration at stationary phase, or starvation in a prolonged period. During these events, the transmembrane protein Atg32 localizes to the mitochondrial surface and plays a specific and essential role in yeast mitophagy. In this chapter, we describe methods to observe transport of mitochondria to the vacuole, a lytic compartment in yeast, using fluorescence microscopy, and semi-quantify the progression of Atg32-mediated mitophagy by Western blotting.

  10. CellSs: Scheduling Techniques to Better Exploit Memory Hierarchy

    Directory of Open Access Journals (Sweden)

    Pieter Bellens

    2009-01-01

    Full Text Available Cell Superscalar's (CellSs main goal is to provide a simple, flexible and easy programming approach for the Cell Broadband Engine (Cell/B.E. that automatically exploits the inherent concurrency of the applications at a task level. The CellSs environment is based on a source-to-source compiler that translates annotated C or Fortran code and a runtime library tailored for the Cell/B.E. that takes care of the concurrent execution of the application. The first efforts for task scheduling in CellSs derived from very simple heuristics. This paper presents new scheduling techniques that have been developed for CellSs for the purpose of improving an application's performance. Additionally, the design of a new scheduling algorithm is detailed and the algorithm evaluated. The CellSs scheduler takes an extension of the memory hierarchy for Cell/B.E. into account, with a cache memory shared between the SPEs. All new scheduling practices have been evaluated showing better behavior of our system.

  11. Endothelial cell density after deep anterior lamellar keratoplasty (Melles technique)

    NARCIS (Netherlands)

    van Dooren, Bart T. H.; Mulder, Paul G. H.; Nieuwendaal, Carla P.; Beekhuis, W. Houdijn; Melles, Gerrit R. J.

    2004-01-01

    To measure the recipient endothelial cell loss after the Melles technique for deep anterior lamellar keratoplasty. In 21 eyes of 21 patients, a deep anterior lamellar keratoplasty procedure was performed. Before surgery and at 6, 12, and 24 months after surgery, specular microscopy was performed to

  12. Introducing Mammalian Cell Culture and Cell Viability Techniques in the Undergraduate Biology Laboratory.

    Science.gov (United States)

    Bowey-Dellinger, Kristen; Dixon, Luke; Ackerman, Kristin; Vigueira, Cynthia; Suh, Yewseok K; Lyda, Todd; Sapp, Kelli; Grider, Michael; Crater, Dinene; Russell, Travis; Elias, Michael; Coffield, V McNeil; Segarra, Verónica A

    2017-01-01

    Undergraduate students learn about mammalian cell culture applications in introductory biology courses. However, laboratory modules are rarely designed to provide hands-on experience with mammalian cells or teach cell culture techniques, such as trypsinization and cell counting. Students are more likely to learn about cell culture using bacteria or yeast, as they are typically easier to grow, culture, and manipulate given the equipment, tools, and environment of most undergraduate biology laboratories. In contrast, the utilization of mammalian cells requires a dedicated biological safety cabinet and rigorous antiseptic techniques. For this reason, we have devised a laboratory module and method herein that familiarizes students with common cell culture procedures, without the use of a sterile hood or large cell culture facility. Students design and perform a time-efficient inquiry-based cell viability experiment using HeLa cells and tools that are readily available in an undergraduate biology laboratory. Students will become familiar with common techniques such as trypsinizing cells, cell counting with a hemocytometer, performing serial dilutions, and determining cell viability using trypan blue dye. Additionally, students will work with graphing software to analyze their data and think critically about the mechanism of death on a cellular level. Two different adaptations of this inquiry-based lab are presented-one for non-biology majors and one for biology majors. Overall, these laboratories aim to expose students to mammalian cell culture and basic techniques and help them to conceptualize their application in scientific research.

  13. An improved method for Southern DNA and Northern RNA blotting using a Mupid-2 Mini-Gel electrophoresis unit.

    Science.gov (United States)

    Furuya, Hirokazu; Yamada, Takeshi; Ikezoe, Koji; Ohyagi, Yasumasa; Fukumaki, Yasuyuki; Fujii, Naoki

    2006-08-31

    An improved method for Southern DNA and Northern RNA blotting using the Mupid-2 Mini-Gel System is described. We get sharp and clear bands in Southern and Northern blotting after only 30 min short gel electrophoresis instead of the several hours large gel electrophoresis of conventional methods. The high electrical voltage with a pulse-like current of the Mupid-2 Mini-Gel System also allows reduction of the amount of formaldehyde, a harmful reagent, from the gel running buffer in RNA blotting. This minor modification of DNA and RNA blotting technique enables us to perform the complete experimental procedure more quickly economically in less space, than conventional Southern and Northern blotting, as well as using an extremely small amount of formaldehyde in RNA blotting.

  14. Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using BrdU-ChIP-Slot-Western Technique.

    Science.gov (United States)

    Bhaskara, Srividya

    2016-01-14

    Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA. Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

  15. Experimental techniques for single cell and single molecule biomechanics

    International Nuclear Information System (INIS)

    Lim, C.T.; Zhou, E.H.; Li, A.; Vedula, S.R.K.; Fu, H.X.

    2006-01-01

    Stresses and strains that act on the human body can arise either from external physical forces or internal physiological environmental conditions. These biophysical interactions can occur not only at the musculoskeletal but also cellular and molecular levels and can determine the health and function of the human body. Here, we seek to investigate the structure-property-function relationship of cells and biomolecules so as to understand their important physiological functions as well as establish possible connections to human diseases. With the recent advancements in cell and molecular biology, biophysics and nanotechnology, several innovative and state-of-the-art experimental techniques and equipment have been developed to probe the structural and mechanical properties of biostructures from the micro- down to picoscale. Some of these experimental techniques include the optical or laser trap method, micropipette aspiration, step-pressure technique, atomic force microscopy and molecular force spectroscopy. In this article, we will review the basic principles and usage of these techniques to conduct single cell and single molecule biomechanics research

  16. Cell separation technique in dilectrophoretic chip with bulk electrode

    Science.gov (United States)

    Iliescu, Ciprian; Tay, Francis E. H.; Xu, Guolin; Yu, Liming

    2006-01-01

    This paper presents a new technique for separation of two cell populations in a dielectrophoretic chip with bulk silicon electrode. A characteristic of the dielectrophoretic chip is its "sandwich" structure: glass/silicon/glass that generates a unique definition of the microfluidic channel with conductive walls (silicon) and isolating floor and ceiling (glass). The structure confers the opportunity to use the electrodes not only to generate a gradient of the electric field but also to generate a gradient of velocity of the fluid inside the channel. This interesting combination gives rise to a new solution for dielectrophoretic separation of two cell populations. The separation method consists of four steps. First, the microchannel is field with the cells mixture. Second, the cells are trapped in different locations of the microfluidic channel, the cell population which exhibits positive dielectrophoresis is trapped in the area where the distance between the electrodes is the minimum whilst, the other population that exhibit negative dielectrophoresis is trapped where the distance between electrodes is the maximum. In the next step, increasing the flow in the microchannel will result in an increased hydrodynamic force that sweeps the cells trapped by positive dielectrophoresis out of the chip. In the last step, the electric field is removed and the second population is sweep out and collected at the outlet. The device was tested for separation of dead yeast cells from live yeast cells. The paper presents analytical aspects of the separation method a comparative study between different electrode profiles and experimental results.

  17. A guide to modern quantitative fluorescent western blotting with troubleshooting strategies.

    Science.gov (United States)

    Eaton, Samantha L; Hurtado, Maica Llavero; Oldknow, Karla J; Graham, Laura C; Marchant, Thomas W; Gillingwater, Thomas H; Farquharson, Colin; Wishart, Thomas M

    2014-11-20

    The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term "western blot" suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many "optimized" western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

  18. Use of Nonradioactive Detection Method for North- and South-Western Blot.

    Science.gov (United States)

    Franke, Claudia; Gräfe, Daniel; Bartsch, Holger; Bachmann, Michael P

    2015-01-01

    Many proteins bind to nucleic acids. For the first characterization of novel proteins, a fast and simple technique for testing their nucleic acid binding capabilities is desirable. Here we describe the use of a North-western and South-western blot protocol for the evaluation of the DNA and RNA binding abilities of a novel putative methyl transferase HSPC133 (METTL5).

  19. Development of a dot blot assay using gene probes for the detection of enteroviruses in water

    International Nuclear Information System (INIS)

    Margolin, A.B.

    1986-01-01

    Enteric viruses are viruses which replicate in the intestinal tract of man and animals. One mode of transmission for enteric viruses is the fecal-oral route. Drinking water which has been contaminated with sewage or sewage effluent has been implicated as a means for the spread of enteric viruses. Current methods for the detection of enteric viruses in water requires the use of animal cell culture. This technique has several drawbacks. More rapid techniques, such as fluorescent antibody or radioimmunoassay do not have the needed sensitivity to detect the low levels of virus found in contaminated water. An alternative technique for the detection of viruses in water was sought. Recent advances in recombinant DNA technology now makes it possible to detect viruses without the use of cell culture or antibodies. Gene probes that hybridize to the RNA of poliovirus and hepatitis A virus were tested for their ability to detect different enteric viruses. The probes were labeled with 32 P dCTP and 32 P dATP to a specific activity greater then 1.0 x 10 9 cpm/ug DNA. One infectious unit of poliovirus and hepatitis A virus was detected using labeled cDNA probes. Upon comparison, the dot blot assay was as sensitive as tissue culture for the detection of poliovirus in beef extract, secondary effluent, and tap water. Environmental samples, such as secondary effluent, reclaimed wastewater and unchlorinated drinking water were also assayed for poliovirus and hepatitis A virus with the use of gene probes. The results presented here offer an alternative method for screening water samples for the presence of enteric viruses

  20. Advances of Single-Cell Sequencing Technique in Tumors

    Directory of Open Access Journals (Sweden)

    Ji-feng FENG

    2017-03-01

    Full Text Available With the completion of human genome project (HGP and the international HapMap project as well as rapid development of high-throughput biochip technology, whole genomic sequencing-targeted analysis of genomic structures has been primarily finished. Application of single cell for the analysis of the whole genomics is not only economical in material collection, but more importantly, the cell will be more purified, and the laboratory results will be more accurate and reliable. Therefore, exploration and analysis of hereditary information of single tumor cells has become the dream of all researchers in the field of basic research of tumors. At present, single-cell sequencing (SCS on malignancies has been widely used in the studies of pathogeneses of multiple malignancies, such as glioma, renal cancer and hematologic neoplasms, and in the studies of the metastatic mechanism of breast cancer by some researchers. This study mainly reviewed the SCS, the mechanisms and the methods of SCS in isolating tumor cells, and application of SCS technique in tumor-related basic research and clinical treatment.

  1. Quantum dot bio-conjugate: as a western blot probe for highly sensitive detection of cellular proteins

    Science.gov (United States)

    Kale, Sonia; Kale, Anup; Gholap, Haribhau; Rana, Abhimanyu; Desai, Rama; Banpurkar, Arun; Ogale, Satishchandra; Shastry, Padma

    2012-03-01

    In the present study, we report a quantum dot (QD)-tailored western blot analysis for a sensitive, rapid and flexible detection of the nuclear and cytoplasmic proteins. Highly luminescent CdTe and (CdTe)ZnS QDs are synthesized by aqueous method. High resolution transmission electron microscopy, Raman spectroscopy, fourier transform infrared spectroscopy, fluorescence spectroscopy and X-ray diffraction are used to characterize the properties of the quantum dots. The QDs are functionalized with antibodies of prostate apoptosis response-4 (Par-4), poly(ADP-ribose) polymerases and β actin to specifically bind with the proteins localized in the nucleus and cytoplasm of the cells, respectively. The QD-conjugated antibodies are used to overcome the limitations of conventional western blot technique. The sensitivity and rapidity of protein detection in QD-based approach is very high, with detection limits up to 10 pg of protein. In addition, these labels provide the capability of enhanced identification and localization of marker proteins in intact cells by confocal laser scanning microscopy.

  2. Quantum dot bio-conjugate: as a western blot probe for highly sensitive detection of cellular proteins

    Energy Technology Data Exchange (ETDEWEB)

    Kale, Sonia [Agharkar Research Institute (India); Kale, Anup [University of Alabama, Center for Materials for Information Technology (United States); Gholap, Haribhau; Rana, Abhimanyu [National Chemical Laboratory, Physical and Materials Chemistry Division (India); Desai, Rama [National Centre for Cell Science (India); Banpurkar, Arun [University of Pune, Department of Physics (India); Ogale, Satishchandra, E-mail: sb.ogale@ncl.res.in [National Chemical Laboratory, Physical and Materials Chemistry Division (India); Shastry, Padma, E-mail: padma@nccs.res.in [National Centre for Cell Science (India)

    2012-03-15

    In the present study, we report a quantum dot (QD)-tailored western blot analysis for a sensitive, rapid and flexible detection of the nuclear and cytoplasmic proteins. Highly luminescent CdTe and (CdTe)ZnS QDs are synthesized by aqueous method. High resolution transmission electron microscopy, Raman spectroscopy, fourier transform infrared spectroscopy, fluorescence spectroscopy and X-ray diffraction are used to characterize the properties of the quantum dots. The QDs are functionalized with antibodies of prostate apoptosis response-4 (Par-4), poly(ADP-ribose) polymerases and {beta} actin to specifically bind with the proteins localized in the nucleus and cytoplasm of the cells, respectively. The QD-conjugated antibodies are used to overcome the limitations of conventional western blot technique. The sensitivity and rapidity of protein detection in QD-based approach is very high, with detection limits up to 10 pg of protein. In addition, these labels provide the capability of enhanced identification and localization of marker proteins in intact cells by confocal laser scanning microscopy.

  3. Simulation of limiting dilution technique in determination of immunocompetent cells frequency in irradiated cell cultures

    International Nuclear Information System (INIS)

    Martini Filho, R.J.; Barlette, V.E.; Goes, E.G.; Covas, D.T.; Orellana, M.

    2001-01-01

    Limiting dilution techniques (LDA) dose-response data have been used to detect immunocompetent T-Cells in microcultures. In this work, LDA frequencies estimates was obtained using χ2 minimization for irradiated cells in a range of 500 to 1,500 cGy. (author)

  4. Western blotting using in-gel protein labeling as a normalization control: stain-free technology.

    Science.gov (United States)

    Gilda, Jennifer E; Gomes, Aldrin V

    2015-01-01

    Western blotting is a commonly used laboratory technique for semi-quantifying protein amounts. It is important when quantifying protein expression to account for differences in the amount of total protein loaded onto the gel using a loading control. Common loading controls include housekeeping proteins, such as β-actin or GAPDH, quantified by Western blot, or total protein, quantified using a stain such as Coomassie Brilliant Blue or Ponceau S. A more recently developed method for total protein quantification utilizes stain-free technology, which has a linear dynamic detection range and allows for protein detection on both gels and membranes. Here, we describe the theory and use of stain-free gels for total protein quantification and normalization of Western blots.

  5. Single Particle Tracking: Analysis Techniques for Live Cell Nanoscopy

    Science.gov (United States)

    Relich, Peter Kristopher, II

    Single molecule experiments are a set of experiments designed specifically to study the properties of individual molecules. It has only been in the last three decades where single molecule experiments have been applied to the life sciences; where they have been successfully implemented in systems biology for probing the behaviors of sub-cellular mechanisms. The advent and growth of super-resolution techniques in single molecule experiments has made the fundamental behaviors of light and the associated nano-probes a necessary concern amongst life scientists wishing to advance the state of human knowledge in biology. This dissertation disseminates some of the practices learned in experimental live cell microscopy. The topic of single particle tracking is addressed here in a format that is designed for the physicist who embarks upon single molecule studies. Specifically, the focus is on the necessary procedures to generate single particle tracking analysis techniques that can be implemented to answer biological questions. These analysis techniques range from designing and testing a particle tracking algorithm to inferring model parameters once an image has been processed. The intellectual contributions of the author include the techniques in diffusion estimation, localization filtering, and trajectory associations for tracking which will all be discussed in detail in later chapters. The author of this thesis has also contributed to the software development of automated gain calibration, live cell particle simulations, and various single particle tracking packages. Future work includes further evaluation of this laboratory's single particle tracking software, entropy based approaches towards hypothesis validations, and the uncertainty quantification of gain calibration.

  6. Advances in PEM fuel cells with CFD techniques

    Energy Technology Data Exchange (ETDEWEB)

    Robalinho, Eric; Cunha, Edgar Ferrari da; Zararya, Ahmed; Linardi, Marcelo [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)], Email: eric@ipen.br; Cekinski, Efrain [Instituto de Pesquisas Tecnologicas (IPT), Sao Paulo, SP (Brazil)

    2010-07-01

    This paper presents some applications of computational fluid dynamics techniques in the optimization of Proton Exchange Membrane Fuel Cell (PEMFC) designs. The results concern: modeling of gas distribution channels, the study for both porous anode and cathode and the three-dimensional modeling of a partial geometry layer containing catalytic Gas Diffusion Layers (GDL) and membrane. Numerical results of the simulations of graphite plates flow channels, using ethanol as fuel, are also presented. Some experimental results are compared to the corresponding numerical ones for several cases, demonstrating the importance and usefulness of this computational tool. (author)

  7. Multistrip Western blotting to increase quantitative data output

    OpenAIRE

    Kiyatkin, Anatoly; Aksamitiene, Edita

    2009-01-01

    The qualitative and quantitative measurements of protein abundance and modification states are essential in understanding their functions in diverse cellular processes. Typical Western blotting, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. Multistrip Western blotting is a modified immunoblotting procedure based on simultaneous electrophoretic transfer of proteins from multiple strips of polyacrylamide gels to a single mem...

  8. Cells and Stripes: A novel quantitative photo-manipulation technique.

    Science.gov (United States)

    Mistrik, Martin; Vesela, Eva; Furst, Tomas; Hanzlikova, Hana; Frydrych, Ivo; Gursky, Jan; Majera, Dusana; Bartek, Jiri

    2016-01-18

    Laser micro-irradiation is a technology widely used in the DNA damage response, checkpoint signaling, chromatin remodeling and related research fields, to assess chromatin modifications and recruitment of diverse DNA damage sensors, mediators and repair proteins to sites of DNA lesions. While this approach has aided numerous discoveries related to cell biology, maintenance of genome integrity, aging and cancer, it has so far been limited by a tedious manual definition of laser-irradiated subcellular regions, with the ensuing restriction to only a small number of cells treated and analyzed in a single experiment. Here, we present an improved and versatile alternative to the micro-irradiation approach: Quantitative analysis of photo-manipulated samples using innovative settings of standard laser-scanning microscopes. Up to 200 cells are simultaneously exposed to a laser beam in a defined pattern of collinear rays. The induced striation pattern is then automatically evaluated by a simple algorithm, which provides a quantitative assessment of various laser-induced phenotypes in live or fixed cells. Overall, this new approach represents a more robust alternative to existing techniques, and provides a versatile tool for a wide range of applications in biomedicine.

  9. Three-dimensional cell culture technique and pathophysiology.

    Science.gov (United States)

    Matsusaki, Michiya; Case, Charles Patrick; Akashi, Mitsuru

    2014-07-01

    Three-dimensional (3D) tissue constructs consisting of human cells have opened a new avenue for tissue engineering, pharmaceutical and pathophysiological applications, and have great potential to estimate the dynamic pharmacological effects of drug candidates, metastasis processes of cancer cells, and toxicity expression of nano-materials, as a 3D-human tissue model instead of in vivo animal experiments. However, most 3D-cellular constructs are a cell spheroid, which is a heterogeneous aggregation, and thus the reconstruction of the delicate and precise 3D-location of multiple types of cells is almost impossible. In recent years, various novel technologies to develop complex 3D-human tissues including blood and lymph capillary networks have demonstrated that physiological human tissue responses can be replicated in the nano/micro-meter ranges. Here, we provide a brief overview on current 3D-tissue fabrication technologies and their biomedical applications. 3D-human tissue models will be a powerful technique for pathophysiological applications. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Mixed lubrication after rewetting of blotted pleural mesothelium.

    Science.gov (United States)

    Bodega, Francesca; Sironi, Chiara; Porta, Cristina; Pecchiari, Matteo; Zocchi, Luciano; Agostoni, Emilio

    2013-01-15

    Coefficient of kinetic friction (μ) of pleural mesothelium blotted with filter paper, and rewetted with Ringer solution markedly increases; this increase is removed if a sufficient amount of sialomucin or hyaluronan is added to Ringer (Bodega et al., 2012. Respiratory Physiology and Neurobiology 180, 34-39). In this research we found that μ of pleural mesothelium blotted, rewetted, and sliding at physiological velocities and loads, decreased with increase of velocity, mainly at low velocities. Despite this decrease, μ at highest velocity was still double that before blotting. With small concentration of sialomucin or hyaluronan μ was markedly smaller at each velocity, decreased less with increase of velocity, and at highest velocity approached preblotting value. These findings indicate a regime of mixed lubrication in post-blotting Ringer, at variance with boundary lubrication occurring before blotting or postblotting with sufficient macromolecule addition. Greater roughness of mesothelial surface, caused by blotting, likely induces zones of elastohydrodynamic lubrication, which increase with velocity, while contact area decreases. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Far-western blotting as a solution to the non-specificity of the anti-erythropoietin receptor antibody.

    Science.gov (United States)

    Fecková, Barbora; Kimáková, Patrícia; Ilkovičová, Lenka; Szentpéteriová, Erika; Debeljak, Nataša; Solárová, Zuzana; Sačková, Veronika; Šemeláková, Martina; Bhide, Mangesh; Solár, Peter

    2016-08-01

    The erythropoietin receptor (EpoR) is a member of the cytokine receptor family. The interaction between erythropoietin (Epo) and EpoR is important for the production and maturation of erythroid cells, resulting in the stimulation of hematopoiesis. The fact that EpoR was also detected in neoplastic cells has opened the question about the relevance of anemia treatment with recombinant Epo in cancer patients. Numerous studies have reported pro-stimulating and anti-apoptotic effects of Epo in cancer cells, thus demonstrating EpoR functionality in these cells. By contrast, a previous study claims the absence of EpoR in tumor cells. This apparent discrepancy is based, according to certain authors, on the use of non-specific anti-EpoR antibodies. With the aim of bypassing the direct detection of EpoR with an anti-EpoR antibody, the present authors propose a far-western blot methodology, which in addition, confirms the interaction of Epo with EpoR. Applying this technique, the presence of EpoR and its interaction with Epo in human ovarian adenocarcinoma A2780 and normal human umbilical vein endothelial cells was confirmed. Furthermore, modified immunoprecipitation of EpoR followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis confirmed a 57 kDa protein as a human Epo-interacting protein in both cell lines.

  12. Effect of fabrication technique on direct methanol fuel cells designed to operate at low airflow

    Science.gov (United States)

    Valdez, T. I.; Narayanan, S. R.

    2002-01-01

    This study investigates the effects of catalyst ink constituents and MEA fabrication techniques on improving cell performance. Particular attention was focused on increasing the overall cell efficiency.

  13. The Use of Biotin to Demonstrate Immunohistochemistry, Western Blotting, and Dot Blots in University Practical Classes

    Science.gov (United States)

    Millar, Thomas James; Knighton, Ronald; Chuck, Jo-Anne

    2012-01-01

    Immunological detection of proteins is an essential method to demonstrate to undergraduate biology students, however, is often difficult in resource and time poor student laboratory sessions. This method describes a failsafe method to rapidly and economically demonstrate this technique using biotinylated proteins or biotin itself as targets for…

  14. Far Western blotting as a rapid and efficient method for detecting interactions between DNA replication and DNA repair proteins.

    Science.gov (United States)

    Walsh, Brian W; Lenhart, Justin S; Schroeder, Jeremy W; Simmons, Lyle A

    2012-01-01

    Protein-protein interactions are required for the proper function of many biological pathways. Numerous biochemical and protein blotting methods are available for probing direct and indirect interactions between two protein-binding partners. Here, we describe the methodology of far Western blotting, or immunodot blotting, as a technique for probing direct interactions between two proteins. We describe the utility of this approach as a rapid, qualitative screen for identifying novel protein-binding partners. We also describe the importance of this technique for measuring differences in interaction between wild-type and mutant forms of a known binding partner. Far Western blotting is a rapid and highly reproducible experimental approach for identifying and understanding the interaction between protein-binding partners leading to new discoveries in the function and regulation of biological pathways.

  15. Western Blot of Stained Proteins from Dried Polyacrylamide Gels

    Science.gov (United States)

    Gruber, Claudia; Stan-Lotter, Helga

    1996-01-01

    Western blotting of proteins is customarily performed following their separation on polyacrylamide gels, either prior to staining (1) or, as recently reported, following staining (2). We describe here Western blotting with stained gels, which had been dried and some of which had been stored for years. This procedure permits immunological analysis of proteins, to which antisera may have become available only later, or where the application of newly developed sensitive detection methods is desired. Once rehydration of the gels is achieved, proteins can be-transferred to blotting membranes by any appropriate protocol. Proteins stained with Coomassie Blue have to be detected with a non-chromogenic method, such as the film-based enhanced chemiluminescence (ECL)2) procedure (3). Silver stained proteins, which transfer in the colorless form, may be visualized by any detection method, although, because of the usually very low amounts of proteins, detection by ECL is preferable. Blotting of stained proteins from rehydrated gels is as rapid and as quantitative as from freshly prepared gels, in contrast to blotting from wet stained gels, which requires extensive washing and results in low transfer efficiency (2). Together with a photographic record of the gel pattern, unambiguous identification of immunoreactive proteins from complex mixtures is possible. Some further applications of this work are discussed.

  16. Western blotting via proximity ligation for high performance protein analysis.

    Science.gov (United States)

    Liu, Yanling; Gu, Jijuan; Hagner-McWhirter, Åsa; Sathiyanarayanan, Poojahrau; Gullberg, Mats; Söderberg, Ola; Johansson, Johan; Hammond, Maria; Ivansson, Daniel; Landegren, Ulf

    2011-11-01

    Western blotting is a powerful and widely used method, but limitations in detection sensitivity and specificity, and dependence upon high quality antibodies to detect targeted proteins, are hurdles to overcome. The in situ proximity ligation assay, based on dual antibody recognition and powerful localized signal amplification, offers increased detection sensitivity and specificity, along with an ability to identify complex targets such as phosphorylated or interacting proteins. Here we have applied the in situ proximity ligation assay mechanism in Western blotting. This combination allowed the use of isothermal rolling circle amplification of DNA molecules formed in target-specific ligation reaction, for 16-fold or greater increase in detection sensitivity. The increased specificity because of dual antibody recognition ensured highly selective assays, detecting the specific band when combinations of two cross-reactive antitubulin antibodies were used (i.e. both producing distinct nonspecific bands in traditional Western blotting). We also demonstrated detection of phosphorylated platelet-derived growth factor receptor β by proximity ligation with one antibody directed against the receptor and another directed against the phosphorylated tyrosine residue. This avoided the need for stripping and re-probing the membrane or aligning two separate traditional blots. We demonstrate that the high-performance in situ proximity ligation-based Western blotting described herein is compatible with detection via enhanced chemiluminescence and fluorescence detection systems, and can thus be readily employed in any laboratory.

  17. Genetic relatedness of orbiviruses by RNA-RNA blot hybridization

    International Nuclear Information System (INIS)

    Bodkin, D.K.

    1985-01-01

    RNA-RNA blot hybridization was developed in order to identify type-specific genes among double-stranded (ds) RNA viruses, to assess the genetic relatedness of dsRNA viruses and to classify new strains. Viral dsRNA segments were electrophoresed through 10% polyacrylamide gels, transferred to membranes, and hybridized to [5' 32 P]-pCp labeled genomic RNA from a related strain. Hybridization was performed at 52 0 C, 50% formamide, 5X SSC. Under these conditions heterologous RNA species must share ≥ 74% sequence homology in order to form stable dsRNA hybrids. Cognate genes of nine members of the Palyam serogroup of orbiviruses were identified and their sequence relatedness to the prototype. Palyam virus, was determined. Reciprocal blot hybridizations were performed using radiolabeled genomic RNA of all members of the Palyam serogroup. Unique and variant genes were identified by lack of cross-homology or by weak homology between segments. Since genes 2 and 6 exhibited the highest degree of sequence variability, response to the vertebrate immune system may be a major cause of sequence divergence among members of a single serogroup. Changuinola serogroup isolates were compared by dot-blot hybridization, while Colorado tick fever (CTF) serogroup isolates were compared by the RNA-RNA blot hybridization procedure described for reovirus and Palyam serogroup isolates. Preliminary blot hybridization data were also obtained on the relatedness of members of different Orbivirus serogroups

  18. Improvement of western blot test specificity for detecting equine serum antibodies to Sarcocystis neurona.

    Science.gov (United States)

    Rossano, M G; Mansfield, L S; Kaneene, J B; Murphy, A J; Brown, C M; Schott, H C; Fox, J C

    2000-01-01

    Equine protozoal myeloencephalitis (EPM) is a neurological disease of horses and ponies caused by the apicomplexan protozoan parasite Sarcocystis neurona. The purposes of this study were to develop the most stringent criteria possible for a positive test result, to estimate the sensitivity and specificity of the EPM Western blot antibody test, and to assess the ability of bovine antibodies to Sarcocystis cruzi to act as a blocking agent to minimize false-positive results in the western blot test for S. neurona. Sarcocystis neurona merozoites harvested from equine dermal cell culture were heat denatured, and the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a 12-20% linear gradient gel. Separated proteins were electrophoretically transferred to polyvinylidene fluoride membranes and blocked in 1% bovine serum albumin and 0.5% Tween-Tris-buffered saline. Serum samples from 6 horses with S. neurona infections (confirmed by culture from neural tissue) and 57 horses without infections (horses from the Eastern Hemisphere, where S. neurona does not exist) were tested by Western blot. Horses from both groups had reactivity to the 62-, 30-, 16-, 13-, 11-, 10.5-, and 10-kD bands. Testing was repeated with another step. Blots were treated with bovine S. cruzi antibodies prior to loading the equine samples. After this modification of the Western blot test, positive infection status was significantly associated with reactivity to the 30- and 16-kD bands (Pblot had a sample sensitivity of 100% and sample specificity of 98%. It is concluded that the specificity of the Western blot test is improved by blocking proteins not specific to S. neurona and using reactivity to the 30- and 16-kD bands as the criterion for a positive test.

  19. A Fast and Inexpensive Western Blot Experiment for the Undergraduate Laboratory

    Science.gov (United States)

    Farrell, Shawn O.; Farrell, Lynn E.

    1995-08-01

    Western blotting is an important, modern technique for transferring proteins from a gel onto nitrocellulose or other suitable support and then detecting a protein of interest using antibodies. We have developed an experiment and optimized the conditions for the undergraduate laboratory. The experiment can be done quickly using an electrophoretic blotter or more cheaply using passive transfer. This experiment allows the student to learn valuable procedures currently used in biochemistry and other biological sciences.

  20. Better management of Western blotting results using professional photo management software.

    Science.gov (United States)

    Iorio-Morin, Christian; Germain, Pascale; Parent, Jean-Luc

    2013-04-01

    Western blotting is a proven technique essential to a significant proportion of molecular biology projects. However, as results accumulate over the years, managing data can become daunting. Recognizing that the needs of a scientist working with Western blotting results are conceptually the same as those of a professional photographer managing a summer's worth of wedding photos, we report here a new workflow for managing Western blotting results using professional photo management software. The workflow involves (i) scanning all film-based results; (ii) importing the scans into the software; (iii) processing the scans; (iv) tagging the files with metadata, and (v) creating appropriate "smart-albums." Advantages of this system include space savings (both on our hard drives and on our desks), safer archival, quicker access, and easier sharing of the results. In addition, metadata-based workflows improve cross-experiment discovery and enable questions like "show me all blots labelled with antibody X" or "show me all experiments featuring protein Y". As project size and breadth increase, workflows delegating results management to the computer will become more and more important so that scientists can keep focussing on science. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Evaluation of an adaptive virtual laboratory environment using Western Blotting for diagnosis of disease.

    Science.gov (United States)

    Polly, Patsie; Marcus, Nadine; Maguire, Danni; Belinson, Zack; Velan, Gary M

    2014-10-20

    Providing large numbers of undergraduate students in scientific disciplines with engaging, authentic laboratory experiences is important, but challenging. Virtual laboratories (vLABs) are a potential means to enable interactive learning experiences. A vLAB focusing on Western Blotting was developed and implemented in a 3rd year undergraduate Pathology course for science students to facilitate learning of technical molecular laboratory skills that are linked to development of diagnostic skills. Such skills are important for undergraduates in building a conceptual understanding of translation of laboratory techniques to changes in human biology due to disease. The Western Blotting vLAB was developed and deployed using the Adaptive eLearning Platform (AeLP) developed by Smart Sparrow (https://www.smartsparrow.com/). The vLAB was evaluated to assess students' perceptions of their laboratory skills relevant to the diagnosis of Muscular Dystrophy. A blended learning rotation model was applied in which wet laboratory and vLAB environments for Western Blotting were both delivered to three consecutive cohorts of 3rd year science undergraduates undertaking a Muscle Diseases practical class. Evaluation questionnaires were administered at the completion of the practical classes. Students indicated in online questionnaires that the Western Blotting vLAB was at least equivalent to the real lab in their perceived development of concepts, laboratory skills and diagnosis of disease. vLABs have great potential for improving students' development of diagnostic skills. Further studies are required to determine the impact of vLABs on student learning.

  2. Miniaturized fluorescent RNA dot blot method for rapid quantitation of gene expression

    Directory of Open Access Journals (Sweden)

    Yadetie Fekadu

    2004-06-01

    Full Text Available Abstract Background RNA dot blot hybridization is a commonly used technique for gene expression assays. However, membrane based RNA dot/slot blot hybridization is time consuming, requires large amounts of RNA, and is less suited for parallel assays of more than one gene at a time. Here, we describe a glass-slide based miniaturized RNA dot blot (RNA array procedure for rapid and parallel gene expression analysis using fluorescently labeled probes. Results RNA arrays were prepared by simple manual spotting of RNA onto amino-silane coated microarray glass slides, and used for two-color fluorescent hybridization with specific probes labeled with Cy3 and 18S ribosomal RNA house-keeping gene probe labeled with Cy5 fluorescent dyes. After hybridization, arrays were scanned on a fluorescent microarray scanner and images analyzed using microarray image analysis software. We demonstrate that this method gives comparable results to Northern blot analysis, and enables high throughput quantification of transcripts from nanogram quantities of total RNA in hundreds of samples. Conclusion RNA array on glass slide and detection by fluorescently labeled probes can be used for rapid and parallel gene expression analysis. The method is particularly well suited for gene expression assays that involve quantitation of many transcripts in large numbers of samples.

  3. Combined use of Western blot/ELISA to improve the serological diagnosis of human tuberculosis

    Directory of Open Access Journals (Sweden)

    S. T. Beck

    Full Text Available Two recombinant antigens and a crude bacterial antigen of a wild M. tuberculosis strain were used to detect specific IgG antibodies in sera from 52 patients with pulmonary tuberculosis, confirmed by an acid-fast smear and serum culture of these patients and that of 25 contacts. The patients were not infected with HIV. We evaluated the sensitivity and specificity of ELISA, based on the recombinant TbF6® and TbF6/DPEP antigen and a search for reactivity patterns in the Western blot technique, using whole mycobacterium antigen. Serum samples from 22 healthy individuals and from 30 patients with lung diseases other than tuberculosis were used as controls. The best ELISA results were obtained with the TbF6/DPEP antigen combination, which gave 85% sensitivity and 91% specificity. ELISA sensitivity improved from 85% to 92% when the Western blot results were used. Western blot specificity was 100% when antibody reactivity with different antigenic bands was analyzed and associated. The association of TbF6/DPEP antigens used in ELISA with specific patterns of reactivity determined by Western blot can help make an identification when classic methods for the diagnosis of pulmonary tuberculosis are not sufficient.

  4. Combined use of Western blot/ELISA to improve the serological diagnosis of human tuberculosis.

    Science.gov (United States)

    Beck, Sandra Trevisan; Leite, O M; Arruda, R S; Ferreira, A W

    2005-02-01

    Two recombinant antigens and a crude bacterial antigen of a wild M. tuberculosis strain were used to detect specific IgG antibodies in sera from 52 patients with pulmonary tuberculosis, confirmed by an acid-fast smear and serum culture of these patients and that of 25 contacts. The patients were not infected with HIV. We evaluated the sensitivity and specificity of ELISA, based on the recombinant TbF6 and TbF6/DPEP antigen and a search for reactivity patterns in the Western blot technique, using whole mycobacterium antigen. Serum samples from 22 healthy individuals and from 30 patients with lung diseases other than tuberculosis were used as controls. The best ELISA results were obtained with the TbF6/DPEP antigen combination, which gave 85% sensitivity and 91% specificity. ELISA sensitivity improved from 85% to 92% when the Western blot results were used. Western blot specificity was 100% when antibody reactivity with different antigenic bands was analyzed and associated. The association of TbF6/DPEP antigens used in ELISA with specific patterns of reactivity determined by Western blot can help make an identification when classic methods for the diagnosis of pulmonary tuberculosis are not sufficient.

  5. Detection of autoimmunity in early primary Epstein-Barr virus infection by Western blot analysis.

    Science.gov (United States)

    Mascia, M T; Sandri, G; Guerzoni, C; Roncaglia, R; Mantovani, G; Ferri, C

    2008-01-01

    The Epstein-Barr virus (EBV) represents a potentially important factor in the pathogenesis of certain autoimmune disorders such as systemic lupus erythematosus (SLE), and Sjögren's syndrome, probably through a molecular mimicry mechanism. Several studies have focused on the relationship between previous EBV infection and clinically overt connective tissue diseases (CTDs), while the aim of this study was to investigate the immunological alterations during the early phase of primary acute EBV infection by means of ENA Western blotting (WB) analysis. This technique is able to detect a wide spectrum of anti-ENA autoantibodies, potentially directed against diverse epitopes of the same antigen. Sera from 54 subjects (F/M=24/30, mean age 17+/-6 SD years) with primary acute EBV infection were analysed using indirect immunofluorescence (IF) on Hep-2 cells for ANA, and both ELISA and WB for ENA. Only 8 ANA+ and no ENA+ were found by means of IF and ELISA techniques, respectively; however, one or more ENA autoantibodies were detected in 24/54 (44%) sera using WB. The autoantibodies were no longer present at the second evaluation. Subjects with immunological alterations had not developed any significant clinical manifestations at a 5-year follow-up. This study demonstrated the appearance of autoantibody production in a high proportion of individuals with primary acute EBV infection; interestingly, the observed serological subsets are quite similar to clinical SLE clusters. Moreover, the absence of immunological disorders during the follow-up reinforces the role of multiple genetic and/or environmental co-factors in the pathogenesis of CTDs.

  6. Routine Western blot to check autophagic flux : Cautions and recommendations

    NARCIS (Netherlands)

    Gomez-Sanchez, Ruben; Pizarro-Estrella, Elisa; Yakhine-Diop, Sokhna M. S.; Rodriguez-Arribas, Mario; Bravo-San Pedro, Jose M.; Fuentes, Jose M.; Gonzalez-Polo, Rosa A.

    2015-01-01

    At present, the analysis of autophagic flux by Western blotting (WB), which measures two of the most important markers of autophagy, i.e., microtubule-associated protein 1 light chain 3 (LC3) and p62, is widely accepted in the scientific community. In this study, we addressed the possible

  7. Effects of Reusing Gel Electrophoresis and Electrotransfer Buffers on Western Blotting.

    Science.gov (United States)

    Heda, Ghanshyam D; Omotola, Oluwabukola B; Heda, Rajiv P; Avery, Jamie

    2016-09-01

    SDS-PAGE and Western blotting are 2 of the most commonly used biochemical methods for protein analysis. Proteins are electrophoretically separated based on their MWs by SDS-PAGE and then electrotransferred to a solid membrane surface for subsequent protein-specific analysis by immunoblotting, a procedure commonly known as Western blotting. Both of these procedures use a salt-based buffer, with the latter procedure consisting of methanol as an additive known for its toxicity. Previous reports present a contradictory view in favor or against reusing electrotransfer buffer, also known as Towbin's transfer buffer (TTB), with an aim to reduce the toxic waste. In this report, we present a detailed analysis of not only reusing TTB but also gel electrophoresis buffer (EB) on proteins of low to high MW range. Our results suggest that EB can be reused for at least 5 times without compromising the electrophoretic separation of mixture of proteins in an MW standard, BSA, and crude cell lysates. Additionally, reuse of EB did not affect the quality of subsequent Western blots. Successive reuse of TTB, on the other hand, diminished the signal of proteins of different MWs in a protein standard and a high MW membrane protein cystic fibrosis transmembrane-conductance regulator (CFTR) in Western blotting.

  8. Investigation of asymptomatic visceral leishmaniasis cases using western blot in an endemic area in Turkey.

    Science.gov (United States)

    Sakru, Nermin; Korkmaz, Metin; Ozbel, Yusuf; Ertabaklar, Hatice; Sengul, Mustafa; Toz, Seray Ozensoy

    2007-01-01

    In Turkey, Leishmania infantum is responsible for human visceral leishmaniasis (VL), which is seen mainly in the Aegean, Mediterranean, and Central Anatolia Regions. This study aimed to determine asymptomatic infections in an endemic area of VL in Turkey using the western blot technique. A total of 82 persons including children and adults were chosen randomly in Denizli province which is one of the endemic sites for VL. Serum samples were collected and screened using indirect immunofluorescent test (IFAT), enzyme-linked immunosorbent assay (ELISA) and western blot (WB). One year later, 35 of the 82 persons were sampled and screened serologically for the second time. Seven out of 82 samples were found to be positive by western blot analysis with the presence of 14 and/or 18 kDa bands. Two of these seven sera were also positive by IFAT, but only one of these two was positive by ELISA. Only one person showing seropositivity with all three tests had clinical symptoms and was diagnosed as VL with the presence of amastigotes in bone marrow aspirate. Because six people, including the one found to be seropositive in all two tests, had no clinical symptoms, they were accepted as asymptomatic carriers. The ratio of asymptomatic infection was calculated as 7.41% (6/81) in the region. In the second sampling, the western blot revealed antibodies against the same antigens in all seven subjects. Our findings showed that the presence of antibodies against 14 and 18 kDa antigens are important for the diagnosis of symptomatic and asymptomatic infections. Western blot was found to be effective in the detection of asymptomatic persons in the epidemiological studies in endemic areas.

  9. Microchamber Western blotting using poly-L-lysine conjugated polyacrylamide gel for blotting of sodium dodecyl sulfate coated proteins.

    Science.gov (United States)

    Chung, Minsub; Kim, Dohyun; Herr, Amy E

    2013-08-20

    We report a novel strategy to immobilize sodium dodecyl sulfate (SDS)-coated proteins for fully integrated microfluidic Western blotting. Polyacrylamide gel copolymerized with a cationic polymer, poly-L-lysine, effectively immobilizes all sized proteins after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and enables SDS-PAGE and subsequent immuno-probing in an automated microfluidic chip. Design of a poly-l-lysine conjugated polyacrylamide gel allows optimization of SDS-protein immobilization strength in the blotting gel region of the microchamber. The dependence of protein capture behavior on both the concentration of copolymerized charges and poly-lysine length is studied and gives important insight into an electrostatic immobilization mechanism. Based on analysis of protein conformation, the immobilized proteins bind with partner antibody after SDS dilution. We demonstrate each step of the microchamber Western blot, including injection, separation, transfer, immobilization, blocking, and immunoblot. The approach advances microfluidic protein immunoblotting, which is directly relevant to the widely-used SDS-PAGE based slab-gel Western blot, while saving sample volume, labor, and assay time.

  10. Ri antibodies in patients with breast, ovarian or small cell lung cancer determined by a sensitive immunoprecipitation technique.

    Science.gov (United States)

    Knudsen, Anette; Monstad, Sissel E; Dørum, Anne; Lønning, Per E; Salvesen, Helga B; Drivsholm, Lars; Aarseth, Jan H; Vedeler, Christian A

    2006-10-01

    The presence of circulating antineuronal antibodies has been associated with paraneoplastic neurological syndromes (PNS). Ri antibodies are often associated with lung or breast cancer, but the prevalence of such antibodies in large cancer materials is largely unknown. We used a highly sensitive immunoprecipitation assay to study the level of Ri antibodies in blood samples from 200 patients with small cell lung cancer (SCLC), 253 patients with breast cancer and 557 patients with ovarian cancer. Two hundred blood donors and six Ri positive PNS patients served as controls. The recombinant antigen used in the immunoprecipitation assay was radiolabeled by a coupled in vitro transcription and translation (ITT) technique, enabling low levels of antibodies to be detected. None of the blood donors contained Ri antibodies, whereas all of the sera from the PNS patients were positive. Ri antibodies were present in 4.5% of the patients with SCLC, 0.8% of the patients with breast cancer and in 0.2% of the patients with ovarian cancer. Retesting of the Ri positive samples with immunofluorescense and immune blot showed that the immunoprecipitation technique was more sensitive than the other immune assays. Ri antibodies were not associated with PNS in the patients with breast or ovarian cancer. Neurological data were not available for the SCLC patients, but in these, Ri antibodies were not associated with survival.

  11. HIV‑2 antibody detection after indeterminate or negative HIV‑1 Western blot in Cuba, 2005-2008.

    Science.gov (United States)

    Díaz, Dervel F; Ortiz, Eva; Martín, Dayamí; Nibot, Carmen; Rizo, Adis; Silva, Eladio

    2012-01-01

    that HIV-2 Western blot be included in the diagnostic algorithm for HIV-1/2 to followup negative or indeterminate HIV-1 Western blot results. KEYWORDS Diagnosis, laboratory techniques and procedures, antibodies, HIV-2, Western blot, enzyme-linked immunosorbent assay, algorithm, Cuba.

  12. Demonstration of functional low-density lipoprotein receptors by protein blotting in fibroblasts from a subject with homozygous receptor-negative familial hypercholesterolemia

    International Nuclear Information System (INIS)

    Semenkovich, C.F.; Ostlund, R.E. Jr.; Yang, J.; Reaban, M.E.

    1985-01-01

    We report the detection of low-density lipoprotein (LDL) receptors by the technique of receptor blotting in fibroblasts from a patient with homozygous familial hypercholesterolemia (FHC) previously classified as ''receptor negative.'' Solubilized receptors were electrophoresed, transferred to nitrocellulose paper, treated with LDL followed by radiolabeled antibody to LDL, and visualized by autoradiography. GM 2000 FHC fibroblasts revealed LDL receptors with an apparent molecular weight of approximately 140,000, the same as in normal cells. LDL receptor activity by blotting in GM 2000 cells was greatly diminished in comparison with normal cells, but was calcium dependent. Receptor activity was also detectable by conventional monolayer binding and degradation assays. Thus, GM 2000 cells have profoundly diminished LDL receptor activity, but retain the genetic capacity to make LDL receptor material of normal molecular weight that is capable of binding LDL. Previous studies have demonstrated the presence of trace amounts of immunoreactive LDL receptor protein in fibroblasts from some receptor-negative FHC homozygotes. These studies are extended by demonstrating the ability of this material to bind LDL

  13. A Technique for Designing Variation Resilient Subthreshold Sram Cell

    Directory of Open Access Journals (Sweden)

    Aminul Islam

    2013-04-01

    Full Text Available This paper presents a technique for designing a variability aware subthreshold SRAM cell. The architecture of the proposed cell is similar to the standard read-decoupled 8-transistor (RD8T SRAM cell with the exception that the access FETS are replaced with transmission gates (TGs. In this work, various design metrics are assessed and compared with RD8T SRAM cell. The proposed design offers 2.14× and 1.75× improvement in TRA (read access time and TWA (write access time respectively compared with RD8T. It proves its robustness against process variations by featuring narrower spread in TRA distribution (2.35× and TWA distribution (3.79× compared with RD8T. The proposed bitcell offers 1.16× higher read current (IREAD and 1.64× lower bitline leakage current (ILEAK respectively compared with RD8T. It also shows its robustness by offering 1.34× (1.58× tighter spread in IREAD (ILEAK compared with RD8T. It exhibits 1.42× larger IREAD to ILEAK ratio. It shows 2.2× higher frequency @ 250 mV with read bitline capacitance of 10 fF. Besides, the proposed bitcell achieves same read stability and write-ability as that of RD8T at the cost of 3 extra transistors. The leakage power of the proposed design is close to that of RD8T.   ABSTRAK: Kertas kerja ini membentangkan teknik merekabentuk sel bawah ambang SRAM yang bolehubah. Senibina sel yang dicadangkan adalah sama dengan sel SRAM 8-transistor (RD8T “pisahan-bacaan” piawai kecuali FET akses  digantikan dengan sel pintu transmisi (TGs. Di dalam kajian ini, beberapa metrik rekabentuk dinilai dan dibandingkan dengan sel RD8T SRAM. Rekabentuk yang dicadangkan menawarkan  peningkatan 2.14× dan 1.75×  dalam TRA (masa akses baca dan TWA (masa akses tulis berbanding dengan RD8T. Ia membuktikan kekukuhan variasi proses dengan menampilkan tebaran yang lebih sempit dalam pengagihan TRA (2.35 × dan pengagihan TWA (3.79 × berbanding dengan RD8T. Sel-Bit yang dicadangkan mempunyai arus baca 1.16

  14. Western Blotting Against Tagged Virulence Determinants to Study Bacterial Pathogenicity.

    Science.gov (United States)

    Aviv, Gili; Gal-Mor, Ohad

    2018-01-01

    Western blotting is a common approach to detect the presence of a target protein in biological samples or proteins mixture using specific antibodies. This method is also useful to study regulation of virulence determinants by analyzing changes in protein expression between different genetic backgrounds or under varying environmental conditions. To avoid the need to raise specific antibodies for each studied protein, commercial antibody against commonly used peptidic epitopes can be utilized if the right target tagged version is constructed. Here we describe a C-terminal fusion between a protein of interest and the two hemagglutinin A (2HA) tag. The tagged protein is cloned into a low-copy number vector and expressed under its native promoter in Salmonella enterica. Then, the expression of the tagged protein can be analyzed by Western blotting and commercially available anti-2HA antibodies.

  15. Fingerprinting of Natural Product by Eastern Blotting Using Monoclonal Antibodies

    OpenAIRE

    Tanaka, Hiroyuki; Putalun, Waraporn; Shoyama, Yukihiro

    2012-01-01

    We succeeded in developing the fingerprint of natural product by eastern blotting using monoclonal antibodies. After developing and separating them on a TLC plate, solasodine glycosides are oxidized by NaIO4 and reacted with a protein to give conjugates which are recognized with anti-solamargine monoclonal antibody (MAb). Anti-solamargine MAb having wide cross-reactivity can stain and detect all solasodine glycosides by fingerprint. Different sensitivity between solamargine and solasonine was...

  16. Standardization of Licorice and TCM Formulations Using Eastern Blot Fingerprinting Analysis

    Directory of Open Access Journals (Sweden)

    Yukihiro Shoyama

    2013-01-01

    Full Text Available To prepare the antiglycyrrhizin (GC monoclonal antibody (MAb, GC was treated with NaIO4 resulting in aldehyde which can be combined with carrier protein. An antigen conjugate was performed by a matrix-assisted laser desorption/ionization TOF mass spectrometry to determine the hapten numbers in the conjugate. Anti-GC MAb was prepared from a hybridoma which was fixed from the spleen cells producing anti-GC MAb and the myeloma cells after immunization. The TCM and licorice extract were developed by TLC and blotted to a polyvinylidene difluoride (PVDF membrane. The membrane was treated by NaIO4 and protein, enzyme labeled secondary MAb, and finally substrate was added. Clear spot appeared on PVDF membrane identifying GC against a background containing large amount of impurities. In eastern blotting, the GC molecule was divided into two functions. The aglycone part is recognized as an epitope and the sugar moiety can be combined to membrane. The specific reactivity of sugar moiety in the GC molecule against anti-GC MAb might be modified by the NaIO4 treatment on the membrane because glycyrrhetic acid 3-O-glucuronide can be stained although the cross-reactivity is only 4.3%. Eastern blotting for GC can not only apply for the standardization of licorice and TCM, but also it can open for the other bioactive products.

  17. V3 stain-free workflow for a practical, convenient, and reliable total protein loading control in western blotting.

    Science.gov (United States)

    Posch, Anton; Kohn, Jonathan; Oh, Kenneth; Hammond, Matt; Liu, Ning

    2013-12-30

    The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a "black box" because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-free technology to address the major concerns associated with the traditional western blot protocol. This workflow allows researchers: 1) to run a gel in about 20-30 min; 2) to visualize sample separation quality within 5 min after the gel run; 3) to transfer proteins in 3-10 min; 4) to verify transfer efficiency quantitatively; and most importantly 5) to validate changes in the level of the protein of interest using total protein loading control. This novel approach eliminates the need of stripping and reprobing the blot for housekeeping proteins such as β-actin, β-tubulin, GAPDH, etc. The V3 stain-free workflow makes the western blot process faster, transparent, more quantitative and reliable.

  18. A new approach to detect small peptides clearly and sensitively by Western blotting using a vacuum-assisted detection method.

    Science.gov (United States)

    Tomisawa, Satoshi; Abe, Chiharu; Kamiya, Masakatsu; Kikukawa, Takashi; Demura, Makoto; Kawano, Keiichi; Aizawa, Tomoyasu

    2013-01-01

    Western blotting is a widely used technique for the detection and quantification of proteins and peptides. However, it is challenging to detect small peptides efficiently by the conventional Western blotting method with shaking, in part because the peptides readily detach from the blotted membrane. Although some modified Western blotting protocols have been developed to overcome this problem, it remains difficult to prevent peptide detachment from the membrane. In this study, we show that the previously developed vacuum-assisted detection method greatly improves the detection of small peptides without additional protocol modification. The vacuum-assisted method was developed to shorten the time required for all immunodetection steps, and all the Western blotting solutions penetrated the membrane quickly and efficiently by this method. By using this vacuum method, we succeeded in detecting small peptides that were completely undetectable by the conventional Western blotting method. We also confirmed that peptide detachment was induced even by gentle shaking in the case of the conventional method, and the detachment was accelerated when detergent was present in the buffer. Unlike in the conventional method, there is no need to shake the membrane in solution in the vacuum method. Therefore, it is thought that the small peptides could be detected sensitively only by the vacuum method.

  19. Stereological Quantification of Leydig and Sertoli Cells: Technique ...

    African Journals Online (AJOL)

    Changes in the numbers or volume of the different cell types in the testis have been widely used to ascertain the effects of environmental and chemical agents on the testis. This study is designed to investigate the direct effects of metronidazole on the testicular cells by quantifying the number of Sertoli and Leydig cells.

  20. Gonadotropin releasing hormone receptor expression in primary breast cancer: comparison of immunohistochemical, radioligand and Western blot analyses.

    Science.gov (United States)

    Mangia, Anita; Tommasi, Stefania; Reshkin, Stephan J; Simone, Giovanni; Stea, Baldassarre; Schittulli, Francesco; Paradiso, Angelo

    2002-01-01

    GnRH biological effect is mediated through specific GnRH membrane receptors (GnRH-receptor, GnRH-R) that have been demonstrated in human breast cancer by molecular and biochemical techniques. The A9E4 monoclonal antibody (moAb) against the epitope 1-29 of N-terminal of human GnRH-R has been proposed, suggesting the possibility to perform retrospective studies for the confirmation of clinical relevance of this receptor. The aim of the present study was to verify the performance of the A9E4 moAb when utilised for immunohistochemical analysis in 71 formalin-fixed paraffin-embedded breast cancer samples; furthermore, a comparison with results obtained with the radioligand biochemical assay (GnRH-Rbca) and with Western blot has been performed. The A9E4 specificity was preliminarily demonstrated by Western blot analysis in both MCF-7 and T47D breast cancer cell lines. In both cell lines, only a protein of 60-64 kDa was demonstrated in the membrane and nuclear compartments. Immuno-reactivity for A9E4 was detected in the cytoplasm of morphologically normal adjacent glandular epithelia and in tumour cells. Cytoplasmic GnRH-R immuno-staining (GnRH-Rica) was shown in 55% of tumours but only 28% of these had a percentage of positive cells higher than >25%. A correlation between the percentage of positive GnRH-Rica cells and femtomoles of the GnRH-Rbca content was shown (c.c.=0.295, p=0.01). The mean content of GnRH-Rbca in the subgroup of tumours with >25% of cell positive at GnRH-Rica was significantly different with respect to that of negative GnRH-Rica tumours (25 fmol vs 11 fmol, respectively; p=0.03 by t-test). The immunohistochemical analysis of GnRH-R by A9E4 moAb in human breast cancer tissues seems to provide information that correlates with the standard biochemical assay. Retrospective clinical studies with GnRH-Rica on archival samples are strongly suggested to verify the prognostic-predictive relevance of this receptor in human breast cancer.

  1. Investigation of HIV-1 infected and uninfected cells using the optical trapping technique

    CSIR Research Space (South Africa)

    Ombinda-Lemboumba, Saturnin

    2017-02-01

    Full Text Available Optical trapping has emerged as an essential tool for manipulating single biological material and performing sophisticated spectroscopy analysis on individual cell. The optical trapping technique has been used to grab and immobilize cells from a...

  2. Optimized solubilization of TRIzol-precipitated protein permits Western blotting analysis to maximize data available from brain tissue.

    Science.gov (United States)

    Kopec, Ashley M; Rivera, Phillip D; Lacagnina, Michael J; Hanamsagar, Richa; Bilbo, Staci D

    2017-03-15

    Techniques simultaneously assessing multiple levels of molecular processing are appealing because molecular signaling underlying complex neural phenomena occurs at complementary levels. The TRIzol method isolates RNA and DNA, but protein retrieval is difficult due to inefficient solubilization of precipitated protein pellets. We optimized a buffer for the efficient solubilization of protein from TRIzol-precipitated brain tissue for Western blotting analysis, which was also more effective at directly homogenizing brain tissue than RIPA buffer. Protein yield during solubilization, in addition to protein yield via direct homogenization, is increased by optimizing concentrations of chemicals in a standard lysis buffer. Effective incubation parameters for both total protein yield and the analysis of post-translational modifications is remarkably flexible. Importantly, different neural cell types and protein classes are represented in solubilized protein samples. Moreover, we used dissociated mouse brain tissue to isolate microglia from other cell types and successfully resolved cell type-specific proteins from these small and difficult to attain samples. Solubilization buffers to date have been comprised primarily of SDS or urea; the data herein demonstrate that components common to lysis buffers can also enhance protein solubilization both after direct homogenization and after precipitation. This method is suitable for assessing gene and protein expression from a single brain sample, allowing for a more comprehensive evaluation of neural phenomena while minimizing the number of subjects. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Immunohistological techniques for studying the Drosophila male germline stem cell.

    Science.gov (United States)

    Singh, Shree Ram; Hou, Steven X

    2008-01-01

    Stem cells are undifferentiated cells that have a remarkable ability to self-renew and produce differentiated cells that support normal development and tissue homeostasis. This unique capacity makes stem cells a powerful tool for future regenerative medicine and gene therapy. Accumulative evidence suggests that stem cell self-renewal or differentiation is controlled by both intrinsic and extrinsic factors, and that deregulation of stem cell behavior results in cancer formation, tissue degeneration, and premature aging. The Drosophila testis provides an excellent in vivo model for studying and understanding the fundamental cellular and molecular mechanisms controlling stem cell behavior and the relationship between niches and stem cells. At the tip of the Drosophila testes, germline stem cells (GSCs) and somatic stem cells (SSCs) contact each other and share common niches (known as a hub) to maintain spermatogenesis. Signaling pathways, such as the Janus kinase (JAK)/signal transducer and activator of transcription (STAT), bone morphogenetic protein (BMP), ras-associated protein-guanine nucleotide exchange factor for small GTPase (Rap-GEF), and epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK), are known to regulate self-renewal or differentiation of Drosophila male germline stem cells. We describe the detailed in vivo immunohistological protocols that mark GSCs, SSCs, and their progeny in Drosophila testes.

  4. A review of the three-dimensional cell culture technique: Approaches, advantages and applications.

    Science.gov (United States)

    Zhang, Weijie; Zhuang, Ai; Gu, Ping; Zhou, Huifang; Fan, Xianqun

    2016-01-01

    Cell culture is a core and basic technique in biotechnology and is widely applied in biology, medicine, drug research and development. Traditional two-dimensional cell culture methods have undergone great developments. However, with in-depth basic research, higher requirements are needed to better mimic the in vivo environment to accurately observe cell behavior and explore its mechanisms. To comply with this situation, the three-dimensional cell culture technique emerged and has made profound advances in sustaining inherent cell properties. Here, we briefly review the development of this technique, including the main approaches to form three-dimensional microtissues, and its application and potential for future clinical therapies.

  5. A microlagoon technique for the culture of mammalian cells

    Science.gov (United States)

    Cone, C. D., Jr.; Peddrew, K. H.

    1968-01-01

    Technique obtains micropartitioning in a simple and reproducible manner by forming a field of tiny ponds or lagoons on the surface of a suitable culturing vessel. The technique allows free access of the common culture to all parts of the field.

  6. Simple and inexpensive technique for measuring oxygen consumption rate in adherent cultured cells.

    Science.gov (United States)

    Takahashi, Eiji; Yamaoka, Yoshihisa

    2017-11-01

    Measurement of cellular oxygen consumption rate (OCR) is essential in assessing roles of mitochondria in physiology and pathophysiology. Classical techniques, in which polarographic oxygen electrode measures the extracellular oxygen concentration in a closed measuring vessel, require isolation and suspension of the cell. Because cell functions depend on the extracellular milieu including the extracellular matrix, isolation of cultured cells prior to the measurement may significantly affect the OCR. More recent techniques utilize optical methods in which oxygen-dependent quenching of fluorophores determines oxygen concentration in the medium at a few microns above the surface of the cultured cells. These techniques allow the OCR measurement in cultured cells adhered to the culture dish. However, this technique requires special equipment such as a fluorescence lifetime microplate reader or specialized integrated system, which are usually quite expensive. Here, we introduce a simple and inexpensive technique for measuring OCR in adherent cultured cells that utilizes conventional fluorescence microscopy and a glassware called a gap cover glass.

  7. New technique for needle-less implantation of eukaryotic cells.

    Science.gov (United States)

    Arenas da Silva, Luis Fernando; Schober, Lena; Sloff, Marije; Traube, Andrea; Hart, Melanie L; Feitz, Wout F J; Stenzl, Arnulf

    2015-11-01

    On review of the use of stem cells in the literature, promissory outcomes for functional organ recovery in many subspecialties in medicine underscore its therapeutic potential. The application of stem cells through the use of a needle can result in additional scar formation, which is undesired for delicate organs. The present work describes the use of a needle-less stem cell injector with the Immediate Drop on Demand Technology (I-DOT) for cell injection in vitro. Mesenchymal stromal cells from human bone marrow were labeled with ethynyl-deoxyuridine (EdU) for 2 days and then were re-suspended. With the use of I-DOT, the cells were applied to type 1 collagen matrices or pig bladder tissue specimens with or without mucosa at different levels of energy. The collagen matrices were analyzed after 4 h and 5 days; bladder tissue specimens were analyzed 4 h after cell implantation. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (MTT) assay was performed immediately after cell application to the collagen matrices. Histological analysis with the use of frozen sections and immunofluorescence was used to localize EdU-labeled cells. A considerable number of cells were detected by use of the MTT assay for collagen matrices. In the collagen matrix, the mean measured depth immediately after application ranged between 210 μm and 489 μm, 220 μm and 270 μm for entire bladder specimens, and 230 μm and 370 μm for bladder without mucosa. Cells survived for up to 5 days in the collagen matrix in both bladder specimens. Cells can survive during I-DOT application, which suggests that the I-DOT device may be a potentially suitable technology for needle-less cell application onto tissues. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  8. Techniques for measuring red cell, platelet, and WBC survival

    International Nuclear Information System (INIS)

    Mayer, K.; Freeman, J.E.

    1986-01-01

    Blood cell survival studies yield valuable information concerning production and destruction of cells circulating in the bloodstream. Methodologies for the measurement of red cell survival include nonisotopic methods such as differential agglutination and hemolysis. The isotopic label may be radioactive or, if not, will require availability of a mass spectrograph. These methods fall into two categories, one where red cells of all ages are labeled ( 51 Cr, DFP32, etc.) and those employing a cohort label of newly formed cells ( 14 C glycine, 75 Se methionine, etc.). Interpretation of results for methodology employed and mechanism of destruction, random or by senescence, are discussed. A similar approach is presented for platelet and leukocyte survival studies. The inherent difficulties and complications of sequestration, storage, and margination of these cells are emphasized and discussed. 38 references

  9. A verified technique for calibrating space solar cells

    Science.gov (United States)

    Anspaugh, Bruce

    1987-01-01

    Solar cells have been flown on high-altitude balloons for over 24 years, to produce solar cell standards that can be used to set the intensity of solar simulators. The events of a typical balloon calibration flight are reported. These are: the preflight events, including the preflight cell measurements and the assembly of the flight cells onto the solar tracker; the activities at the National Scientific Balloon Facility in Palestine, Texas, including the preflight calibrations, the mating of the tracker and cells onto the balloon, preparations for launch, and the launch; the payload recovery, which includes tracking the balloon by aircraft, terminating the flight, and retrieving the payload. In 1985, the cells flow on the balloon were also flown on a shuttle flight and measured independently. The two measurement methods are compared and shown to agree within 1 percent.

  10. Normalized Quantitative Western Blotting Based on Standardized Fluorescent Labeling.

    Science.gov (United States)

    Faden, Frederik; Eschen-Lippold, Lennart; Dissmeyer, Nico

    2016-01-01

    Western blot (WB) analysis is the most widely used method to monitor expression of proteins of interest in protein extracts of high complexity derived from diverse experimental setups. WB allows the rapid and specific detection of a target protein, such as non-tagged endogenous proteins as well as protein-epitope tag fusions depending on the availability of specific antibodies. To generate quantitative data from independent samples within one experiment and to allow accurate inter-experimental quantification, a reliable and reproducible method to standardize and normalize WB data is indispensable. To date, it is a standard procedure to normalize individual bands of immunodetected proteins of interest from a WB lane to other individual bands of so-called housekeeping proteins of the same sample lane. These are usually detected by an independent antibody or colorimetric detection and do not reflect the real total protein of a sample. Housekeeping proteins-assumed to be constitutively expressed mostly independent of developmental and environmental states-can greatly differ in their expression under these various conditions. Therefore, they actually do not represent a reliable reference to normalize the target protein's abundance to the total amount of protein contained in each lane of a blot.Here, we demonstrate the Smart Protein Layers (SPL) technology, a combination of fluorescent standards and a stain-free fluorescence-based visualization of total protein in gels and after transfer via WB. SPL allows a rapid and highly sensitive protein visualization and quantification with a sensitivity comparable to conventional silver staining with a 1000-fold higher dynamic range. For normalization, standardization and quantification of protein gels and WBs, a sample-dependent bi-fluorescent standard reagent is applied and, for accurate quantification of data derived from different experiments, a second calibration standard is used. Together, the precise quantification of

  11. Serological follow-up of patients with paracoccidioidomycosis treated with itraconazole using Dot-blot, ELISA and Western-blot

    Directory of Open Access Journals (Sweden)

    Rosana MARTINS

    1997-09-01

    Full Text Available Twenty-seven mycologically proven cases of paracoccidioidomycosis (PCM were treated with itraconazole (100-200 mg/day in month 1 and 100 mg/day until month 6-8 and evaluated clinically and serologically, up to 3.5 years post-therapy, using Dot-blot and ELISA for measuring the titers of IgG, IgA and IgM anti-P. brasiliensis antibodies and Western-blot for determining IgG, IgA and IgM antibodies against the antigen components of the fungus. Before treatment, 81.5% (Dot-blot and 84% (ELISA of the patients presented elevated IgG anti-P. brasiliensis antibody titers which dropped slightly with treatment. On the other hand, the percentages of pre-treatment high-titered sera for IgA and IgM anti-P.brasiliensis were lower (5l.9% and 5l.8%: Dot-blot; 16.5 and 36%: ELISA, respectively but the titers tended to become negative more frequently with treatment. Prior to treatment, the percentages of positivity for IgG, IgA and IgM anti-P.brasiliensis antibodies in Western-blot were 96%, 20.8% and 41.6%, respectively. Antigens with molecular weights varying from 16-78 kDa, from 21-76 kDa and from 27-78 kDa were reactive for IgG, IgA and IgM antibodies, respectively. The most frequently reactive antigenic components had molecular weights of 27, 33 and 43 kDa for IgG, and 70 for IgA and IgM antibodies. During the period of study, the patients responded well to treatment. The present data confirm the diversity and complexity of the humoral response in PCM, and the importance of utilizing different serological tests to detect IgG, IgA and IgM anti-P. brasiliensis antibodiesVinte e sete pacientes portadores de paracoccidioidomicose (PCM foram tratados com itraconazole (100-200 mg/dia no primeiro mês e 100 mg/dia até 6-8 meses e avaliados sob o ponto de vista clínico e sorológico, até 3 e meio anos após o início do tratamento, utilizando-se os testes de Dot-blot e ELISA para medir os títulos de anticorpos IgG, IgA e IgM anti-P. brasiliensis, e Western-blot

  12. Biotechnology Apprenticeship for Secondary-Level Students: Teaching Advanced Cell Culture Techniques for Research.

    Science.gov (United States)

    Lewis, Jennifer R.; Kotur, Mark S.; Butt, Omar; Kulcarni, Sumant; Riley, Alyssa A.; Ferrell, Nick; Sullivan, Kathryn D.; Ferrari, Mauro

    2002-01-01

    Discusses small-group apprenticeships (SGAs) as a method for introducing cell culture techniques to high school participants. Teaches cell culture practices and introduces advance imaging techniques to solve various biomedical engineering problems. Clarifies and illuminates the value of small-group laboratory apprenticeships. (Author/KHR)

  13. Optical Trapping Techniques Applied to the Study of Cell Membranes

    Science.gov (United States)

    Morss, Andrew J.

    Optical tweezers allow for manipulating micron-sized objects using pN level optical forces. In this work, we use an optical trapping setup to aid in three separate experiments, all related to the physics of the cellular membrane. In the first experiment, in conjunction with Brian Henslee, we use optical tweezers to allow for precise positioning and control of cells in suspension to evaluate the cell size dependence of electroporation. Theory predicts that all cells porate at a transmembrane potential VTMof roughly 1 V. The Schwann equation predicts that the transmembrane potential depends linearly on the cell radius r, thus predicting that cells should porate at threshold electric fields that go as 1/r. The threshold field required to induce poration is determined by applying a low voltage pulse to the cell and then applying additional pulses of greater and greater magnitude, checking for poration at each step using propidium iodide dye. We find that, contrary to expectations, cells do not porate at a constant value of the transmembrane potential but at a constant value of the electric field which we find to be 692 V/cm for K562 cells. Delivering precise dosages of nanoparticles into cells is of importance for assessing toxicity of nanoparticles or for genetic research. In the second experiment, we conduct nano-electroporation—a novel method of applying precise doses of transfection agents to cells—by using optical tweezers in conjunction with a confocal microscope to manipulate cells into contact with 100 nm wide nanochannels. This work was done in collaboration with Pouyan Boukany of Dr. Lee's group. The small cross sectional area of these nano channels means that the electric field within them is extremely large, 60 MV/m, which allows them to electrophoretically drive transfection agents into the cell. We find that nano electroporation results in excellent dose control (to within 10% in our experiments) compared to bulk electroporation. We also find that

  14. Construction of multiple-epitope tag sequence by PCR for sensitive Western blot analysis.

    OpenAIRE

    Nakajima, K; Yaoita, Y

    1997-01-01

    Epitope tagging is a powerful technique to characterize a recombinantly expressed protein encoded by cDNA without the purification of the protein and the immunization of animals. In some cases, however, the expression of a tagged protein is too low to analyze by Western blot. We have developed a simple method to generate tandem repetitive nucleotide sequence by PCR, which allows us to label a protein of interest with a multiple-epitope tag. When five myc epitopes were attached to vaccinia vir...

  15. Western blotting revisited: critical perusal of underappreciated technical issues.

    Science.gov (United States)

    Gorr, Thomas A; Vogel, Johannes

    2015-04-01

    The most commonly used semiquantitative analysis of protein expression still employs protein separation by denaturing SDS-PAGE with subsequent Western blotting and quantification of the resulting ODs of bands visualized with specific antibodies. However, many questions regarding this procedure are usually ignored, although still in need of answering: Does isolation or separation procedure harm the integrity or affect modifications (e.g., phosphorylation) of the protein of interest? Does denaturation reduce binding of antibodies used for detection? Should denaturation be performed or should a native gel be run? How can artificial degradations or aggregations be distinguished from biological relevant ones? If the antibody detects multiple bands (which is not uncommon), which one(s) should be taken into account for quantification and why? Which loading control protein should be chosen and is it really "housekeeping" and how can this be verified? Is the image acquisition system linear and does it come with a sufficient dynamic range? How to account and control for background staining? This article is intended to address these questions and raise the readers awareness to possible Western blot alternatives in the attempt of minimizing possible pitfalls that might loom anywhere from protein isolation to acquisition of final quantitative data. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Determination of Diagnostic Antigens in Cattle Amphistomiasis Using Western Blotting

    Directory of Open Access Journals (Sweden)

    A Halajian

    2009-05-01

    Full Text Available "nBackground: Mixed infection with amphistomes seems common in native cattle of Iran. The aim of this study was to determine diagnostic antigens in cattle mixed amphistomiasis."nMethods: Specific antigens of Cotylophoron cotylophorum, Gastrothylax crumenifer and Paramphisto­mum cervi (mixed infection, the most common species, were collected from cattle was deter­mined. Adult trematodes were collected from the rumen of naturally infected cattle at meat inspec­tion. After their homogenization and centrifugation, somatic antigens were prepared and ana­lyzed by SDS-PAGE. Specific antigens were determinated by western blot with homologous and heterolo­gous sera. SDS-PAGE of whole worms extract was performed at different concentrations and subse­quent gels staining. Immunoblotting analysis using sera from cattle naturally infected with am­phistomes, Dicrocoelium dendriticum, Fasciola spp. and hydatid cyst was performed."nResults: Electrophorese analysis of somatic antigens revealed the presence of 10 and 21 protein bands at 4 µgr/ml and 8 µgr/ml with molecular weights ranging from 25-120 and 25-150 kDa, respectively. The best result was taken at 8 mg/ml concentration. Although western blot of these proteins demon­strate 5 major antigenic polypeptides ranging from 50 to 100 kDa which were recognized by serum of cat­tle naturally infected with mixed amphistomes.

  17. Sensitive Western-Blot Analysis of Azide-Tagged Protein Post Translational Modifications Using Thermoresponsive Polymer Self-Assembly.

    Science.gov (United States)

    Liu, Tong; Zhang, Wanjun; Zhang, Zheng; Chen, Mingli; Wang, Jianhua; Qian, Xiaohong; Qin, Weijie

    2018-02-06

    Western-blot (WB) is a powerful analytical technique for protein identification in complex biological samples and has been widely used in biological studies for decades. Detection specificity and sensitivity of WB largely relies on quality of the antibodies and performance of the conjugated HRP. However, the application of WB analysis for the detection of protein post-translational modifications (PTMs) is hampered by the low abundance of protein PTMs and by the limited availability of antibodies that specifically differentiate various kinds of PTMs from their protein substrates. Therefore, new recognition mechanisms and signal amplification strategies for WB analysis of protein PTMs is in high demand. In this work, we prepared a soluble polymer that detects various azide-tagged PTM proteins in WB analysis using triarylphosphine and HRP modified thermoresponsive polymer. Specific and efficient detection of azide-tagged PTM protein is achieved via the bioorthogonal reaction between azide and triarylphosphine. More importantly, the chemiluminiscent signal in the WB analysis is largely amplified by the temperature induced self-assembly of numerous thermoresponsive polymer chains carrying multiple HRPs. As a result, approximately 100 times more sensitive detection than commercial antibodies is achieved by this method using standard PTM proteins. Though, this new reagent does not directly detect native PTMs in cell, tissue or blood samples, it still has important application potential in protein PTM studies, considering the wide availability of azide-tagging techniques to a variety of PTMs.

  18. Growth techniques used to develop CDS/CDTE thin film solar cells ...

    African Journals Online (AJOL)

    Growth techniques used to develop CDS/CDTE thin film solar cells: a review. ... Techniques such as molecular beam epitaxy (MBE), metal organic chemical vapour deposition (MOCVD) called melt growth or Bridgman are well known as high quality semiconductor growth techniques. One of the limitations of these ...

  19. Measurement of Hepatic Protein Fractional Synthetic Rate with Stable Isotope Labeling Technique in Thapsigargin Stressed HepG2 Cells

    Science.gov (United States)

    Song, Juquan; Zhang, Xiao-jun; Boehning, Darren; Brooks, Natasha C.; Herndon, David N.; Jeschke, Marc G.

    2012-01-01

    Severe burn-induced liver damage and dysfunction is associated with endoplasmic reticulum (ER) stress. ER stress has been shown to regulate global protein synthesis. In the current study, we induced ER stress in vitro and estimated the effect of ER stress on hepatic protein synthesis. The aim was two-fold: (1) to establish an in vitro model to isotopically measure hepatic protein synthesis and (2) to evaluate protein fractional synthetic rate (FSR) in response to ER stress. Human hepatocellular carcinoma cells (HepG2) were cultured in medium supplemented with stable isotopes 1,2-13C2-glycine and L-[ring-13C6]phenylalanine. ER stress was induced by exposing the cells to 100 nM of thapsigargin (TG). Cell content was collected from day 0 to 14. Alterations in cytosolic calcium were measured by calcium imaging and ER stress markers were confirmed by Western blotting. The precursor and product enrichments were detected by GC-MS analysis for FSR calculation. We found that the hepatic protein FSR were 0.97±0.02 and 0.99±0.05%/hr calculated from 1,2-13C2-glycine and L-[ring-13C6]phenylalanine, respectively. TG depleted ER calcium stores and induced ER stress by upregulating p-IRE-1 and Bip. FSR dramatically decreased to 0.68±0.03 and 0.60±0.06%/hr in the TG treatment group (pisotope tracer incorporation technique is a useful method for studying the effects of ER stress on hepatic protein synthesis. PMID:22298954

  20. Stain-free detection as loading control alternative to Ponceau and housekeeping protein immunodetection in Western blotting.

    Science.gov (United States)

    Rivero-Gutiérrez, B; Anzola, A; Martínez-Augustin, O; de Medina, F Sánchez

    2014-12-15

    It is currently a routine practice to require a measurement of a housekeeping reference, including actin, glyceraldehyde-3-phosphate dehydrogenase, β-tubulin, among others, in Western blots, as it is the rule in RNA blots. Reversible Ponceau staining has been applied successfully to check equal loading of gels. Here we test a new technique, with the Stain-Free gels from Bio-Rad, against both Ponceau staining and housekeeping protein immunodetection under different conditions. Our results show that Stain-Free gels outperform Ponceau staining and that both are more consistent than housekeeping proteins as a loading control. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  1. Chikungunya virus isolation using simplified cell culture technique in Mauritius.

    Science.gov (United States)

    Pyndiah, M N; Pursem, V; Meetoo, G; Daby, S; Ramuth, V; Bhinkah, P; Chuttoo, R; Paratian, U

    2012-03-01

    During the chikungunya outbreak of 2005 - 2006, the only laboratory facilities available in Mauritius were virus isolation in cell culture tubes and serology. The laboratory was submerged with large numbers of blood samples. Comparative isolation was made in human embryonic lung (HEL) and VERO cells grown in 96-well plate. Culture on HEL cells was found to be more sensitive and presence of cytopathic effect (CPE) was observed earlier than in VERO cells. Out of the 18 300 blood samples inoculated on HEL, 11 165 were positive. This virus isolation method was of great help for the surveillance and control of the vectors. In cases of an outbreak a cheap, rapid and simple method of isolating chikungunya virus is described.

  2. Technique for Outdoor Test on Concentrating Photovoltaic Cells

    Directory of Open Access Journals (Sweden)

    Paola Sansoni

    2015-01-01

    Full Text Available Outdoor experimentation of solar cells is essential to maximize their performance and to assess utilization requirements and limits. More generally tests with direct exposure to the sun are useful to understand the behavior of components and new materials for solar applications in real working conditions. Insolation and ambient factors are uncontrollable but can be monitored to know the environmental situation of the solar exposure experiment. A parallel characterization of the photocells can be performed in laboratory under controllable and reproducible conditions. A methodology to execute solar exposure tests is proposed and practically applied on photovoltaic cells for a solar cogeneration system. The cells are measured with concentrated solar light obtained utilizing a large Fresnel lens mounted on a sun tracker. Outdoor measurements monitor the effects of the exposure of two multijunction photovoltaic cells to focused sunlight. The main result is the continuous acquisition of the V-I (voltage-current curve for the cells in different conditions of solar concentration and temperature of exercise to assess their behavior. The research investigates electrical power extracted, efficiency, temperatures reached, and possible damages of the photovoltaic cell.

  3. Characterization of lithium-thionyl chloride cells by impedance techniques

    Science.gov (United States)

    Walsh, F.; Pozin, M.; Cherniy, A.; Tikhonov, K.

    The main contributor to voltage drop observed on initial discharge of lithium-thionyl chloride cells is the resistance of the passive layer on the lithium anode, as can be determined from the Nyquist plot of a lithium-thionyl chloride cell. At extremely low discharge currents, initial voltage drop corresponds to the value found from the impedance measurements; at higher current, an empirical correction based on the experimental results is required. The dispersion in the values of the impedance parameters and thus in initial voltage drop of individual cells was analyzed. The condition of the lithium surface after assembly was shown not to be the only reason for high dispersion in impedance parameter values.

  4. Stem cell clonality -- theoretical concepts, experimental techniques, and clinical challenges.

    Science.gov (United States)

    Glauche, Ingmar; Bystrykh, Leonid; Eaves, Connie; Roeder, Ingo

    2013-04-01

    Here we report highlights of discussions and results presented at an International Workshop on Concepts and Models of Stem Cell Organization held on July 16th and 17th, 2012 in Dresden, Germany. The goal of the workshop was to undertake a systematic survey of state-of-the-art methods and results of clonality studies of tissue regeneration and maintenance with a particular emphasis on the hematopoietic system. The meeting was the 6th in a series of similar conceptual workshops, termed StemCellMathLab,(2) all of which have had the general objective of using an interdisciplinary approach to discuss specific aspects of stem cell biology. The StemCellMathLab 2012, which was jointly organized by the Institute for Medical Informatics and Biometry, Medical Faculty Carl Gustav Carus, Dresden University of Technology and the Institute for Medical Informatics, Statistics and Epidemiology, Medical Faculty, University of Leipzig, brought together 32 scientists from 8 countries, with scientific backgrounds in medicine, cell biology, virology, physics, computer sciences, bioinformatics and mathematics. The workshop focused on the following questions: (1) How heterogeneous are stem cells and their progeny? and (2) What are the characteristic differences in the clonal dynamics between physiological and pathophysiological situations? In discussing these questions, particular emphasis was placed on (a) the methods for quantifying clones and their dynamics in experimental and clinical settings and (b) general concepts and models for their description. In this workshop summary we start with an introduction to the current state of clonality research and a proposal for clearly defined terminology. Major topics of discussion include clonal heterogeneity in unperturbed tissues, clonal dynamics due to physiological and pathophysiological pressures and conceptual and technical issues of clone quantification. We conclude that an interactive cross-disciplinary approach to research in this

  5. Experimental Verification of Interference Mitigation Techniques for 5G Small Cells

    DEFF Research Database (Denmark)

    Assefa, Dereje; Berardinelli, Gilberto; Tavares, Fernando Menezes Leitão

    2015-01-01

    Inter-cell interference is the main performance limiting factor in the dense deployment of small cells targeted by the upcoming 5th Generation (5G) radio access technology. In this paper, we present an experimental evaluation of inter-cell interference mitigation techniques in a real indoor office...

  6. Applications of large cell remote handling techniques in nuclear plants

    International Nuclear Information System (INIS)

    Issel, W.; Leister, P.

    1989-01-01

    A comprehensive demonstration project in a special remote handling test facility was performed in parallel to the design of, and the basic engineering work for, the planned reprocessing plant at Wackersdorf. The aim of this project was to demonstrate the feasibility of a completely remote maintenance of the components of the PUREX process. These components were to be arranged as modules in large cells. Remote handling transporters, manipulators and tools (FEMO) for preplanned and unscheduled repair work were constructed and tested. The results of the successful demonstration project are summarized, and potential applications of the remote handling tools in hot cells and other nuclear plants are outlined. (orig./HP) [de

  7. Acid-Urea Gel Electrophoresis and Western Blotting of Histones.

    Science.gov (United States)

    Hazzalin, Catherine A; Mahadevan, Louis C

    2017-01-01

    Acid-urea gel electrophoresis offers significant advantages over SDS-PAGE for analysis of post-translational protein modifications, being capable of resolving proteins of similar size but varying in charge. Hence, it can be used to separate protein variants with small charge-altering differences in primary sequence, and is particularly useful in the analysis of histones whose charge variation arises from post-translational modification, such as phosphorylation or acetylation. On acid-urea gels, histones that carry multiple modifications, each with a characteristic charge, are resolved into distinct bands, the so-called "histone ladder." Thus, the extent and distribution of different modification states of histones can be visualized. Here, we describe the analysis of histone H3 by acid-urea gel electrophoresis and western blotting.

  8. Defining thermostability of membrane proteins by western blotting.

    Science.gov (United States)

    Ashok, Y; Nanekar, R; Jaakola, V-P

    2015-12-01

    Membrane proteins are relatively challenging targets for structural and other biophysical studies. Insufficient expression in various expression systems, inherent flexibility, and instability in the detergents that are required for membrane extraction are the main reasons for this limited success. Therefore, identification of suitable conditions and membrane protein variants that can help stabilize functional protein for extended periods of time is critical for structural studies. Here, we describe a western blot-based assay that simplifies identification of thermostabilizing conditions for membrane proteins. We show successful testing of a variety of parameters such as additive lipids, ligands and detergents. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  9. Identification and validation of rice reference proteins for western blotting.

    Science.gov (United States)

    Li, Xiaoming; Bai, Hui; Wang, Xianyun; Li, Liyun; Cao, Yinghao; Wei, Jian; Liu, Yumeng; Liu, Lijuan; Gong, Xiaodong; Wu, Lin; Liu, Siqi; Liu, Guozhen

    2011-10-01

    Studies of rice protein expression have increased considerably with the development of rice functional genomics. In order to obtain reliable expression results in western blotting, information on appropriate reference proteins is necessary for data normalization. To date, no published study has identified and systematically validated reference proteins suitable for the investigation of rice protein expression. In this study, nine candidate proteins were selected and their specific antibodies were obtained through immunization of rabbits with either recombinant proteins expressed in Escherichia coli or synthesized peptides. Western blotting was carried out to detect the expression of target proteins in a set of 10 rice samples representing different rice tissues/organs at different developmental stages. The expression stability of the proteins was analysed using geNorm and Microcal Origin 6.0 software. The results indicated that heat shock protein (HSP) and elongation factor 1-α (eEF-1α) were the most constantly expressed among all rice proteins tested throughout all developmental stages, while the proteins encoded by conventional internal reference genes fluctuated in amount. Comparison among the profiling of translation and transcription [expressed sequence tags (EST) and massively parallel signature sequencing (MPSS)] revealed that a correlation existed. Based on the standard curves derived from the antigen-antibody reaction, the concentrations of HSP and eEF-1α proteins in rice leaves were ∼0.12%. Under the present experimental conditions, the lower limits of detection for HSP and eEF-1α proteins in rice were 0.24 ng and 0.06 ng, respectively. In conclusion, the reference proteins selected in this study, and the corresponding antibodies, can be used in qualitative and quantitative analysis of rice proteins.

  10. Proteomic techniques for characterisation of mesenchymal stem cell secretome.

    Czech Academy of Sciences Publication Activity Database

    Kupcová Skalníková, Helena

    2013-01-01

    Roč. 95, č. 12 (2013), s. 2196-2211 ISSN 0300-9084 R&D Projects: GA MŠk ED2.1.00/03.0124; GA TA ČR TA01011466 Institutional support: RVO:67985904 Keywords : mesenchymal stem cells * secretome * exosome * conditioned medium * proteomics Subject RIV: CE - Biochemistry Impact factor: 3.123, year: 2013

  11. A cell culture technique for human epiretinal membranes to describe cell behavior and membrane contraction in vitro.

    Science.gov (United States)

    Wertheimer, Christian; Eibl-Lindner, Kirsten H; Compera, Denise; Kueres, Alexander; Wolf, Armin; Docheva, Denitsa; Priglinger, Siegfried G; Priglinger, Claudia; Schumann, Ricarda G

    2017-11-01

    To introduce a human cell culture technique for investigating in-vitro behavior of primary epiretinal cells and membrane contraction of fibrocellular tissue surgically removed from eyes with idiopathic macular pucker. Human epiretinal membranes were harvested from ten eyes with idiopathic macular pucker during standard vitrectomy. Specimens were fixed on cell culture plastic using small entomological pins to apply horizontal stress to the tissue, and then transferred to standard cell culture conditions. Cell behavior of 400 epiretinal cells from 10 epiretinal membranes was observed in time-lapse microscopy and analyzed in terms of cell migration, cell velocity, and membrane contraction. Immunocytochemistry was performed for cell type-specific antigens. Cell specific differences in migration behavior were observed comprising two phenotypes: (PT1) epiretinal cells moving fast, less directly, with small round phenotype and (PT2) epiretinal cells moving slowly, directly, with elongated large phenotype. No mitosis, no outgrowth and no migration onto the plastic were seen. Horizontal contraction measurements showed variation between specimens. Masses of epiretinal cells with a myofibroblast-like phenotype expressed cytoplasmatic α-SMA stress fibers and correlated with cell behavior characteristics (PT2). Fast moving epiretinal cells (PT1) were identified as microglia by immunostaining. This in-vitro technique using traction application allows for culturing surgically removed epiretinal membranes from eyes with idiopathic macular pucker, demonstrating cell behavior and membrane contraction of primary human epiretinal cells. Our findings emphasize the abundance of myofibroblasts, the presence of microglia and specific differences of cell behavior in these membranes. This technique has the potential to improve the understanding of pathologies at the vitreomacular interface and might be helpful in establishing anti-fibrotic treatment strategies.

  12. Detection of alien genetic introgressions in bread wheat using dot-blot genomic hybridisation.

    Science.gov (United States)

    Rey, María-Dolores; Prieto, Pilar

    2017-01-01

    Simple, reliable methods for the identification of alien genetic introgressions are required in plant breeding programmes. The use of genomic dot-blot hybridisation allows the detection of small Hordeum chilense genomic introgressions in the descendants of genetic crosses between wheat and H. chilense addition or substitution lines in wheat when molecular markers are difficult to use. Based on genomic in situ hybridisation, DNA samples from wheat lines carrying putatively H. chilense introgressions were immobilised on a membrane, blocked with wheat genomic DNA and hybridised with biotin-labelled H. chilense genomic DNA as a probe. This dot-blot screening reduced the number of plants necessary to be analysed by molecular markers or in situ hybridisation, saving time and money. The technique was sensitive enough to detect a minimum of 5 ng of total genomic DNA immobilised on the membrane or about 1/420 dilution of H. chilense genomic DNA in the wheat background. The robustness of the technique was verified by in situ hybridisation. In addition, the detection of other wheat relative species such as Hordeum vulgare , Secale cereale and Agropyron cristatum in the wheat background was also reported .

  13. An overview of technical considerations for Western blotting applications to physiological research.

    Science.gov (United States)

    Bass, J J; Wilkinson, D J; Rankin, D; Phillips, B E; Szewczyk, N J; Smith, K; Atherton, P J

    2017-01-01

    The applications of Western/immunoblotting (WB) techniques have reached multiple layers of the scientific community and are now considered routine procedures in the field of physiology. This is none more so than in relation to skeletal muscle physiology (i.e., resolving the mechanisms underpinning adaptations to exercise). Indeed, the inclusion of WB data is now considered an essential aspect of many such physiological publications to provide mechanistic insight into regulatory processes. Despite this popularity, and due to the ubiquitous and relatively inexpensive availability of WB equipment, the quality of WB in publications and subsequent analysis and interpretation of the data can be variable, perhaps resulting in spurious conclusions. This may be due to poor laboratory technique and/or lack of comprehension of the critical steps involved in WB and what quality control procedures should be in place to ensure robust data generation. The present review aims to provide a detailed description and critique of WB procedures and technicalities, from sample collection through preparation, blotting and detection, to analysis of the data collected. We aim to provide the reader with improved expertise to critically conduct, evaluate, and troubleshoot the WB process, to produce reproducible and reliable blots. © 2016 The Authors. Scandinavian Journal of Medicine & Science in Sports published by John Wiley & Sons Ltd.

  14. Fabrication and processing of polymer solar cells: A review of printing and coating techniques

    DEFF Research Database (Denmark)

    Krebs, Frederik C

    2009-01-01

    Polymer solar cells are reviewed in the context of the processing techniques leading to complete devices. A distinction is made between the film-forming techniques that are used currently such as spincoating, doctor blading and casting and the, from a processing point of view, more desirable film...... suited, but little explored in the context of polymer solar cells. A further distinction is made between printing and coating when a film is formed. The entire process leading to polymer solar cells is broken down into the individual steps and the available techniques and materials for each step...

  15. [The cell micro-encapsulation techniques and its advancement in the field of gene therapy].

    Science.gov (United States)

    Li, Xiaoling; Cai, Shaohui

    2006-12-01

    It is no doubt that the gene therapy using recombinant engineering cells provides a novel approach to many refractory diseases. However, the transplant rejection from the host's immune system against heterogeneous cells has been the main handicap of its clinical application. The modern cell micro-encapsulation technique with good immune isolation makes it possible to overcome this problem and has shown potential application foreground in clinical therapies for a lot of diseases such as Parkinson's disease and Hemophiliac disease. This article reviews mainly the relative materials and techniques in processing micro-encapsulation, the host cells used to construct the recombinant genetic engineering cells and application of cell micro-encapsulation technique in the field of gene therapy.

  16. Deformation of filamentous Escherichia coli cells in a microfluidic device: a new technique to study cell mechanics.

    Directory of Open Access Journals (Sweden)

    Yaron Caspi

    Full Text Available The mechanical properties of bacterial cells are determined by their stress-bearing elements. The size of typical bacterial cells, and the fact that different time and length scales govern their behavior, necessitate special experimental techniques in order to probe their mechanical properties under various spatiotemporal conditions. Here, we present such an experimental technique to study cell mechanics using hydrodynamic forces in a microfluidic device. We demonstrate the application of this technique by calculating the flexural rigidity of non-growing Escherichia coli cells. In addition, we compare the deformation of filamentous cells under growing and non-growing conditions during the deformation process. We show that, at low forces, the force needed to deform growing cells to the same extent as non-growing cells is approximately two times smaller. Following previous works, we interpret these results as the outcome of the difference between the elastic response of non-growing cells and the plastic-elastic response of growing cells. Finally, we observe some heterogeneity in the response of individual cells to the applied force. We suggest that this results from the individuality of different bacterial cells.

  17. Design, preparation and use of ligated phosphoproteins: a novel approach to study protein phosphatases by dot blot array, ELISA and Western blot assays.

    Science.gov (United States)

    Sun, Luo; Ghosh, Inca; Barshevsky, Tanya; Kochinyan, Samvel; Xu, Ming-Qun

    2007-07-01

    The study of substrate specificity of protein phosphatases (PPs) is very challenging since it is difficult to prepare a suitable phosphorylated substrate. Phosphoproteins, phosphorylated by a protein kinase, or chemically synthesized phosphopeptides are commonly used substrates for PPs. Both types of these substrates have their advantages and limitations. Phosphoproteins mimic more closely the physiologically relevant PP substrates, but their preparation is technically demanding. Synthetic phosphopeptides present advantages over proteins because they can be easily produced in large quantity and their amino acid sequence can be designed to contain potential determinants of substrate specificity. However, short peptides are less optimal compared to in vivo PP substrates and often display poor and variable binding to different matrices, resulting in low sensitivity in analysis of PP activity on solid support. In this work we utilize the intein-mediated protein ligation (IPL) technique to generate substrates for PPs, combining the advantages of proteins and synthetic peptides in one molecule. The ligation of a synthetic phosphopeptide to an intein-generated carrier protein (CP) with a one-to-one stoichiometry results in the formation of a ligated phosphoprotein (LPP). Three widely used assays, dot blot array, Western blot and ELISA were employed to study the PP activity on LPP substrates. Dephosphorylation was measured by detection of the remaining phosphorylation, or lack of it, with a phospho-specific antibody. The data show the advantage of LPPs over free peptides in assays on solid supports. LPPs exhibited enhanced binding to the matrices used in the study, which significantly improved sensitivity and consistency of the assays. In addition, saturation of the signal was circumvented by serial dilution of the assay samples. This report describes detailed experimental procedures for preparation of LPP substrates and their use in PP assays based on immobilization on

  18. A novel method that improves sensitivity of protein detection in PAGE and western blot

    Science.gov (United States)

    Vallejo-Illarramendi, Ainara; Marciano, Denise K.; Reichardt, Louis F.

    2013-01-01

    We have developed a simple and inexpensive method that improves sensitivity of protein and antigen detection in standard PAGE procedures. Our technique uses a sample microloader device with a funnel-like structure, filled with a 4% stacking gel. When attach to the top of a polyacrylamide slab gel the proteins in a sample are concentrated by electrophoresis into a small volume as they emerge from the device's narrow outlet. Our microloader has several advantages over previous devices, including simple assembly, high versatility and absence of cross-contamination between lanes. Addition of this device to a slab gel results in a 5-fold increase in the sensitivity of antigen detection in a western blot. As a result less protein is required for loading and signal detection. Our protocol is a straightforward modification of a standard experimental technique, and is especially useful when only limited sample quantities are available. PMID:23400834

  19. A novel method that improves sensitivity of protein detection in PAGE and Western blot.

    Science.gov (United States)

    Vallejo-Illarramendi, Ainara; Marciano, Denise K; Reichardt, Louis F

    2013-04-01

    We have developed a simple and inexpensive method that improves sensitivity of protein and antigen detection in standard PAGE procedures. Our technique uses a sample microloader device with a funnel-like structure, filled with a 4% stacking gel. When attach to the top of a polyacrylamide slab gel, the proteins in a sample are concentrated by electrophoresis into a small volume as they emerge from the device's narrow outlet. Our microloader has several advantages over previous devices, including simple assembly, high versatility, and absence of cross-contamination between lanes. Addition of this device to a slab gel results in a fivefold increase in the sensitivity of antigen detection in a Western blot. As a result, less protein is required for loading and signal detection. Our protocol is a straightforward modification of a standard experimental technique, and is especially useful when only limited sample quantities are available. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. A novel histological technique for distinguishing between epithelial cells in forensic casework.

    Science.gov (United States)

    French, Claire E V; Jensen, Cynthia G; Vintiner, Susan K; Elliot, Douglas A; McGlashan, Susan R

    2008-06-10

    There are a number of forensic cases in which the identification of the epithelial cell type from which DNA originated would provide important probative evidence. This study aimed to develop a technique using histological staining of fixed cells to distinguish between skin, buccal and vaginal epithelium. First, 11 different stains were screened on formalin-fixed, wax-embedded cells from five women. Samples were analysed qualitatively by examining staining patterns (colour) and morphology (absence or presence of nuclei). Three of the staining methods--Dane's, Csaba's and Ayoub-Shklar--were successful in distinguishing skin epithelial cells from buccal and vaginal. Second, cells were smeared directly onto slides, fixed with one of five fixatives and stained with one of the three stains mentioned above. Methanol fixation, coupled with the Dane's staining method, specific to keratin, was the only technique that distinguished between all three cell types. Skin cells stained magenta, red and orange and lacked nuclei; buccal cells stained predominantly orange-pink with red nuclei; while vaginal cells stained bright orange with orange nuclei and a blue extracellular hue. This staining pattern in vaginal cells was consistent in samples collected from 50 women aged between 18 and 67. Identification of cell type from unlabelled micrographs by 10 trained observers showed a mean success rate of 95%. The results of this study demonstrate that histological staining may provide forensic scientists with a technique for distinguishing between skin, buccal and vaginal epithelial cells and thus would enable more conclusive analyses when investigating sexual assault cases.

  1. Detection of Autophagy in Caenorhabditis elegans by Western Blotting Analysis of LGG-1.

    Science.gov (United States)

    Palmisano, Nicholas J; Meléndez, Alicia

    2016-02-01

    A common way to measure the induction of autophagy in yeast and mammalian cells is to compare the amount of Atg8/LC3-I with that of Atg8-PE/LC3-II by using western blot analysis. This is because changes in the amount of LC3-II correlate closely with changes in the number of autophagosomes present in cells. Atg8/LC3 is initially synthesized as an unprocessed form, which is proteolytically processed to form Atg8/LC3-I, and then this is modified into the phosphatidylethanolamine (PE)-conjugated Atg8-PE/LC3-II form. Atg8/LC3-II is membrane bound, whereas Atg8-PE/LC3-I is cytosolic. By associating with both the inner and outer membranes of the autophagosome, Atg8-PE/LC3-II is the only autophagy reporter that is reliably associated with completed autophagosomes. In the nematode Caenorhabditis elegans, the ortholog of Atg8/LC3 is LGG-1. Here, we discuss how changes in the levels of LGG-1-II (and the paralog LGG-2) protein can, with appropriate controls, be used to monitor autophagy activity in nematodes and present a protocol for monitoring changes in the protein levels of different forms of LGG-1 by western blotting. © 2016 Cold Spring Harbor Laboratory Press.

  2. Ex vivo hyperpolarized MR spectroscopy on isolated renal tubular cells: A novel technique for cell energy phenotyping.

    Science.gov (United States)

    Juul, Troels; Palm, Fredrik; Nielsen, Per Mose; Bertelsen, Lotte Bonde; Laustsen, Christoffer

    2017-08-01

    It has been demonstrated that hyperpolarized 13 C MR is a useful tool to study cultured cells. However, cells in culture can alter phenotype, which raises concerns regarding the in vivo significance of such findings. Here we investigate if metabolic phenotyping using hyperpolarized 13 C MR is suitable for cells isolated from kidney tissue, without prior cell culture. Isolation of tubular cells from freshly excised kidney tissue and treatment with either ouabain or antimycin A was investigated with hyperpolarized MR spectroscopy on a 9.4 Tesla preclinical imaging system. Isolation of tubular cells from less than 2 g of kidney tissue generally resulted in more than 10 million live tubular cells. This amount of cells was enough to yield robust signals from the conversion of 13 C-pyruvate to lactate, bicarbonate and alanine, demonstrating that metabolic flux by means of both anaerobic and aerobic pathways can be quantified using this technique. Ex vivo metabolic phenotyping using hyperpolarized 13 C MR in a preclinical system is a useful technique to study energy metabolism in freshly isolated renal tubular cells. This technique has the potential to advance our understanding of both normal cell physiology as well as pathological processes contributing to kidney disease. Magn Reson Med 78:457-461, 2017. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine.

  3. Application of the wavelet image analysis technique to monitor cell concentration in bioprocesses

    Directory of Open Access Journals (Sweden)

    G. J. R. Garófano

    2005-12-01

    Full Text Available The growth of cells of great practical interest, such as, the filamentous cells of bacterium Streptomyces clavuligerus, the yeast Saccharomyces cerevisiae and the insect Spodoptera frugiperda (Sf9 cell, cultivated in shaking flasks with complex media at appropriate temperatures and pHs, was quantified by the new wavelet transform technique. This image analysis tool was implemented using Matlab 5.2 software to process digital images acquired of samples taken of these three types of cells throughoot their cultivation. The values of the average wavelet coefficients (AWCs of simplified images were compared with experimental measurements of cell concentration and with computer-based densitometric measurements. AWCs were shown to be directly proportional to measurements of cell concentration and to densitometric measurements, making evident the great potential of the wavelet transform technique to quantitatively estimate the growth of several types of cells.

  4. Rapid detection and differentiation of important Campylobacter spp. in poultry samples by dot blot and PCR.

    Science.gov (United States)

    Fontanot, Marco; Iacumin, Lucilla; Cecchini, Francesca; Comi, Giuseppe; Manzano, Marisa

    2014-10-01

    The detection of Campylobacter, the most commonly reported cause of foodborne gastroenteritis in the European Union, is very important for human health. The most commonly recognised risk factor for infection is the handling and/or consumption of undercooked poultry meat. The methods typically applied to evaluate the presence/absence of Campylobacter in food samples are direct plating and/or enrichment culture based on the Horizontal Method for Detection and Enumeration of Campylobacter spp. (ISO 10272-1B: 2006) and PCR. Molecular methods also allow for the detection of cells that are viable but cannot be cultivated on agar media and that decrease the time required for species identification. The current study proposes the use of two molecular methods for species identification: dot blot and PCR. The dot blot method had a sensitivity of 25 ng for detection of DNA extracted from a pure culture using a digoxigenin-labelled probe for hybridisation; the target DNA was extracted from the enrichment broth at 24 h. PCR was performed using a pair of sensitive and specific primers for the detection of Campylobacter jejuni and Campylobacter coli after 24 h of enrichment in Preston broth. The initial samples were contaminated by 5 × 10 C. jejuni cells/g and 1.5 × 10(2)C. coli cells/g, thus the number of cells present in the enrichment broth at 0 h was 1 or 3 cell/g, respectively. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Quantitative Phase Imaging Techniques for the Study of Cell Pathophysiology: From Principles to Applications

    Directory of Open Access Journals (Sweden)

    Hyunjoo Park

    2013-03-01

    Full Text Available A cellular-level study of the pathophysiology is crucial for understanding the mechanisms behind human diseases. Recent advances in quantitative phase imaging (QPI techniques show promises for the cellular-level understanding of the pathophysiology of diseases. To provide important insight on how the QPI techniques potentially improve the study of cell pathophysiology, here we present the principles of QPI and highlight some of the recent applications of QPI ranging from cell homeostasis to infectious diseases and cancer.

  6. Applied Protein and Molecular Techniques for Characterization of B Cell Neoplasms in Horses

    Science.gov (United States)

    Badial, Peres R.; Tallmadge, Rebecca L.; Miller, Steven; Stokol, Tracy; Richards, Kristy; Borges, Alexandre S.

    2015-01-01

    Mature B cell neoplasms cover a spectrum of diseases involving lymphoid tissues (lymphoma) or blood (leukemia), with an overlap between these two presentations. Previous studies describing equine lymphoid neoplasias have not included analyses of clonality using molecular techniques. The objective of this study was to use molecular techniques to advance the classification of B cell lymphoproliferative diseases in five adult equine patients with a rare condition of monoclonal gammopathy, B cell leukemia, and concurrent lymphadenopathy (lymphoma/leukemia). The B cell neoplasms were phenotypically characterized by gene and cell surface molecule expression, secreted immunoglobulin (Ig) isotype concentrations, Ig heavy-chain variable (IGHV) region domain sequencing, and spectratyping. All five patients had hyperglobulinemia due to IgG1 or IgG4/7 monoclonal gammopathy. Peripheral blood leukocyte immunophenotyping revealed high proportions of IgG1- or IgG4/7-positive cells and relative T cell lymphopenia. Most leukemic cells lacked the surface B cell markers CD19 and CD21. IGHG1 or IGHG4/7 gene expression was consistent with surface protein expression, and secreted isotype and Ig spectratyping revealed one dominant monoclonal peak. The mRNA expression of the B cell-associated developmental genes EBF1, PAX5, and CD19 was high compared to that of the plasma cell-associated marker CD38. Sequence analysis of the IGHV domain of leukemic cells revealed mutated Igs. In conclusion, the protein and molecular techniques used in this study identified neoplastic cells compatible with a developmental transition between B cell and plasma cell stages, and they can be used for the classification of equine B cell lymphoproliferative disease. PMID:26311245

  7. Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique.

    Science.gov (United States)

    Sato, Kazuhide; Choyke, Peter L; Hisataka, Kobayashi

    2016-03-14

    Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

  8. Quantitative dot blot analysis (QDB), a versatile high throughput immunoblot method.

    Science.gov (United States)

    Tian, Geng; Tang, Fangrong; Yang, Chunhua; Zhang, Wenfeng; Bergquist, Jonas; Wang, Bin; Mi, Jia; Zhang, Jiandi

    2017-08-29

    Lacking access to an affordable method of high throughput immunoblot analysis for daily use remains a big challenge for scientists worldwide. We proposed here Quantitative Dot Blot analysis (QDB) to meet this demand. With the defined linear range, QDB analysis fundamentally transforms traditional immunoblot method into a true quantitative assay. Its convenience in analyzing large number of samples also enables bench scientists to examine protein expression levels from multiple parameters. In addition, the small amount of sample lysates needed for analysis means significant saving in research sources and efforts. This method was evaluated at both cellular and tissue levels with unexpected observations otherwise would be hard to achieve using conventional immunoblot methods like Western blot analysis. Using QDB technique, we were able to observed an age-dependent significant alteration of CAPG protein expression level in TRAMP mice. We believe that the adoption of QDB analysis would have immediate impact on biological and biomedical research to provide much needed high-throughput information at protein level in this "Big Data" era.

  9. [Western blot, ELISA and indirect immunofluorescence test evaluation of Leishmania (Leishmania) infantum-infected dogs].

    Science.gov (United States)

    Vargas-Duarte, Jimmy J; López-Páez, Myriam C; Escovar-Castro, Jesús E; Fernández-Manrique, José

    2009-08-01

    Evaluating canine visceral leishmaniasis diagnostic test performance in Colombia and adapting the Western blot test in naturally and experimentally infected dogs. Sera were obtained from 10 experimentally L. Infantum-infected dogs, 5 naturally infected dogs, 16 healthy dogs, 26 Babesia canis, Erhlichia canis, Dirofilaria immitis, Trypanosoma cruzi and Leishmania (Viannia) spp infected dogs, 40 dogs from non-endemic areas and 150 from endemic areas. Sera were tested for L. infantum infection using immunofluorescent antibody (IFAT), ELISA and Western blot (WB) tests. Positives results were obtained for 73 % of known infected dogs by the IFAT test and false positives were obtained for 2.5 % of non-infected dogs using WB. ELISA was not efficient for diagnosis. 24 antigenic fractions were recognised in tested sera using WB; however, 29, 34, 50, 69, 75, 86, 99 and 123 kDa bands were recognised in sera from dogs from non-endemic areas, healthy dogs and Trypanosoma cruzi, Erhlichia canis, Dirofilaria immitis and Babesia canis infected dogs. The 13 kDa fraction proved potentially useful for diagnosing canine visceral leishmaniasis. The separate use of parasitological and serological test could lead to misdiagnosis of Leishmania infection; using both kinds of technique simultaneously is thus highly recommended.

  10. Expression of glutamate decarboxylase isoform, GAD65, in human mononuclear leucocytes: a possible implication of C-terminal end deletion by Western blot and RT-PCR study.

    Science.gov (United States)

    Matsukawa, Satoko; Ueno, Hiroshi

    2007-11-01

    Human peripheral blood leucocyte was examined for the expression of glutamate decarboxylase (GAD). Peripheral blood from healthy individuals was fractionated into polynuclear leucocytes and mononuclear leucocytes. Cell lysate from the mononuclear leucocytes was analysed by SDS-PAGE and Western blot. With antibody raised against unique C-terminal sequence for GAD65, two protein bands at 30 and 80 kDa were detected. However, with anti-GAD65/67 antibody recognizing very end of C-terminal, about 40 residues toward C-terminal, no protein bands were observed. Expression of mRNA coding for the epitope of these two antibodies was examined by using PCR technique. Results showed an evidence that mononuclear leucocytes express GAD65 with its C-terminal segment truncated. Our results have suggested an expression of GAD with the novel molecular weight may be caused by possible mononuclear leucocyte specific splicing errors.

  11. Two dimensional numerical simulation of gas discharges: comparison between particle-in-cell and FCT techniques

    Energy Technology Data Exchange (ETDEWEB)

    Soria-Hoyo, C; Castellanos, A [Departamento de Electronica y Electromagnetismo, Facultad de Fisica, Universidad de Sevilla, Avda. Reina Mercedes s/n, 41012 Sevilla (Spain); Pontiga, F [Departamento de Fisica Aplicada II, EUAT, Universidad de Sevilla, Avda. Reina Mercedes s/n, 41012 Sevilla (Spain)], E-mail: cshoyo@us.es

    2008-10-21

    Two different numerical techniques have been applied to the numerical integration of equations modelling gas discharges: a finite-difference flux corrected transport (FD-FCT) technique and a particle-in-cell (PIC) technique. The PIC technique here implemented has been specifically designed for the simulation of 2D electrical discharges using cylindrical coordinates. The development and propagation of a streamer between two parallel electrodes has been used as a convenient test to compare the performance of both techniques. In particular, the phase velocity of the cathode directed streamer has been used to check the internal consistency of the numerical simulations. The results obtained from the two techniques are in reasonable agreement with each other, and both techniques have proved their ability to follow the high gradients of charge density and electric field present in this type of problems. Moreover, the streamer velocities predicted by the simulation are in accordance with the typical experimental values.

  12. Single-cell analysis of dihydroartemisinin-induced apoptosis through reactive oxygen species-mediated caspase-8 activation and mitochondrial pathway in ASTC-a-1 cells using fluorescence imaging techniques

    Science.gov (United States)

    Lu, Ying-Ying; Chen, Tong-Sheng; Wang, Xiao-Ping; Li, Li

    2010-07-01

    Dihydroartemisinin (DHA), a front-line antimalarial herbal compound, has been shown to possess promising anticancer activity with low toxicity. We have previously reported that DHA induced caspase-3-dependent apoptosis in human lung adenocarcinoma cells. However, the cellular target and molecular mechanism of DHA-induced apoptosis is still poorly defined. We use confocal fluorescence microscopy imaging, fluorescence resonance energy transfer, and fluorescence recovery after photobleaching techniques to explore the roles of DHA-elicited reactive oxygen species (ROS) in the DHA-induced Bcl-2 family proteins activation, mitochondrial dysfunction, caspase cascade, and cell death. Cell Counting Kit-8 assay and flow cytometry analysis showed that DHA induced ROS-mediated apoptosis. Confocal imaging analysis in a single living cell and Western blot assay showed that DHA triggered ROS-dependent Bax translocation, mitochondrial membrane depolarization, alteration of mitochondrial morphology, cytochrome c release, caspase-9, caspase-8, and caspase-3 activation, indicating the coexistence of ROS-mediated mitochondrial and death receptor pathway. Collectively, our findings demonstrate for the first time that DHA induces cell apoptosis by triggering ROS-mediated caspase-8/Bid activation and the mitochondrial pathway, which provides some novel insights into the application of DHA as a potential anticancer drug and a new therapeutic strategy by targeting ROS signaling in lung adenocarcinoma therapy in the future.

  13. Biophysical Techniques for Detection of cAMP and cGMP in Living Cells

    Directory of Open Access Journals (Sweden)

    Viacheslav O. Nikolaev

    2013-04-01

    Full Text Available Cyclic nucleotides cAMP and cGMP are ubiquitous second messengers which regulate myriads of functions in virtually all eukaryotic cells. Their intracellular effects are often mediated via discrete subcellular signaling microdomains. In this review, we will discuss state-of-the-art techniques to measure cAMP and cGMP in biological samples with a particular focus on live cell imaging approaches, which allow their detection with high temporal and spatial resolution in living cells and tissues. Finally, we will describe how these techniques can be applied to the analysis of second messenger dynamics in subcellular signaling microdomains.

  14. Detection of irregular red cell antibodies: more than 3 years of experience with a gel technique and pooled screening cells.

    Science.gov (United States)

    Titlestad, K; Georgsen, J; Andersen, H; Kristensen, T

    1997-01-01

    The purpose of this study was to evaluate more than 3 years of experience with a gel technique in combination with pooled screening cells for the detection of irregular red cell antibodies. Conventional serologic methods were used for blood typing, antibody screening and cross-matching until the end of 1992. We introduced the gel technique as a routine assay for antibody detection and identification in 1993. After the tube technique had been abandoned, the number of false-positive antibody screening tests was reduced by 71%, positive antibody screening tests by 33%, enzyme agglutination by 100% and rouleaux reactions and cold-reacting antibodies by more than 50%. There was a 40% increase in first-time detection of clinically relevant antibodies. We saw no increase in delayed haemolytic transfusion reactions. For the detection of irregular red cell antibodies, pooled screening cells in combination with a gel technique are at least as efficient and safe as a conventional tube technique with unpooled test cells.

  15. Modified Western blotting for insulin and other diabetes-associated peptide hormones.

    Science.gov (United States)

    Okita, Naoyuki; Higami, Yoshikazu; Fukai, Fumio; Kobayashi, Masaki; Mitarai, Miku; Sekiya, Takao; Sasaki, Takashi

    2017-07-31

    Now, the quantification of proinsulin/insulin contents within organisms tends to be evaluated only by enzyme-linked immunosorbent assay (ELISA), although assessing the adequacy of results by some quantification method is important. Remarkably, few scientific papers use detection by Western blotting (WB), another immunological assay, of proinsulin/insulin. We found two problems with quantification of insulin and proinsulin by general WB: the shape of an insulin band in gel electrophoresis is distorted, and the retention potency to a blotting membrane of the peptide hormones (mainly insulin) is low. We solved the first problem by optimizing the sodium dodecyl sulfate concentration in the sample buffer and the second problem by glutaraldehyde fixation following treatment with a blocking solution for a short time. The improvements were confirmed by quantification of proinsulin/insulin in standards, MIN6c4 cell lysates, and MIN6c4 culture supernatants. Furthermore, we showed that the modified WB is applicable to other diabetes-associated peptide hormones: insulin analogs, glucagon, GLP-1s, somatostatins, ghrelins, and pancreatic polypeptide. Our data showed that the modified WB can contribute to qualitative or quantitative analyses of diabetes-associated peptides by providing analytical information based on electrophoresis, although ELISA, which is an almost exclusive method in the quantification of peptide hormones, supplies only numerical data.

  16. A Rapid Culture Technique Produces Functional Dendritic-Like Cells from Human Acute Myeloid Leukemia Cell Lines

    Directory of Open Access Journals (Sweden)

    Jian Ning

    2011-01-01

    Full Text Available Most anti-cancer immunotherapeutic strategies involving dendritic cells (DC as vaccines rely upon the adoptive transfer of DC loaded with exogenous tumour-peptides. This study utilized human acute myeloid leukemia (AML cells as progenitors from which functional dendritic-like antigen presenting cells (DLC were generated, that constitutively express tumour antigens for recognition by CD8+ T cells. DLC were generated from AML cell lines KG-1 and MUTZ-3 using rapid culture techniques and appropriate cytokines. DLC were evaluated for their cell-surface phenotype, antigen uptake and ability to stimulate allogeneic responder cell proliferation, and production of IFN-γ; compared with DC derived from normal human PBMC donors. KG-1 and MUTZ-3 DLC increased expression of CD80, CD83, CD86, and HLA-DR, and MUTZ-3 DLC downregulated CD14 and expressed CD1a. Importantly, both KG-1 and MUTZ-3-derived DLC promoted proliferation of allogeneic responder cells more efficiently than unmodified cells; neither cells incorporated FITC-labeled dextran, but both stimulated IFN-γ production from responding allogeneic CD8+ T cells. Control DC produced from PBMC using the FastDC culture also expressed high levels of critical cell surface ligands and demonstrated good APC function. This paper indicates that functional DLC can be cultured from the AML cell lines KG-1 and MUTZ-3, and FastDC culture generates functional KG-1 DLC.

  17. Northern blot analysis to investigate the abundance of microorganisms

    International Nuclear Information System (INIS)

    Krause, D.O.

    2005-01-01

    areas known as hyper-variable regions which have a high degree of sequence variation. As a result of this structure, it is possible to design signature oligonucleotide probes varying in length from about 15 to 30 nucleotides that are diagnostic of microorganisms at the kingdom, domain, genus and even species level. These signature sequences can be used in a variety of applications such as PCR analysis, construction of clone libraries or direct probing of bulk rRNA. In this chapter, I provide detailed protocols for the analysis of extracted rRNA and give detailed procedures that must be followed to do northern blot analysis of bulk RNA extracted from the rumen

  18. Study on production of useful metabolites by development of advanced cell culture techniques using radiation

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Byung Yeoup; Lee, Seung Sik; Bai, Hyounwoo; Singh, Sudhir; Lee, Eun Mi; Hong, Sung Hyun; Park, Chul Hong; Srilatha, B.; Kim, Mi Ja; Lee, Ohchul

    2012-01-15

    The purpose of this project is improvement of investigation, materialization and evaluation techniques on effectiveness for functional natural compounds throughout development of tissue/cell culture techniques for mass production of useful metabolites using radiation. Research scope includes Development of a technique for radiation tissue and cell culture, Database construction for radiation response in plants and radiation effects, Construction of general-purpose national based techniques of cell culture technique using radiation. Main results are as follow: Development of a technique for radiation tissue and cell culture for Erigeron breviscapus (Vant.) Hand. Mazz.; Identification and functional analysis of AtTDX (chaperone and peroxidase activities); Functional analysis of radiation(gamma ray, electron beam, and proton beam) induced chaperon protein activities (AtTDX); Determine the action mechanism of yPrx2; Development of transgenic plant with bas I gene from Arabidopsis; Development of transgenic plant with EoP gene from centipedegrass; Identification of radiation induced multi functional compounds from Aloe; Determination of the effects of radiation on removing undesirable color and physiological activities (Schizandra chinensis baillon, centipedegrass); Determine the action mechanism of transgenic plant with 2-Cys Prx for heat stress resistance; Determination of the effects of centipedegrass extracts on anti-cancer activities; Functional analysis of centipedegrass extracts (anti-virus effects)

  19. The value of the Lugol's iodine staining technique for the identification of vaginal epithelial cells.

    Science.gov (United States)

    Hausmann, R; Pregler, C; Schellmann, B

    1994-01-01

    This paper reports on the specificity of the Lugol's iodine staining technique for the detection of vaginal epithelial cells on penile swabs. Air-dried swabs taken from the glans of the penis of 153 hospital patients and from 50 healthy volunteers, whose last sexual intercourse had taken place at least 5 days previously, were stained with Lugol's solution. Glycogenated cells were found in more than 50% of the cases studied, even in healthy volunteers without urethritis. In almost all of these cases the smear contained at least a few polygonal nucleated epithelial cells showing an unequivocal positive Lugol reaction. These cells cannot be distinguished from superficial or intermediate vaginal cells, by cytomorphology or staining. Urinary tract infections had no influence on the glycogen content of male squamous epithelial cells. On the basis of these results the Lugol's method can no longer be assumed to prove the presence of vaginal cells in penile swabs.

  20. The study of preparation for immobilized cells membranes of E. Coli. by radiation technique

    International Nuclear Information System (INIS)

    Cao Jin; Chen Pin; Yu Yi

    1991-01-01

    The paper described the preparation of immobilized cells membranes with E. Coli by radiation technique. The nylon 6 was grafted with HEMA, which as a matrix to prepare immobilized cells membranes with E. Coli. by radiation entrapment at low temperature. The results showed that the retentive activity possessed a maximum value for membranes with E. Coli. when the irradiation dose was at 10-12 kGy, the entrapped cells has 2.3 g/ml at 50% HEMA concentration, the optimum pH and optimum temperature for membranes with E. Coli. are as same the original cells

  1. Isolation and Western Blotting of Latex-Bead Phagosomes to Track Phagosome Maturation.

    Science.gov (United States)

    Härtlova, Anetta; Peltier, Julien; Bilkei-Gorzo, Orsolya; Trost, Matthias

    2017-01-01

    Phagocytosis plays an essential role in the immune system for the defense against invading microorganisms and the clearing of apoptotic cells. After internalization, the newly formed phagosome is constantly remodeled by fusion with early endosomes, late endosomes, and lysosomes. These changes ultimately deliver the engulfed material into the terminal degradative compartments known as phagolysosomes. However, defective phagosome maturation can result in inflammatory or autoimmune disease depending on the type of phagosome cargo. Therefore, characterization of the components involved in phagosome formation and maturation is important for a better understanding of macrophage physiological and pathological functions. In this chapter we describe a step-by-step protocol for the isolation of large-scale latex/polystyrene bead phagosome preparations with high degrees of purity for Western blotting analysis of phagosome maturation.

  2. [Effects on proliferation ability of vascular smooth muscle cells by static and/or dynamic cell culture: utility of pre-seeding technique for dynamic cell culture].

    Science.gov (United States)

    Yokomuro, Hiroki; Ozawa, Tsukasa; Fujii, Takeshiro; Shiono, Noritsugu; Watanabe, Yoshinori; Yoshihara, Katsunori; Koyama, Nobuya; Okada, Mitsumasa

    2007-11-01

    Conventional biomaterials are not viable, do not grow, and do not provide contractile effects in cardiac tissue. Foreign synthetic material may become thrombogenic or infected. The most recent cardiac constructs consist of biodegradable material which has the potential to solve these problems. However, dynamic three-dimensional cell culture is necessary because conventional culture is limited to construct tough biografts. Vascular smooth muscle cells derived from rat aorta were seeded to poly-L-lactide-epsilon-capro-lactone copolymer in three groups; static culture group (static cell seeding + static cell culture), dynamic culture group (dynamic cell seeding + dynamic cell culture), and pre-seeding group [static cell seeding and culture for 1 week (pre-seeding) + dynamic cell culture]. The dynamic cell culture system used an original spinner flask. The pre-seeding technique used static cell seeding and culture before dynamic culture. The three groups were evaluated by cell proliferation and histologic studies. Vascular smooth muscle cells could be proliferated in/on the biodegradable materials. The pre-seeding group cells grew much more efficiently than the other groups. Very few cells were found in the biodegradable materials with the dynamic groups. However, there were many cells in the materials with the static culture group and pre-seeding group, especially the pre-seeding group. Dynamic culture is useful for constructing tough biografts by the pre-seeding technique.

  3. Studying cell-surface interactions in vitro: a survey of experimental approaches and techniques.

    Science.gov (United States)

    Michaelis, Stefanie; Robelek, Rudolf; Wegener, Joachim

    2012-01-01

    A better understanding of the interactions of animal (or human) cells with in vitro surfaces is the key to the successful development, improvement and optimization of biomaterials for biomedical or biotechnological purposes. State-of-the-art experimental approaches and techniques are a prerequisite for further and deeper insights into the mechanisms and processes involved in cell-surface adhesion. This chapter provides a brief but not complete survey of optical, mechanical, electrochemical and acoustic devices that are currently used to study the structural and functional properties of the cell-surface junction. Each technique is introduced with respect to the underlying principles before example data are discussed. At the end of the chapter all techniques are compared in terms of their strengths, limitations and technical requirements.

  4. Fluorescence exclusion: A simple versatile technique to calculate cell volumes and local heights (Conference Presentation)

    Science.gov (United States)

    Thouvenin, Olivier; Fink, Mathias; Boccara, A. Claude

    2017-02-01

    Understanding volume regulation during mitosis is technically challenging. Indeed, a very sensitive non invasive imaging over time scales ranging from seconds to hours and over large fields is required. Therefore, Quantitative Phase Imaging (QPI) would be a perfect tool for such a project. However, because of asymmetric protein segregation during mitosis, an efficient separation of the refractive index and the height in the phase signal is required. Even though many strategies to make such a separation have been developed, they usually are difficult to implement, have poor sensitivity, or cannot be performed in living cells, or in a single shot. In this paper, we will discuss the use of a new technique called fluorescence exclusion to perform volume measurements. By coupling such technique with a simultaneous phase measurement, we were also able to recover the refractive index inside the cells. Fluorescence exclusion is a versatile and powerful technique that allows the volume measurement of many types of cells. A fluorescent dye, which cannot penetrate inside the cells, is mixed with the external medium in a confined environment. Therefore, the fluorescent signal depends on the inverse of the object's height. We could demonstrate both experimentally and theoretically that fluorescence exclusion can accurately measure cell volumes, even for cells much higher than the depth of focus of the objective. A local accurate height and RI measurement can also be obtained for smaller cells. We will also discuss the way to optimize the confinement of the observation chamber, either mechanically or optically.

  5. A technique for in vitro culture of canine valvular interstitial cells.

    Science.gov (United States)

    Heaney, Allison M; Bulmer, Barret J; Ross, Christopher R; Schermerhorn, Thomas

    2009-06-01

    To develop a method for in vitro culture of canine valvular interstitial cells (VICs). Canine VICs were isolated from the distal third of the anterior mitral valve leaflet using an explant technique and maintained in cell culture. Molecular phenotyping of the cultured cells was performed using reverse transcription polymerase chain reaction and immunocytochemistry. Cells resembling fibroblasts migrated from canine mitral valve explants and were maintained in culture for up to eight passages. Establishment of the valve explant required collagen but once established, subsequent passages grew on non-coated plastic plates. At confluence the cultured cells exhibited the characteristic whorled pattern of fibroblasts in culture. The isolated valve cells expressed vimentin but not platelet endothelial cell adhesion molecule or von Willebrand's factor, consistent with the molecular phenotype of VICs. VICs can be readily isolated from canine mitral valve leaflets and successfully maintained in culture using standard culture techniques. The described techniques permit the study of bioactive VICs in a controlled environment and may be a useful in vitro model for investigation of cellular and molecular alterations associated with canine chronic degenerative valve disease.

  6. Direct tissue blot immunoassay for detection of Xylella fastidiosa in olive trees

    Directory of Open Access Journals (Sweden)

    Khaled DJELOUAH

    2015-01-01

    Full Text Available A direct tissue blot immunoassay (DTBIA technique has been compared with ELISA and PCR for detection of Xylella fastidiosa in olive trees from Apulia (southern Italy. Fresh cross-sections of young twigs and leaf petioles were printed onto nitrocellulose membranes and analyzed in the laboratory. Analyses of a first group of 61 samples gave similar efficiency for the three diagnostic techniques for detection the bacterium (24 positive and 36 negative samples, except for a single sample which was positive only with DTBIA and PCR. Similar results were obtained by separately analyzing suckers and twigs collected from different sectors of tree canopies of a second group of 20 olive trees (ten symptomatic and ten symptomless. In this second test the three diagnostic techniques confirmed the irregular distribution of the bacterium in the tree canopies and erratic detectability of the pathogen in the young suckers. It is therefore necessary to analyse composite samples per tree which should be prepared with twigs collected from different sides of the canopy. The efficiency comparable to ELISA and PCR, combined with the advantages of easier handling, speed and cost, make DTBIA a valid alternative to ELISA in large-scale surveys for occurrence of X. fastidiosa. Moreover, the printing of membranes directly in the field prevents infections spreading to Xylella-free areas, through movement of plant material with pathogen vectors for laboratory testing.

  7. Recent Advances in Genetic Technique of Microbial Report Cells and Their Applications in Cell Arrays

    Directory of Open Access Journals (Sweden)

    Do Hyun Kim

    2015-01-01

    Full Text Available Microbial cell arrays have attracted consistent attention for their ability to provide unique global data on target analytes at low cost, their capacity for readily detectable and robust cell growth in diverse environments, their high degree of convenience, and their capacity for multiplexing via incorporation of molecularly tailored reporter cells. To highlight recent progress in the field of microbial cell arrays, this review discusses research on genetic engineering of reporter cells, technologies for patterning live cells on solid surfaces, cellular immobilization in different polymers, and studies on their application in environmental monitoring, disease diagnostics, and other related fields. On the basis of these results, we discuss current challenges and future prospects for novel microbial cell arrays, which show promise for use as potent tools for unraveling complex biological processes.

  8. Study on production of useful metabolites by development of advanced cell culture techniques using radiation

    International Nuclear Information System (INIS)

    Chung, Byung Yeoup; Kim, Jinhong; Lee, Seung Sik; Bai, Hyounwoo; An, Byung Chull; Lee, Eun Mi; Lee, Jae Taek; Kim, Mi Ja

    2010-12-01

    The purpose of this project is improvement of investigation, materialization and evaluation techniques on effectiveness for functional natural compounds throughout development of tissue/cell culture techniques for mass production of useful metabolites using radiation. Research scope includes Development of a technique for radiation tissue and cell culture, Database construction for radiation response in plants and radiation effects, Construction of general-purpose national based techniques of cell culture technique using radiation. Main results are as follow: Isolation and identification of radiation induced basI gene; Determination of stresses sensitivities by transformating basI gene into arabidopsis; Isolation and identification of radiation induced chaperon proteins (PaAhpC and yPrxII) from Pseudomonas and yeast, and structural and functional analysis of the proteins; Determination of oxidative and heat resistance by transformating PaAhpC; Isolation and identification of maysin and its derivatives from centipedgrass; Investigation of enhancement technique for improving maysin and its derivatives production using radiation; Investigation of removing undesirable color in maysin and its derivatives using radiation; Determination of the effect of radiation on physiological functions of centipedgrass extracts; Identification of H 2 O 2 removing enzyme in radiation irradiated plant (Spinach); Determination of the effects of centipedgrass extracts on anti-obesity and anti-cancer activities

  9. Measurement of the cell membrane capacitance and conductance of colonic crypt cells of the rat using the patch clamp technique

    CERN Document Server

    Schill, C

    2005-01-01

    Using the patch clamp technique the membrane capacitance and membrane conductance of colonic crypt cells of the rat was measured. The influence of the intracellular agonists Ca++, cAMP and of osmotic changes on the membrane capacitance and conductance was studied.

  10. Synthesis on power electronics for large fuel cells: From power conditioning to potentiodynamic analysis technique

    International Nuclear Information System (INIS)

    De Bernardinis, Alexandre

    2014-01-01

    Highlights: • Active load for fuel cell managing electrical drive constraints: frequency and current ripple can be adjusted independently. • Multi-port resonant soft-switched topology for power management of a thirty kilowatt segmented PEM fuel cell. • Splitting current control strategy for power segmented PEM fuel cell in case of a segment is under fault. • Reversible Buck topology for large fuel cell with control of the fuel cell potential linked to current density nonlinearity. - Abstract: The work addressed in this paper deals with a synthesis on power electronic converters used for fuel cells. The knowledge gap concerns conceptually different electronic converter architectures for PEM (Proton Exchange Membrane) fuel cells able to perform three types of functionalities: The first one is the capacity of emulating an active load representative of electrical drive constraints. In that case, frequency and fuel cell current ripple can be set independently to investigate the dynamic behavior of the fuel cell. The second one is power conditioning applied to large high power and segmented fuel cell systems (“Large” represents several tens of cells and multi-kilowatt stacks), which is a non trivial consideration regarding the topological choices to be made for improving efficiency, compactness and ensure operation under faulty condition. A multi-port resonant isolated boost topology is analyzed enabling soft switching over a large operating range for a thirty kilowatt segmented fuel cell. A splitting current control strategy in case of a segment is under fault is proposed. Each considered converter topologies meet specific constraints regarding fuel cell stack design and power level. The third functionality is the ability for the power electronics to perform analysis and diagnosis techniques, like the cyclic voltammetry on large PEM fuel cell assemblies. The latter technique is an uncommon process for large fuel cell stacks since it is rather performed on

  11. The effects of radiation on p53-mutated glioma cells using cDNA microarray technique

    International Nuclear Information System (INIS)

    Ngo, F.Q.H.; Hsiao, Y.-Y.H.

    2003-01-01

    Full text: In this study, we investigated the effects of 10-Gy irradiation on cell-cycle arrest, apoptosis and clonogenic death in the p53-mutated human U138MG (malignant glioblastoma) cell line. In order to evaluate time-dependent events in cellular responses to radiation, we did a time course study by incubating cells ranging from 0.5 to 48 hours after irradiation. Cell-cycle distribution and apoptosis were evaluated by flow cytometry using propidium iodide (PI) and annexin-V plus PI staining. Cell viability and proliferative capacity were studied by colony formation assay. Dual fluorescence cDNA microarray technique was used to examine the differential expression patterns of the irradiated cells. The cDNA microarray chips used contained DNA sequences corresponding to 12,814 human genes. From the flow cytometry data, it can be observed that radiation induced G2/M phase arrest and that late apoptosis was more evident following G2/M arrest. After 36 hours, some cells underwent senescence and the remains continued on with the cell cycle. Microarray analyses revealed changes in the expression of a small number of cell-cycle-related genes (p21, cyclin B1, etc.) and cell-death genes (tumor necrosis factors, DDB2, etc.) suggesting their involvement in radiation-induced cell-cycle arrest and apoptosis. In silico interpretations of the molecular mechanisms responsible for these radiation effects are in progress

  12. Techniques for remote maintenance of in-cell material-handling system in the HFEF/N main cell

    International Nuclear Information System (INIS)

    Tobias, D.A.; Frickey, C.A.

    1975-01-01

    Operations in the main cell of HFEF/N have required development of remote handling equipment and unique techniques for maintaining the in-cell material-handling system. Specially designed equipment is used to remove a disabled crane or electromechanical manipulator bridge from its support rails and place it on floor stands for repair or maintenance. Support areas for the main cell, such as the spray chamber and hot repair area, provide essential decontamination, repair, and staging areas for the in-cell material-handling-system equipment and tools. A combined engineering and technical effort in upgrading existing master-slave manipulators has definitely reduced the requirements for their maintenance. The cell is primarily for postirradiation examination of LMFBR materials and fuel elements

  13. Standard loading controls are not reliable for Western blot quantification across brain development or in pathological conditions.

    Science.gov (United States)

    Goasdoue, Kate; Awabdy, Doreen; Bjorkman, Stella Tracey; Miller, Stephanie

    2016-02-01

    A frequently utilized method of data quantification in Western blot analysis is comparison of the protein of interest with a house keeping gene or control protein. Commonly used proteins include β-actin, glyceraldehyde 3 phosphate dehydrogenase (GAPDH), and α-tubulin. Various reliability issues have been raised when using this technique for data analysis-particularly when investigating protein expression changes during development and in disease states. In this study, we have demonstrated that β-actin, GAPDH, and α-tubulin are not appropriate controls in the study of development and hypoxic-ischemic induced damage in the piglet brain. We have also shown that using an in-house pooled standard, loaded on all blots is a reliable method for controlling interassay variability and data normalization in protein expression analysis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.

    Directory of Open Access Journals (Sweden)

    Samantha L Eaton

    Full Text Available Western blotting has been a key technique for determining the relative expression of proteins within complex biological samples since the first publications in 1979. Recent developments in sensitive fluorescent labels, with truly quantifiable linear ranges and greater limits of detection, have allowed biologists to probe tissue specific pathways and processes with higher resolution than ever before. However, the application of quantitative Western blotting (QWB to a range of healthy tissues and those from degenerative models has highlighted a problem with significant consequences for quantitative protein analysis: how can researchers conduct comparative expression analyses when many of the commonly used reference proteins (e.g. loading controls are differentially expressed? Here we demonstrate that common controls, including actin and tubulin, are differentially expressed in tissues from a wide range of animal models of neurodegeneration. We highlight the prevalence of such alterations through examination of published "-omics" data, and demonstrate similar responses in sensitive QWB experiments. For example, QWB analysis of spinal cord from a murine model of Spinal Muscular Atrophy using an Odyssey scanner revealed that beta-actin expression was decreased by 19.3±2% compared to healthy littermate controls. Thus, normalising QWB data to β-actin in these circumstances could result in 'skewing' of all data by ∼20%. We further demonstrate that differential expression of commonly used loading controls was not restricted to the nervous system, but was also detectable across multiple tissues, including bone, fat and internal organs. Moreover, expression of these "control" proteins was not consistent between different portions of the same tissue, highlighting the importance of careful and consistent tissue sampling for QWB experiments. Finally, having illustrated the problem of selecting appropriate single protein loading controls, we demonstrate

  15. Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.

    Science.gov (United States)

    Eaton, Samantha L; Roche, Sarah L; Llavero Hurtado, Maica; Oldknow, Karla J; Farquharson, Colin; Gillingwater, Thomas H; Wishart, Thomas M

    2013-01-01

    Western blotting has been a key technique for determining the relative expression of proteins within complex biological samples since the first publications in 1979. Recent developments in sensitive fluorescent labels, with truly quantifiable linear ranges and greater limits of detection, have allowed biologists to probe tissue specific pathways and processes with higher resolution than ever before. However, the application of quantitative Western blotting (QWB) to a range of healthy tissues and those from degenerative models has highlighted a problem with significant consequences for quantitative protein analysis: how can researchers conduct comparative expression analyses when many of the commonly used reference proteins (e.g. loading controls) are differentially expressed? Here we demonstrate that common controls, including actin and tubulin, are differentially expressed in tissues from a wide range of animal models of neurodegeneration. We highlight the prevalence of such alterations through examination of published "-omics" data, and demonstrate similar responses in sensitive QWB experiments. For example, QWB analysis of spinal cord from a murine model of Spinal Muscular Atrophy using an Odyssey scanner revealed that beta-actin expression was decreased by 19.3±2% compared to healthy littermate controls. Thus, normalising QWB data to β-actin in these circumstances could result in 'skewing' of all data by ∼20%. We further demonstrate that differential expression of commonly used loading controls was not restricted to the nervous system, but was also detectable across multiple tissues, including bone, fat and internal organs. Moreover, expression of these "control" proteins was not consistent between different portions of the same tissue, highlighting the importance of careful and consistent tissue sampling for QWB experiments. Finally, having illustrated the problem of selecting appropriate single protein loading controls, we demonstrate that normalisation

  16. Microphotographs of cyanobacteria documenting the effects of various cell-lysis techniques

    Science.gov (United States)

    Rosen, Barry H.; Loftin, Keith A.; Smith, Christopher E.; Lane, Rachael F.; Keydel, Susan P.

    2011-01-01

    Cyanotoxins are a group of organic compounds biosynthesized intracellularly by many species of cyanobacteria found in surface water. The United States Environmental Protection Agency has listed cyanotoxins on the Safe Drinking Water Act's Contaminant Candidate List 3 for consideration for future regulation to protect public health. Cyanotoxins also pose a risk to humans and other organisms in a variety of other exposure scenarios. Accurate and precise analytical measurements of cyanotoxins are critical to the evaluation of concentrations in surface water to address the human health and ecosystem effects. A common approach to total cyanotoxin measurement involves cell membrane disruption to release the cyanotoxins to the dissolved phase followed by filtration to remove cellular debris. Several methods have been used historically, however no standard protocols exist to ensure this process is consistent between laboratories before the dissolved phase is measured by an analytical technique for cyanotoxin identification and quantitation. No systematic evaluation has been conducted comparing the multiple laboratory sample processing techniques for physical disruption of cell membrane or cyanotoxins recovery. Surface water samples collected from lakes, reservoirs, and rivers containing mixed assemblages of organisms dominated by cyanobacteria, as well as laboratory cultures of species-specific cyanobacteria, were used as part of this study evaluating multiple laboratory cell-lysis techniques in partnership with the U.S. Environmental Protection Agency. Evaluated extraction techniques included boiling, autoclaving, sonication, chemical treatment, and freeze-thaw. Both treated and untreated samples were evaluated for cell membrane integrity microscopically via light, epifluorescence, and epifluorescence in the presence of a DNA stain. The DNA stain, which does not permeate live cells with intact membrane structures, was used as an indicator for cyanotoxin release into the

  17. Live cell imaging techniques to study T cell trafficking across the blood-brain barrier in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Coisne Caroline

    2013-01-01

    Full Text Available Abstract Background The central nervous system (CNS is an immunologically privileged site to which access for circulating immune cells is tightly controlled by the endothelial blood–brain barrier (BBB located in CNS microvessels. Under physiological conditions immune cell migration across the BBB is low. However, in neuroinflammatory diseases such as multiple sclerosis, many immune cells can cross the BBB and cause neurological symptoms. Extravasation of circulating immune cells is a multi-step process that is regulated by the sequential interaction of different adhesion and signaling molecules on the immune cells and on the endothelium. The specialized barrier characteristics of the BBB, therefore, imply the existence of unique mechanisms for immune cell migration across the BBB. Methods and design An in vitro mouse BBB model maintaining physiological barrier characteristics in a flow chamber and combined with high magnification live cell imaging, has been established. This model enables the molecular mechanisms involved in the multi-step extravasation of T cells across the in vitro BBB, to be defined with high-throughput analyses. Subsequently these mechanisms have been verified in vivo using a limited number of experimental animals and a spinal cord window surgical technique. The window enables live observation of the dynamic interaction between T cells and spinal cord microvessels under physiological and pathological conditions using real time epifluorescence intravital imaging. These in vitro and in vivo live cell imaging methods have shown that the BBB endothelium possesses unique and specialized mechanisms involved in the multi-step T cell migration across this endothelial barrier under physiological flow. The initial T cell interaction with the endothelium is either mediated by T cell capture or by T cell rolling. Arrest follows, and then T cells polarize and especially CD4+ T cells crawl over long distances against the direction of

  18. Novel bandgap grading technique for enhancing the limiting efficiency of solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Habib, S.E.-D.; Rafat, N.H. [Cairo Univ., Giza (Egypt). Faculty of Engineering

    1997-02-01

    The efficiency of solar cells is limited by radiative and/or Auger recombination losses. Radiative recombination can be reduced by limiting the escape angle of the re-emitted rays. Auger recombination can be reduced by limiting the cells` thickness. A novel technique for reducing the Auger recombination limit is proposed in this work. We show that bandgap grading can be effectively utilized to suppress the Auger recombination limit. The optimum bandgap grading profile that maximises the limiting efficiency for an idealized, one dimensional solar cell is hence calculated under AMO irradiation conditions. (author)

  19. Study on production of useful metabolites by development of advanced cell culture techniques using radiation

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Byung Yeoup; Kim, Jin Hong; Lee, Seung Sik; Kim, Jae Sung; An, Byung Chull; Moon, Yu Ran; Lee, Eun Mi; Lee, Min Hee; Lee, Jae Tack [KAERI, Daejeon (Korea, Republic of)

    2010-02-15

    The purpose of this project is improvement of investigation, materialization and evaluation techniques on effectiveness for functional natural compounds throughout development of tissue/cell culture techniques for mass production of useful metabolites using radiation. Research scope includes 1) Development of a technique for radiation tissue and cell culture, 2) Database construction for radiation response in plants and radiation effects, 3) Construction of general-purpose national based techniques of cell culture technique using radiation. Main results are as follow: mass culture of the adventitious roots of mountain ginseng (Panax ginseng C. A. Meyer) roots using rare earth elements in bioreactor: characterization of a transcription factor EoP gene from centipedegrass and the transcription regulation of LexA from Synechocystis sp PCC6803 and E. coli: identification of gamma-ray induced hydrogenase synthesis in hox gene transformed E. coli: transformation and the selection of the EoP transgene from Arabidopsis, rice and lettuce: Identification of the maysin and maysin derivatives in centipedegrass: characterization of gamma-ray induced color change in Taxus cuspidata: verification of the expression of antioxidant proteins (POD, APX and CAT) to gamma-ray in Arabidopsis: comparison of the response of the expression level to gamma-ray or H{sub 2}O{sub 2} in Arabidopsis; verification of the responses and effects to gamma-ray from plants (analysis of NPQ and ROS levels): the development method for rapidly enhancing maysin content of centipede grass; establishment of mass culture system for red beet

  20. Progress in emerging techniques for characterization of immobilized viable whole-cell biocatalysts

    Czech Academy of Sciences Publication Activity Database

    Bučko, M.; Vikartovská, A.; Schenkmayerová, A.; Tkáč, J.; Filip, J.; Chorvát Jr., D.; Neděla, Vilém; Ansorge-Schumacher, M.B.; Gemeiner, P.

    2017-01-01

    Roč. 71, č. 11 (2017), s. 2309-2324 ISSN 0366-6352 Institutional support: RVO:68081731 Keywords : bioelectrocatalysis * imaging techniques * immobilized whole-cell biocatalyst * multienzyme cascade reactions * online kinetics Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering OBOR OECD: Bioprocessing technologies (industrial processes relying on biological agents to drive the process) biocatalysis, fermentation Impact factor: 1.258, year: 2016

  1. Differential detection of cytoplasmic Wilms tumor 1 expression by immunohistochemistry, western blotting and mRNA quantification.

    Science.gov (United States)

    Maki, Takehiro; Ikeda, Hiroaki; Kuroda, Aki; Kyogoku, Noriaki; Yamamura, Yoshiyuki; Tabata, Yukiko; Abiko, Takehiro; Tsuchikawa, Takahiro; Hida, Yasuhiro; Shichinohe, Toshiaki; Tanaka, Eiichi; Kaga, Kichizo; Hatanaka, Kanako; Matsuno, Yoshihiro; Imai, Naoko; Hirano, Satoshi

    2017-01-01

    Wilms tumor 1 (WT1) is considered to be a promising target of cancer treatment because it has been reported to be frequently expressed at high levels in various malignancies. Although WT1-targeted cancer treatment has been initiated, conclusive detection methods for WT1 are not established. The present study aimed to consolidate immunohistochemistry for WT1 with statistical basis. Transfected cells with forced WT1 expression yielded specific western blot bands and nuclear immunostaining; cytoplasmic immunostaining was not specifically recognized. Immunohistochemistry, western blotting, and quantitative reverse transcriptase-polymerase chain reaction were performed in 35 human cell lines using multiple WT1 antibodies and their results were quantified. Relationships among the quantified results were statistically analyzed; the nuclear immunostaining positively correlated with western blot bands and mRNA expression levels, whereas cytoplasmic immunostaining did not. These results indicate that nuclear immunostaining reflects WT1 expression but cytoplasmic immunostaining does not. The nuclear immunostaining was barely (3/541) observed in primary cancer of esophagus, bile duct, pancreas and lung. Although the present study has some limitations, the results indicate that the cytoplasmic immunostaining does not correlate with actual WT1 expression and prompts researchers to carefully evaluate target molecule expression in treatment of cancer.

  2. Development of a monoclonal antibody-based colony blot immunoassay for detection of thermotolerant Campylobacter species.

    Science.gov (United States)

    Huang, Hongsheng; Phipps-Todd, Beverley; McMahon, Tanis; Elmgren, Catherine L; Lutze-Wallace, Cheryl; Todd, Zoe A; Garcia, Manuel M

    2016-11-01

    Campylobacter species, particularly thermotolerant Campylobacter spp., such as C. jejuni, are major human foodborne pathogens. Culture methods have been routinely used for the detection of this organism in various types of samples. An alternative, simple and rapid confirmation test(s) without further tedious biochemical tests would be useful. Meanwhile, Campylobacter-like colonies can be difficult to identify on agar plates overgrown with competitive bacteria, which can lead to false-negative results. This study was to develop a simple colony blot immunoassay using a new monoclonal antibody (Mab) produced in the present study for rapid screening, confirmation and quantification of campylobacters on culture agar plates. The procedure developed in this study was able to specifically detect thermotolerant Campylobacter spp., but not other non-thermotolerant Campylobacter and non-Campylobacter reference strains tested. This assay could detect 10 5 cells in a single dot. This assay showed 100% correlation with the culture method for the blotted membranes from 21 either chicken meat or vegetable samples experimentally inoculated with thermotolerant campylobacters. Among 101 natural samples of chicken meat (n=44), chicken feces (n=20) and vegetables (n=37), this assay also showed positive for 23 chicken meat and 14 fecal samples that were positive for thermotolerant campylobacters by culture method, and identified four additional suspects that were culture negative. Membranes stored at 4°C for at least 4years could also be used for this assay. The assay developed in this study can be used in quantitative study for immediate or archival usage, and for diagnostic test to preliminarily confirm the presence of thermotolerant Campylobacter on agar plates. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

  3. A multi-slice sliding cell technique for diffusion measurements in liquid metals

    Science.gov (United States)

    Zhong, Langxiang; Hu, Jinliang; Geng, Yongliang; Zhu, Chunao; Zhang, Bo

    2017-09-01

    The long capillary and shear-cell techniques are traditionally used for diffusion measurements in liquid metals. Inspired by the idea of the shear-cell method, we have built a multi-slice sliding cell device for inter-diffusion measurements in liquid metals. The device is designed based on a linear sliding movement rather than a rotational shearing as used in the traditional shear-cell method. Compared with the normal shear-cell method, the present device is a more compact setup thus easier to handle. Also, it is expected to be easier to monitor with X-rays or neutrons if used in in situ experiments. A series of benchmark time-dependent diffusion experiments in Al-Cu melts carried out with the present technique reveal that accurate diffusion constants can be achieved only after a sufficient time. For short annealing times, the initial shearing process causing convective flow dominates the measurement and leads to an increase of the measured diffusion coefficient by a factor three. The diffusion data obtained for Al-Cu liquids are consistent with the most accurate data measured by the in situ X-ray radiography method under well controlled conditions of no temperature gradient or other perturbation. High accuracy and easy handling as well as superior adaptability make the present technique suitable for diffusion studies in liquid metals.

  4. Impact of bone harvesting techniques on cell viability and the release of growth factors of autografts.

    Science.gov (United States)

    Miron, Richard J; Gruber, Reinhard; Hedbom, Erik; Saulacic, Nikola; Zhang, Yufeng; Sculean, Anton; Bosshardt, Dieter D; Buser, Daniel

    2013-08-01

    Autogenous bone grafts obtained by different harvesting techniques behave differently during the process of graft consolidation; the underlying reasons are however not fully understood. One theory is that harvesting techniques have an impact on the number and activity of the transplanted cells which contribute to the process of graft consolidation. To test this assumption, porcine bone grafts were harvested with four different surgical procedures: bone mill, piezosurgery, bone drilling (bone slurry), and bone scraper. After determining cell viability, the release of molecules affecting bone formation and resorption was assessed by reverse transcription polymerase chain reaction and immunoassay. The mitogenic and osteogenic activity of the conditioned media was evaluated in a bioassay with isolated bone cells. Cell viability and the release of molecules affecting bone formation were higher in samples harvested by bone mill and bone scraper when compared with samples prepared by bone drilling and piezosurgery. The harvesting procedure also affected gene expression, for example, bone mill and bone scraper samples revealed significantly higher expression of growth factors such as bone morphogenetic protein-2 and vascular endothelial growth factor compared with the two other modalities. Receptor activator of nuclear factor kappa B ligand expression was lowest in bone scraper samples. These data can provide a scientific basis to better understand the impact of harvesting techniques on the number and activity of transplanted cells, which might contribute to the therapeutic outcome of the augmentation procedure. © 2012 Wiley Periodicals, Inc.

  5. Coin-Cell-Based In Situ Characterization Techniques for Li-Ion Batteries

    Directory of Open Access Journals (Sweden)

    Liao Zhang

    2018-03-01

    Full Text Available In situ characterization techniques have made a significant progress in recent years, especially in the electrochemical field. For Li-ion batteries, in situ characterization techniques refer to using analytical equipment to directly characterize electrode materials during electrochemical measurements. At present, most in situ batteries are developed from commercial simulated batteries, of which the cost is very high and the cycle life is quite short. In this work, two kinds of coin-cell-based in situ batteries were designed as in situ X-ray diffraction (XRD and Raman coin cells which exhibit many admirable advantages, such as low cost, long cycle life, easy to carry, and so on. In the designing process, in situ XRD and Raman coin cell have been tested with two electrode materials of Li4Ti5O12 and LiFePO4, and we solved many technical problems of assembling and measuring these two kinds of cells. Finally, in situ coin cells could be improved to investigate a variety of electrode materials, and this technique would arouse wide interests in the electrochemical field.

  6. Aloe vera is non-toxic to cells: A microculture tetrazolium technique colorimetric assay study

    Directory of Open Access Journals (Sweden)

    Devi Gopakumar

    2014-01-01

    Full Text Available Introduction: Aloe vera (Av, a succulent of Liliaceae family is now a widely used medicinal plant. Its′ application covers a wide spectrum of human diseases, including oral mucosa, gastric mucosa and skin. Aloe vera preparations in the form of gel, mouth washes and cream are applied topically for many oral diseases. The applications include oral lichen planus, candidiasis, oral submucous fibrosis, geographic tongue, etc. Aims and Objectives: To evaluate the cytotoxicity of Av on human fibroblasts. Materials and Methods: Aloe vera preparation (70% was applied on the fibroblast cell lineage and the cell viability was evaluated by microculture tetrazolium technique (MTT colorimetric assay. Results: The cell viability at different concentrations was measured. The cells have maintained their viability at different concentrations used in the study. Conclusion: Our study shows the cell viability at different sample concentrations of Av. This could open up wide clinical applications of Av for reactive, inflammatory and potentially malignant oral and other mucocutaneous diseases.

  7. Experimental methods and modeling techniques for description of cell population heterogeneity

    DEFF Research Database (Denmark)

    Lencastre Fernandes, Rita; Nierychlo, M.; Lundin, L.

    2011-01-01

    With the continuous development, in the last decades, of analytical techniques providing complex information at single cell level, the study of cell heterogeneity has been the focus of several research projects within analytical biotechnology. Nonetheless, the complex interplay between...... environmental changes and cellular responses is yet not fully understood, and the integration of this new knowledge into the strategies for design, operation and control of bioprocesses is far from being an established reality. Indeed, the impact of cell heterogeneity on productivity of large scale cultivations...... methods for monitoring cell population heterogeneity as well as model frameworks suitable for describing dynamic heterogeneous cell populations. We will furthermore underline the highly important coordination between experimental and modeling efforts necessary to attain a reliable quantitative description...

  8. Complementary techniques for solid oxide cell characterisation on micro- and nano-scale

    International Nuclear Information System (INIS)

    Wiedenmann, D.; Hauch, A.; Grobety, B.; Mogensen, M.; Vogt, U.

    2009-01-01

    High temperature steam electrolysis by solid oxide electrolysis cells (SOEC) is a way with great potential to transform clean and renewable energy from non-fossil sources to synthetic fuels such as hydrogen, methane or dimethyl ether, which have been identified as promising alternative energy carriers. Also, as SOEC can operate in the reverse mode as solid oxide fuel cells (SOFC), during high peak hours e.g. hydrogen can be used in a very efficient way to reconvert chemically stored energy into electrical energy. As solid oxide cells (SOC) are working at high temperatures (700-900 o C), material degradation and evaporation can occur e.g. from the cell sealing material, leading to poisoning effects and aging mechanisms which are decreasing the cell efficiency and long-term durability. In order to investigate such cell degradation processes, thorough examination on SOC often requires the chemical and structural characterisation on the microscopic and the nanoscopic level. The combination of different microscope techniques like conventional scanning electron microscopy (SEM), electron-probe microanalysis (EPMA) and the focused ion-beam (FIB) preparation technique for transmission electron microscopy (TEM) allows performing post mortem analysis on a multi scale level of cells after testing. These complementary techniques can be used to characterize structural and chemical changes over a large and representative sample area (micro-scale) on the one hand, and also on the nano-scale level for selected sample details on the other hand. This article presents a methodical approach for the structural and chemical characterisation of changes in aged cathode-supported electrolysis cells produced at Riso DTU, Denmark. Also, results from the characterisation of impurities at the electrolyte/hydrogen interface caused by evaporation from sealing material are discussed. (author)

  9. Abnormalities in structure and expression of the retinoblastoma gene in small cell lung cancer cell lines and xenografts in nude mice

    DEFF Research Database (Denmark)

    Rygaard, K; Sorenson, G D; Pettengill, O S

    1990-01-01

    the Rb protein, we investigated the expression of the Mr 110,000-116,000 Rb protein in SCLC tumors grown as xenografts in nude mice and/or as cell lines. Rb messenger RNA expression was determined by Northern blotting, and gross structural gene alterations were investigated by Southern blotting. Tumors...... small cell lung cancer (SCLC). Absence of the 4.7 kilobase mRNA has been found to be frequent in SCLC, and it has been reported that the Rb Mr 110,000-116,000 protein product is always absent, even in tumors expressing Rb mRNA. Using Western blotting technique with a monoclonal antibody directed against...

  10. Western blotting as a tool for the serodiagnosis of farmer's lung disease: validation with Lichtheimia corymbifera protein extracts.

    Science.gov (United States)

    Rognon, Bénédicte; Reboux, Gabriel; Roussel, Sandrine; Barrera, Coralie; Dalphin, Jean-Charles; Fellrath, Jean-Marc; Monod, Michel; Millon, Laurence

    2015-04-01

    Electrosyneresis and double diffusion are immunoprecipitation techniques commonly used in the serological diagnosis of Farmer's lung disease (FLD). These techniques are reliable but lack standardization. The aim of this study was to evaluate Western blotting for the serodiagnosis of FLD. We carried out Western blotting with an antigenic extract of Lichtheimia corymbifera, an important aetiological agent of the disease. The membranes were probed with sera from 21 patients with FLD and 21 healthy exposed controls to examine the IgG antibody responses against purified somatic antigens. Given the low prevalence of the disease, 21 patients could be considered as a relevant series. Four bands were significantly more frequently represented in membranes probed with FLD sera (bands at 27.7, 40.5, 44.0 and 50.5 kDa) than those probed with control sera. We assessed the diagnostic value of different criteria alone or in combination. The diagnostic accuracy of the test was highest with the inclusion of at least two of the following criteria: at least five bands on the strip and the presence of one band at 40.5 or 44.0 kDa. Sensitivity, specificity and positive and negative predictive values were all 81%, and the odds ratio was 18.06. Inclusion of bands of high intensity diminished rather than improved the diagnostic value of the test. We concluded that Western blotting is a valuable technique for the serodiagnosis of FLD. The industrial production of ready-to-use membranes would enable the routine use of this technique in laboratories, and provide reliable and standardized diagnostic results within a few hours. © 2015 The Authors.

  11. Research Techniques Made Simple: High-Throughput Sequencing of the T-Cell Receptor.

    Science.gov (United States)

    Matos, Tiago R; de Rie, Menno A; Teunissen, Marcel B M

    2017-06-01

    High-throughput sequencing (HTS) of the T-cell receptor (TCR) is a rapidly advancing technique that allows sensitive and accurate identification and quantification of every distinct T-cell clone present within any biological sample. The relative frequency of each individual clone within the full T-cell repertoire can also be studied. HTS is essential to expand our knowledge on the diversity of the TCR repertoire in homeostasis or under pathologic conditions, as well as to understand the kinetics of antigen-specific T-cell responses that lead to protective immunity (i.e., vaccination) or immune-related disorders (i.e., autoimmunity and cancer). HTS can be tailored for personalized medicine, having the potential to monitor individual responses to therapeutic interventions and show prognostic and diagnostic biomarkers. In this article, we briefly review the methodology, advances, and limitations of HTS of the TCR and describe emerging applications of this technique in the field of investigative dermatology. We highlight studying the pathogenesis of T cells in allergic dermatitis and the application of HTS of the TCR in diagnosing, detecting recurrence early, and monitoring responses to therapy in cutaneous T-cell lymphoma. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Positive selection of Wharton's jelly-derived CD105(+) cells by MACS technique and their subsequent cultivation under suspension culture condition: A simple, versatile culturing method to enhance the multipotentiality of mesenchymal stem cells.

    Science.gov (United States)

    Amiri, Fatemeh; Halabian, Raheleh; Dehgan Harati, Mozhgan; Bahadori, Marzie; Mehdipour, Ahmad; Mohammadi Roushandeh, Amaneh; Habibi Roudkenar, Mehryar

    2015-05-01

    Wharton's jelly (WJ), an appropriate source of mesenchymal stem cells (MSCs), has been shown to have a wide array of therapeutic applications. However, the WJ-derived MSCs are very heterogeneous and have limited expression of pluripotency markers. Hence, improvement of their culture condition would promote the efficiency of WJ-MSCs. This study aims to employ a simple method of cultivation to obtain WJ-MSCs which express more pluripotency markers. CD105(+) cells were separated by magnetic-associated (activated) cell sorting from umbilical cord mucous tissue. CD105(+) cells were added to Methocult medium diluted in α-minimum essential medium (α-MEM) and seeded in poly(2-hydroxyethyl methacrylate) (poly-HEMA)-coated plates for suspension culture preparation. Differentiation capacity of isolated cells was evaluated in the presence of differentiation-inducing media. The expression of pluripotency markers such as Oct3/4, Nanog, and Sox2 was also analyzed by RT-PCR and western blot techniques. Moreover, immunocytochemistry was performed to detect alpha-smooth muscle actin (antigene) (α-SMA) protein. WJ-MSCs grew homogeneously and formed colonies when cultured under suspension culture conditions (Non-adhesive WJ-MSCs). They maintained their growth ability in both adherent and suspension cultures for several passages. Non-adhesive WJ-MSCs expressed Oct3/4, Nanog, and Sox2 both at transcriptional and translational levels in comparison to those cultured in conventional adherent cultures. They also expressed α-SMA protein. In this study, we isolated WJ-MSCs using a slightly modified culture condition. Our simple non-genetic method resulted in a homogeneous population of WJ-MSCs, which highly expressed pluripotency markers. In the future, more multipotent WJ-MSCs can be harnessed as a non-embryonic source of MSCs in MSC-based cell therapy.

  13. Western blot can distinguish natural and acquired antibodies to Mycoplasma agassizii in the desert tortoise (Gopherus agassizii).

    Science.gov (United States)

    Hunter, Kenneth W; Dupré, Sally A; Sharp, Tiffanny; Sandmeier, Franziska C; Tracy, C Richard

    2008-12-01

    Mycoplasma agassizi has been identified as a cause of upper respiratory tract disease (URTD) in the threatened Mojave population of the desert tortoise (Gopherus agassizii), and anti-M. agassizii antibodies have been found by ELISA in as many as 15% of these animals across their geographic range. Here we report that a cohort of 16 egg-reared desert tortoises never exposed to M. agassizii had ELISA antibody titers to this organism that overlapped with titers obtained from some M. agassizii-infected tortoises. These natural antibodies were predominantly of the IgM class. Western blots of plasma from these non-infected tortoises produced a characteristic banding pattern against M. agassizii antigens. A group of 38 wild-caught desert tortoises was tested by ELISA, and although some of these tortoises had antibody titers significantly higher than the non-infected tortoises, there was considerable overlap at the lower titer levels. However, Western blot analysis revealed distinct banding patterns that could readily distinguish between the non-infected tortoises and tortoises with acquired antibodies, regardless of ELISA antibody titers. We conclude that desert tortoises have natural antibodies to M. agassizii that can compromise the determination of infection status by ELISA. However, the Western blot technique can distinguish between natural and acquired antibody patterns and can be used to confirm the diagnosis of M. agassizii infections in the desert tortoise.

  14. Zinc blotting assay for detection of zinc binding prolamin in barley (Hordeum vulgare) grain

    DEFF Research Database (Denmark)

    Uddin, Mohammad Nasir; Nielsen, Ane Langkilde-Lauesen; Vincze, Eva

    2014-01-01

    zinc blotting method with a zinc-sensing dye, dithizone. Hordeins were extracted from mature barley grain, separated by SDS-PAGE, blotted on a membrane, renatured, overlaid, and probed with zinc; subsequently, zinc-binding specificity of certain proteins was detected either by autoradiography or color...

  15. When less is more: a simple Western blotting amendment allowing data acquisition on human single fibers

    DEFF Research Database (Denmark)

    Jensen, Thomas Elbenhardt; Richter, Erik

    2011-01-01

    This editorial discusses a simple western blotting-amendment allowing rapid data-acquisition on single fibers obtained from freeze-dried human skeletal muscle biopsies.......This editorial discusses a simple western blotting-amendment allowing rapid data-acquisition on single fibers obtained from freeze-dried human skeletal muscle biopsies....

  16. Microfluidic integration of Western blotting is enabled by electrotransfer-assisted sodium dodecyl sulfate dilution.

    Science.gov (United States)

    Hou, Chenlu; Herr, Amy E

    2013-01-07

    We integrate sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with subsequent antibody probing in a single, monolithic microdevice to realize microfluidic Western blotting. A hurdle to successful on-chip Western blotting lies in restoring antibody recognition of previously sized (denatured, reduced) proteins. To surmount this hurdle, we locally dilute free SDS from SDS-protein complexes using differential electromigration of the species during electrotransfer between SDS-PAGE and blotting regions of a microchamber. Local dilution of SDS minimizes re-association of SDS with proteins offering means to restore antibody binding affinity to proteins after SDS-PAGE. To achieve automated, programmable operation in a single instrument, we utilize a 1 × 2 mm(2) glass microchamber photopatterned with spatially distinct, contiguous polyacrylamide regions for SDS-PAGE, electrotransfer, and antibody blotting. Optimization of both the SDS-PAGE and electrotransfer conditions yields transfer distances of Western blot is completed in 180 s, with fully automated assay operation using programmable voltage control. After SDS-PAGE and electrotransfer, we observe ~80% capture of protein band mass on the blotting region for a model protein, C-reactive protein. This novel microfluidic Western blot approach introduces fine transport control for in-transit protein handling to form the basis for an automated, rapid alternative to conventional slab-gel Western blotting.

  17. A Laboratory Exercise Illustrating the Sensitivity and Specificity of Western Blot Analysis

    Science.gov (United States)

    Chang, Ming-Mei; Lovett, Janice

    2011-01-01

    Western blot analysis, commonly known as "Western blotting," is a standard tool in every laboratory where proteins are analyzed. It involves the separation of polypeptides in polyacrylamide gels followed by the electrophoretic transfer of the separated polypeptides onto a nitrocellulose or polyvinylidene fluoride membrane. A replica of the…

  18. The role of printing techniques for large-area dye sensitized solar cells

    Science.gov (United States)

    Mariani, Paolo; Vesce, Luigi; Di Carlo, Aldo

    2015-10-01

    The versatility of printing technologies and their intrinsic ability to outperform other techniques in large-area deposition gives scope to revolutionize the photovoltaic (PV) manufacturing field. Printing methods are commonly used in conventional silicon-based PVs to cover part of the production process. Screen printing techniques, for example, are applied to deposit electrical contacts on the silicon wafer. However, it is with the advent of third generation PVs that printing/coating techniques have been extensively used in almost all of the manufacturing processes. Among all the third generation PVs, dye sensitized solar cell (DSSC) technology has been developed up to commercialization levels. DSSCs and modules can be fabricated by adopting all of the main printing techniques on both rigid and flexible substrates. This allows an easy tuning of cell/module characteristics to the desired application. Transparency, colour, shape, layout and other DSSC’s features can be easily varied by changing the printing parameters and paste/ink formulations used in the printing process. This review focuses on large-area printing/coating technologies for the fabrication of DSSCs devices. The most used and promising techniques are presented underlining the process parameters and applications.

  19. The role of printing techniques for large-area dye sensitized solar cells

    International Nuclear Information System (INIS)

    Mariani, Paolo; Vesce, Luigi; Di Carlo, Aldo

    2015-01-01

    The versatility of printing technologies and their intrinsic ability to outperform other techniques in large-area deposition gives scope to revolutionize the photovoltaic (PV) manufacturing field. Printing methods are commonly used in conventional silicon-based PVs to cover part of the production process. Screen printing techniques, for example, are applied to deposit electrical contacts on the silicon wafer. However, it is with the advent of third generation PVs that printing/coating techniques have been extensively used in almost all of the manufacturing processes. Among all the third generation PVs, dye sensitized solar cell (DSSC) technology has been developed up to commercialization levels. DSSCs and modules can be fabricated by adopting all of the main printing techniques on both rigid and flexible substrates. This allows an easy tuning of cell/module characteristics to the desired application. Transparency, colour, shape, layout and other DSSC’s features can be easily varied by changing the printing parameters and paste/ink formulations used in the printing process. This review focuses on large-area printing/coating technologies for the fabrication of DSSCs devices. The most used and promising techniques are presented underlining the process parameters and applications. (paper)

  20. Validating the use of short interfering RNA as a novel technique for cell-specific target gene knockdown in lung ischemia-reperfusion injury.

    Science.gov (United States)

    Merry, Heather E; Phelan, Patrick; Hwang, Billanna; Mulligan, Michael S

    2016-02-01

    Short interfering RNA is an effective method for target gene knockdown. However, concerns surround the design, administration, efficacy, specificity, and immunostimulatory potential. Although uptake by alveolar macrophages has been demonstrated, studies have not examined its use in lung ischemia-reperfusion injury. We describe the validation of short interference RNA as a novel technique for cell-specific target gene knockdown in our model of lung ischemia-reperfusion injury. Dose-response experiments were performed, and 3 distinct sequences of toll-like receptor-4, toll-like receptor-2, and myeloid differentiation factor-88 short interference RNA were tested for efficacy of knockdown. Saline, lipid vector, and noncoding short interference RNA controls were used. Similar experiments were performed in primary cultures of resident pulmonary cells. Target protein knockdown was assessed by Western blot. Rat serum and cell culture media were assessed for interferon and cytokine production. Biotin labeling was used to assess short interference RNA uptake. Target protein expression was significantly reduced using short interference RNA. However, toll-like receptor-4 knockdown was isolated to alveolar macrophages, and biotin labeling confirmed toll-like receptor-4 short interference RNA localization to alveolar macrophages. There was significant knockdown of toll-like receptor-4 expression in cultured cells treated with toll-like receptor-4 short interference RNA. There was no significant change in interferon production after short interference RNA treatment. There was effective target protein knockdown with each sequence used. Short interference RNA is a valid method for achieving target protein knockdown in alveolar macrophages and is an important tool in the evaluation of its role in the development of lung ischemia-reperfusion injury. Copyright © 2016 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

  1. Novel Technique for Sampling of Breast Implant–associated Seroma in Anaplastic Large Cell Lymphoma

    Science.gov (United States)

    T’Kindt, Johan; Mertens, Marianne; Colpaert, Steven D. M.

    2016-01-01

    Summary: We describe a novel technique for the sampling of breast implant–associated seroma. Using a blunt-tip lipofilling cannula, we have the freedom of movement to sample all fluid collections and prevent the misfortunes of damaging the implant. Also, we have demonstrated the inability of the Coleman style I lipofilling cannula to perforate a silicone breast implant. This practical and reliable technique will prove to be useful in managing the breast implant–associated seroma, especially with the rising incidence of the anaplastic large cell lymphoma, where the sampling of seroma is mandatory. PMID:27200250

  2. Human umbilical cord-derived mesenchymal stem cells differentiate into epidermal-like cells using a novel co-culture technique.

    Science.gov (United States)

    Li, Dongjie; Chai, Jiake; Shen, Chuanan; Han, Yanfu; Sun, Tianjun

    2014-08-01

    Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) isolated from human umbilical Wharton's Jelly are a population of primitive and pluripotent cells. In specific conditions, hUCMSCs can differentiate into various cells, including adipocytes, osteoblasts, chondrocytes, neurocytes, and endothelial cells. However, few studies have assessed their differentiation into epidermal cells in vitro. To assess the potential of hUCMSCs to differentiate into epidermal cells, a microporous membrane-based indirect co-culture system was developed in this study. Epidermal stem cells (ESCs) were seeded on the bottom of the microporous membrane, and hUCMSCs were seeded on the top of the microporous membrane. Cell morphology was assessed by phase contrast microscopy, and the expression of early markers of epidermal cell lineage, P63, cytokeratin19 (CK19), and β1-integrin, was determined by immunofluorescence, Western blot, and quantitative real-time PCR (Q-PCR) analyses. hUCMSC morphology changed from spindle-like to oblate or irregular with indirect co-culture with ESCs; they also expressed greater levels P63, CK19, and β1-integrin mRNA and protein compared to the controls (p cultures, indirect co-culture expressed significantly greater CK19 protein (p culture model.

  3. Analyzing Protein Changes in Guinea Pig Tissue Lysates Using Non-guinea Pig Specific Antibodies: Procedures for Western Blotting and Examples Using 16 Individual Antibodies for Common CNS Proteins

    National Research Council Canada - National Science Library

    Johnson, Erik A; Daugherty, Kelly S

    2006-01-01

    .... Common Western blotting techniques were used to compare immunostaining patterns of tissue lysates between a known species, rat, and the guinea pig using antibodies to several common CNS proteins...

  4. Drifter technique: a new method to obtain metaphases in Hep-2 cell line cultures

    Directory of Open Access Journals (Sweden)

    Eleonidas Moura Lima

    2005-07-01

    Full Text Available The Hep-2 cell line is derived from laryngeal carcinoma cells and is often utilized as a model in carcinogenesis and mutagenesis tests. To evaluate the proliferative potential of this line, we developed a cytogenetic methodology (drifter technique to obtain metaphases from cells that loose cellular adhesion when they underwent mitosis in culture. By this procedure, 2000 cells were counted, resulting in a mitotic index (MI of 22.2%. Although this MI was not statistically different from the one obtained using either a classical cytogenetic method or a cell synchronization technique, the drifter technique has the advantage of not requiring the use of some reagents for the obtention of metaphases and also of diminishing the consumption of maintenance reagents for this cell line.A linhagem celular Hep-2 é formada por células de carcinoma da laringe e é muito utilizada em modelos de carcinogênese e mutagenêse. Para avaliar o potencial proliferativo desta linhagem, desenvolvemos uma metodologia citogenética (técnica do sobrenadante para obtenção de metáfases a partir de células que, ao entrarem em mitose, perdem adesão celular, ficando em suspensão no meio de cultura. Através deste procedimento, foram contadas 2000 células, correspondendo a um índice mitótico (IM de 22.2% . Apesar de o IM obtido por esta técnica não ter sido estatisticamente diferente do IM obtido por outras metodologias citogenéticas clássicas, a técnica do sobrenadante é vantajosa porque elimina o uso de alguns reagentes utilizados na obtenção de metáfases e também diminui o consumo de reagentes de manutenção desta linhagem.

  5. An analysis of endothelial microparticles as a function of cell surface antibodies and centrifugation techniques.

    Science.gov (United States)

    Venable, Adam S; Williams, Randall R; Haviland, David L; McFarlin, Brian K

    2014-04-01

    Chronic vascular disease is partially characterized by the presence of lesions along the vascular endothelial wall. Current FDA-approved clinical techniques lack the ability to measure very early changes in endothelial cell health. When endothelial cells are damaged, they release endothelial microparticles (EMPs) into circulation. Thus, blood EMP concentration may represent a useful cardiovascular disease biomarker. Despite the potential value of EMPs, current flow cytometry techniques may not consistently distinguish EMPs from other small cell particles. The purpose of this study was to use imaging flow cytometry to modify existing methods of identifying EMPs based on cell-surface receptor expression and visual morphology. Platelet poor plasma (PPP) was isolated using four different techniques, each utilizing a two-step serial centrifugation process. The cell-surface markers used in this study were selected based on those that are commonly reported in the literature. PPP (100μL) was labeled with CD31, CD42a, CD45, CD51, CD66b, and CD144 for 30-min in dark on ice. Based on replicated experiments, EMPs were best identified by cell-surface CD144 expression relative to other commonly reported EMP markers (CD31 & CD51). It is important to note that contaminating LMPs, GMPs, and PMPs were thought to be removed in the preparation of PPP. However, upon analysis of prepared samples staining CD31 against CD51 revealed a double-positive population that was less than 1% EMPs. In contrast, when using CD144 to identify EMPs, ~87% of observed particles were free of contaminating microparticles. Using a counterstain of CD42a, this purity can be improved to over 99%. More research is needed to understand how our improved EMP measurement method can be used in experimental models measuring acute vascular responses or chronic vascular diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Nonlinear modelling of polymer electrolyte membrane fuel cell stack using nonlinear cancellation technique

    International Nuclear Information System (INIS)

    Barus, R. P. P.; Tjokronegoro, H. A.; Leksono, E.; Ismunandar

    2014-01-01

    Fuel cells are promising new energy conversion devices that are friendly to the environment. A set of control systems are required in order to operate a fuel cell based power plant system optimally. For the purpose of control system design, an accurate fuel cell stack model in describing the dynamics of the real system is needed. Currently, linear model are widely used for fuel cell stack control purposes, but it has limitations in narrow operation range. While nonlinear models lead to nonlinear control implemnetation whos more complex and hard computing. In this research, nonlinear cancellation technique will be used to transform a nonlinear model into a linear form while maintaining the nonlinear characteristics. The transformation is done by replacing the input of the original model by a certain virtual input that has nonlinear relationship with the original input. Then the equality of the two models is tested by running a series of simulation. Input variation of H2, O2 and H2O as well as disturbance input I (current load) are studied by simulation. The error of comparison between the proposed model and the original nonlinear model are less than 1 %. Thus we can conclude that nonlinear cancellation technique can be used to represent fuel cell nonlinear model in a simple linear form while maintaining the nonlinear characteristics and therefore retain the wide operation range

  7. Diagnostic efficacy of Brucella abortus strain RB51 in experimentally inoculated Sprague-Dawley rats using western blot assay.

    Science.gov (United States)

    Rahman, Siddiqur; Baek, Byeong Kirl

    2008-10-01

    To investigate the diagnosis and efficacy of Brucella abortus strain RB51 (SRB51) in experimentally inoculated Sprague-Dawley (SD) rat using western blot assay. Female SD rats were orally administered with 1.0 x 10(7) colony forming unit (cfu) suspension of SRB51 and half of these SD rats were challenged at 4 weeks post inoculation with 1.0 x 10(9) cfu suspension of B. abortus biotype 1 isolated in South Korea. Sera of SD rats were monitored at regular intervals by western blot assay using whole cell antigen of B. abortus strain 1119-3 (S1119-3). The bacteriological examination of blood and clinical examination of the rats were also performed. There were several bands at 120, 70, 45, 30, 20 kDa and clear specific bands were found after vaccination (20, 70 kDa) and challenge (15, 20, 45, 70, 120 kDa). The highest immune response was observed in sera 4 weeks post SRB51 vaccination. SRB51 was recovered from the blood of all of SRB51 inoculated rats until one week post vaccination and there were no clinical signs in that inoculated rats. It is concluded that the SRB51 elicits antigen specific immunity in SD rats based on western blot assay.

  8. Monitoring programmed cell death of living plant tissues in microfluidics using electrochemical and optical techniques

    DEFF Research Database (Denmark)

    Mark, Christina; Zor, Kinga; Heiskanen, Arto

    This project focuses on developing and applying a tissue culture system with electrochemical and optical detection techniques for tissue culture of barley aleurone layer to increase understanding of the underlying mechanisms of programmed cell death (PCD) in plants. The major advantage of electro...... an optical double-fluorescent probe-system[4]. Currently, we are working on integrating both detection methods into a tissue culture system for immobilised plant tissues.......This project focuses on developing and applying a tissue culture system with electrochemical and optical detection techniques for tissue culture of barley aleurone layer to increase understanding of the underlying mechanisms of programmed cell death (PCD) in plants. The major advantage...... of electrochemical detection systems is that they can be miniaturized, multiplexed and automated without losing their performance[1,2]. Combining tissue culture with electrochemical and optical detection allows implementation of a wide range of assays for online, real-time, parallel analysis of important parameters...

  9. Detection of Rickettsia in Rhipicephalus sanguineus Ticks and Ctenocephalides felis Fleas from Southeastern Tunisia by Reverse Line Blot Assay

    Science.gov (United States)

    Khrouf, Fatma; M'Ghirbi, Youmna; Znazen, Abir; Ben Jemaa, Mounir; Hammami, Adnene

    2014-01-01

    Ticks (n = 663) and fleas (n = 470) collected from domestic animals from southeastern Tunisia were screened for Rickettsia infection using reverse line blot assay. Evidence of spotted fever group Rickettsia was obtained. We detected Rickettsia felis in fleas, Rickettsia massiliae Bar 29 and the Rickettsia conorii Israeli spotted fever strain in ticks, and Rickettsia conorii subsp. conorii and Rickettsia spp. in both arthropods. The sensitivity of the adopted technique allowed the identification of a new association between fleas and R. conorii subsp. conorii species. The presence of these vector-borne Rickettsia infections should be considered when diagnosing this disease in humans in Tunisia. PMID:24226919

  10. The study of the structures of the white blood cells using pattern recognition technique

    International Nuclear Information System (INIS)

    Arquisa, M.

    1976-03-01

    It is aimed that through machine recognition, a significant quantitative description of the white blood cells be obtained. This technique will give the characterization of the normal and abnormal white blood cells which may eventually lead to exact and efficient blood examinations and to the possibility of using white blood cells as an effective biological monitor in the assessment of radiation damage and other pathological disorders. Described are the preparation of blood stains and staining procedure with Giemsa and Wright stains, photomicrography of white blood cells with the use of Kodak Dektol Developer and Kodak Acid-Bath Fixer. The film rolls were then scanned. The scanner is used to scan photographic transparencies of white blood cells. This instrument gathers information and converts cell features such as size, shape, ash and granulation into a series of parameters whose values are descriptive of the minute but essential structural characteristics of the cells. From July 1 -December 31, 1975, a total of 51 blood smears were collected and stained. From these blood samples, a total of 103 neutrophils, 30 lymphocytes and 12 monocytes were added to the film library

  11. Re-purposing of histological tissue sections for corroborative western blot analysis of hypothalamic metabolic neuropeptide expression following delineation of transactivated structures by Fos immuno-mapping.

    Science.gov (United States)

    Alenazi, Fahaad S H; Ibrahim, Baher A; Briski, Karen P

    2015-04-01

    Fos immunocytochemistry is a valuable anatomical mapping tool for distinguishing cells within complex tissues that undergo genomic activation, but it is seldom paired with corroborative molecular analytical techniques. Due to preparatory requirements that include protein cross-linking for specimen sectioning, histological tissue sections are regarded as unsuitable for those methods. Our studies show that pharmacological activation of the hindbrain energy sensor AMPK by AICAR elicits estradiol (E)-dependent patterns of Fos immunolabeling of hypothalamic metabolic loci. Here, Western blotting was applied to hypothalamic tissue removed from histological sections of E- versus oil (O)-implanted ovariectomized (OVX) female rat brain to measure levels of metabolic transmitters associated with Fos-positive structures. In both E and O rats, AICAR treatment elicited alterations in pro-opiomelanocortin, neuropeptide Y, SF-1, and orexin-A neuropeptide expression that coincided with patterns of Fos labeling of structures containing neurons that synthesize these neurotransmitters, e.g. arcuate and ventromedial nuclei and lateral hypothalamic area. O, but not E animals also exhibited parallel augmentation of tissue corticotropin-releasing hormone neuropeptide levels and paraventricular nucleus Fos staining. Data demonstrate the utility of immunoblot analysis as a follow-through technique to capitalize on Fos mapping of transactivation sites in the brain. Findings that induction of Fos immunoreactivity coincides with adjustments in hypothalamic metabolic neuropeptide expression affirms that this functional indicator reflects changes in neurotransmission in pathways governing metabolic outflow. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Recent advances in cell-free PrPSc amplification technique.

    Science.gov (United States)

    Atarashi, Ryuichiro

    2009-01-01

    The development of amplification technology for abnormal forms of prion protein in vitro has had a great impact on the field of prion research. This novel technology has generated new possibilities for understanding the molecular basis of prions and for developing an early diagnostic test for prion diseases. This review provides an overview of recent progress in cell-free PrPSc amplification techniques.

  13. Applications of an Automated and Quantitative CE-Based Size and Charge Western Blot for Therapeutic Proteins and Vaccines.

    Science.gov (United States)

    Rustandi, Richard R; Hamm, Melissa; Lancaster, Catherine; Loughney, John W

    2016-01-01

    Capillary Electrophoresis (CE) is a versatile and indispensable analytical tool that can be applied to characterize proteins. In recent years, labor-intensive SDS-PAGE and IEF slab gels have been replaced with CE-SDS (CGE) and CE-IEF methods, respectively, in the biopharmaceutical industry. These two CE-based methods are now an industry standard and are an expectation of the regulatory agencies for biologics characterization. Another important and traditional slab gel technique is the western blot, which detects proteins using immuno-specific reagents after SDS-PAGE separation. This technique is widely used across industrial and academic laboratories, but it is very laborious, manual, time-consuming, and only semi-quantitative. Here, we describe the applications of a relatively new CE-based western blot technology which is automated, fast, and quantitative. We have used this technology for both charge- and size-based CE westerns to analyze biotherapeutic and vaccine products. The size-based capillary western can be used for fast antibody screening, clone selection, product titer, identity, and degradation while the charge-based capillary western can be used to study product charge heterogeneity. Examples using this technology for monoclonal antibody (mAb), Enbrel, CRM197, and Clostridium difficile (C. difficile) vaccine proteins are presented here to demonstrate the utility of the capillary western techniques. Details of sample preparation and experimental conditions for each capillary western mode are described in this chapter.

  14. Identification of toxocara canis antigens by Western blot in experimentally infected rabbits

    Directory of Open Access Journals (Sweden)

    MORALES Olga Lucía

    2002-01-01

    Full Text Available Toxocariasis is a frequent helminthiasis that can cause visceral and ocular damage in humans specially in children. The identification of specific antigens of Toxocara canis is important in order to develop better diagnostic techniques. Ten rabbits were infected orally with a dose of 5000 Toxocara canis embryonated eggs. Rabbits were bled periodically and an ELISA assay was performed to determine levels of specific Toxocara IgG antibodies. ELISA detected antibodies at day 15 after infection. Western blot (WB assay was performed using excretory/secretory antigens (E/S of T. canis second stage larvae. Different antigen concentrations were evaluated: 150, 200, 250 and 300 µg/mL. The concentration of 250 µg/mL was retained for analysis. Rabbit sera were diluted 1:100. Secondary antibody was used at a dilution of 1:1000. Results of WB indicated that in the first month after infection specific antibodies against the 200 KDa, 116 KDa, 92 KDa and 35 KDa antigens were detected; antibodies against the 92 KDa, 80 KDa, 66 KDa, 45 KDa, 31 KDa and 28 KDa antigens appeared later. All positive sera in the ELISA test were also positive in WB. Two antigen bands, 92 KDa and 35 KDa, were identified since the beginning and throughout the course of infection. These antigens merit further evaluation as candidates for use in diagnosis.

  15. An alternative strategy to western blot as a confirmatory diagnostic test for HIV infection.

    Science.gov (United States)

    Feng, Xia; Wang, Jibao; Gao, Zhiyun; Tian, Yu; Zhang, Ling; Chen, Huichao; Zhang, Tong; Xiao, Lin; Yao, Jun; Xing, Wenge; Qiu, Maofeng; Jiang, Yan

    2017-03-01

    In China, western blot (WB) is the recommended procedure for the diagnosis of HIV infection. However, this technique is time consuming and labor intensive, and its complexity restricts wide application in resource-limited regions. The aim of this study was to evaluate the efficacy of a dry blood spots (DBS)-urine paired enzyme-linked immunosorbent assay (ELISA) test, instead of WB, for HIV antibody detection. Plasma, DBS, and urine samples were collected from 1213 subjects from different populations. Two diagnostic testing strategies were conducted in parallel. The equivalence of the paired ELISA and WB strategies was assessed. A diagnosis of HIV was determined in 250 subjects according to the paired ELISA test, and in 249 according to the WB strategy. The discordant case was judged HIV-positive during follow-up. In total, 18 subjects were diagnosed with possible HIV using the paired ELISA test, among whom, 11 subjects tested negative with WB, and one was confirmed to be HIV-positive during follow-up. For the remaining 945 subjects, both strategies indicated a negative result. The kappa test indicated good conformity (kappa=0.954) between the two diagnostic strategies. The DBS-urine paired ELISA could be applied as an alternative to WB in HIV diagnosis, which would be valuable in resource-limited regions owing to the associated affordability and ease of use. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  16. Cell biological and biomechanical evaluation of two different fixation techniques for rotator cuff repair.

    Science.gov (United States)

    Klinger, H-M; Koelling, S; Baums, M H; Kahl, E; Steckel, H; Smith, M M; Schultz, W; Miosge, N

    2009-06-01

    Our objective was to evaluate the cell biology and biomechanical aspects of the healing process after two different techniques in open rotator cuff surgery - double-loaded bio-absorbable suture anchors combined with so-called arthroscopic Mason-Allen stitches (AAMA) and a trans-osseous suture technique combined with traditional modified Mason-Allen stitches (SMMA). Thirty-six mature sheep were randomized into two repair groups. After 6, 12, or 26 weeks, evaluation of the reinsertion site of the infraspinatus tendon was performed. The mechanical load-to-failure and stiffness results did not indicate a significant difference between the two groups. After 26 weeks, fibrocartilage was sparse in the AAMA group, whereas the SMMA group showed the most pronounced amount of fibrocartilage. We found no ultrastructural differences in collagen fiber organization between the two groups. The relative expression of collagen type II mRNA in the normal group was 1.11. For the AAMA group, 6 weeks after surgery, the relative expression was 55.47, whereas for the SMMA group it was 1.90. This in vivo study showed that the AAMA group exhibited a tendon-to-bone healing process more favorable in its cell biology than that of the traditional SMMA technique. Therefore, the AAMA technique might also be more appropriate for arthroscopic repair.

  17. Application of green chemistry techniques to prepare electrocatalysts for direct methanol fuel cells.

    Science.gov (United States)

    Shimizu, Kenichi; Wang, Joanna S; Wai, Chien M

    2010-03-25

    A series of green techniques for synthesizing carbon nanotube-supported platinum nanoparticles and their high electrocatalytic activity toward methanol fuel cell applications are reported. The techniques utilize either the supercritical fluid carbon dioxide or water as a medium for depositing platinum nanoparticles on surfaces of multiwalled or single-walled carbon nanotubes. The catalytic properties of the carbon nanotubes-supported Pt nanoparticle catalysts prepared by four different techniques are compared for anodic oxidation of methanol and cathodic reduction of oxygen using cyclic voltammetry. One technique using galvanic exchange of Pt(2+) in water with zerovalent iron present on the surfaces of as-grown single-walled carbon nanotubes produces a Pt catalyst that shows an unusually high catalytic activity for reduction of oxygen but a negligible activity for oxidation of methanol. This fuel-selective catalyst may have a unique application as a cathode catalyst in methanol fuel cells to alleviate the problems caused by crossover of methanol through the polymer electrolyte membrane.

  18. Sensitivity improvement of rapid Vibrio harveyi detection with an enhanced chemiluminescent-based dot blot.

    Science.gov (United States)

    Li, H; Xiao, J; Zhou, Y; Wang, Q; Zhang, Y

    2017-09-01

    Vibrio harveyi is an opportunistic pathogen in seawater and can cause severe vibriosis. It is prevalent in hatcheries worldwide and can lead to severe economic losses. Therefore, there is an urgent need to develop a rapid detection method for monitoring this pathogen. In this study, to increase the detection sensitivity of our assay with monoclonal antibodies (Mabs) against V. harveyi, the conditions of the dot blot assay were optimized, and enhanced chemiluminescent (ECL) substrate replaced the traditional tetramethylbenzidine (TMB) substrate. Based on the optimization results, an ECL-based novel dot blot assay was developed for the rapid and sensitive detection of V. harveyi. Compared with the traditional dot blot assay, the incubation time was shortened from 8 to 2 h. The limit of detection (LOD) for V. harveyi was 2 × 10 5  CFU per ml (10 3  CFU per spot) in pure bacterial suspension, which was 50-fold more sensitive than the traditional dot blot assay (1 × 10 7  CFU per ml). Furthermore, when compared with indirect ELISA, the dot blot assay showed approximately 1000-fold higher sensitivity (CFU/CFU). After the test sample was pre-enriched in turbot homogenates for 6 h before the dot blot analysis, the LOD for V. harveyi was 10 CFU per ml. Vibrio harveyi is one of the most opportunistic pathogens that can cause high mortality in hatcheries worldwide. To detect this pathogen, a novel dot blot based on enhanced chemiluminescent (ECL) has been established. This ECL-based dot blot was found to be more sensitive and rapid for V. harveyi detection than traditional dot blot, and this technology is recommended as an applied protocol for monitoring V. harveyi in seawater to reduce economic losses. © 2017 The Society for Applied Microbiology.

  19. Southern blot analysis of skin biopsies for human papillomavirus DNA: renal allograft recipients in south-eastern Queensland.

    Science.gov (United States)

    Trenfield, K; Salmond, C A; Pope, J H; Hardie, I R

    1993-01-01

    The 104 skin biopsies from 34 patients who attended a Renal Transplant Unit in Brisbane over 12 months included 40 squamous cell carcinoma (SCC), 22 solar keratoses, 4 hyperkeratoses, 18 warts and 11 basal cell carcinoma (BCC). Human papillomavirus (HPV) DNA was identified by Southern blot hybridisation using, as individual probes, purified insert DNA from recombinant HPV 1, 2, 3 or 3/10, 4, 5 or 5/8, 7, 11, 16, 18 and 41 under relaxed conditions and characterised by restriction enzyme analysis and Southern blot hybridisation under more stringent conditions. Genomic HPV DNA was characterised in 7 skin biopsies from 4 renal allograft recipients (RARs): HPV 1A in a SCC (20 copies/cell) and a BCC (10 copies/cell) from the one patient, HPV 36 (20 copies/cell) in a SCC, HPV 1A [symbol: see text] 1000 copies/cell) in a wart and HPV 2B (200-800 copies/cell) in 3 warts from the one patient. Only HPV 1A in the SCC exhibited a significant degree of subtype variation. HPV DNA was identified in another 5 skin biopsies from another 4 RARs: HPV 3A in a wart and a hyperkeratosis, HPV 3/10-related DNA in 2 solar keratoses and HPV 5/8-related DNA in another (20-50 copies/cell). The incidence of HPV 5 (or 5-related HPVs) in RAR SCC was very low and that of HPV DNA in RAR warts was lower than that recorded elsewhere but this was not due to insensitivity of the assays. There was no evidence for a role for HPV in the aetiology of skin cancer in RARs in south-eastern Queensland but the possibility remains that as yet unidentified HPV types are involved.

  20. Analysis and optimization of a proton exchange membrane fuel cell using modeling techniques

    International Nuclear Information System (INIS)

    Torre Valdés, Ing. Raciel de la; García Parra, MSc. Lázaro Roger; González Rodríguez, MSc. Daniel

    2015-01-01

    This paper proposes a three-dimensional, non-isothermal and steady-state model of Proton Exchange Membrane Fuel Cell using Computational Fluid Dynamic techniques, specifically ANSYS FLUENT 14.5. It's considered multicomponent diffusion and two-phasic flow. The model was compared with experimental published data and with another model. The operation parameters: reactants pressure and temperature, gases flow direction, gas diffusion layer and catalyst layer porosity, reactants humidification and oxygen concentration are analyzed. The model allows the fuel cell design optimization taking in consideration the channels dimensions, the channels length and the membrane thickness. Furthermore, fuel cell performance is analyzed working with SPEEK membrane, an alternative electrolyte to Nafion. In order to carry on membrane material study, it's necessary to modify the expression that describes the electrolyte ionic conductivity. It's found that the device performance has got a great sensibility to pressure, temperature, reactant humidification and oxygen concentration variations. (author)

  1. SDS-Polyacrylamide Electrophoresis and Western Blotting Applied to the Study of Asthma.

    Science.gov (United States)

    García-Solaesa, Virginia; Abad, Sara Ciria

    2016-01-01

    Western blotting is used to analyze proteins after being separated by electrophoresis and subsequently electro-transferred to a membrane. Once immobilized, a specific protein can be identified through its reaction with a labeled antibody or antigen. It is a methodology commonly used in biomedical research such as asthma studies, to assess the pathways of inflammatory mediators involved in the disease.Here, we describe an example of western blotting to determine the factors involved in asthma. In this chapter, the methodology of western blotting is reviewed, paying attention on potential problems and giving interesting recommendations.

  2. Method for resolution and western blotting of very large proteins using agarose electrophoresis.

    Science.gov (United States)

    Greaser, Marion L; Warren, Chad M

    2015-01-01

    Proteins larger than 200 kDa are difficult to separate electrophoretically using polyacrylamide gels, and their transfer during western blotting is typically incomplete. A vertical SDS agarose gel system was developed that has vastly improved resolving power for very large proteins. Complete transfer of proteins as large as titin (Mr 3,000-3,700 kDa) onto blots can be achieved. The addition of a sulfhydryl reducing agent in the upper reservoir buffer and transfer buffer markedly improves the blotting of large proteins.

  3. ESR technique for noninvasive way to quantify cyclodextrins effect on cell membranes

    International Nuclear Information System (INIS)

    Grammenos, A.; Mouithys-Mickalad, A.; Guelluy, P.H.; Lismont, M.; Piel, G.; Hoebeke, M.

    2010-01-01

    Research highlights: → ESR: a new tool for cyclodextrins study on living cells. → Cholesterol and phospholipid extraction by Rameb in a dose- and time-dependent way. → Extracted phospholipids and cholesterol form stable aggregates. → ESR spectra show that lipid rafts are damaged by Rameb. → Quantification of the cholesterol extraction on cell membranes in a noninvasive way. -- Abstract: A new way to study the action of cyclodextrin was developed to quantify the damage caused on cell membrane and lipid bilayer. The Electron Spin Resonance (ESR) spectroscopy was used to study the action of Randomly methylated-beta-cyclodextrin (Rameb) on living cells (HCT-116). The relative anisotropy observed in ESR spectrum of nitroxide spin probe (5-DSA and cholestane) is directly related to the rotational mobility of the probe, which can be further correlated with the microviscosity. The use of ESR probes clearly shows a close correlation between cholesterol contained in cells and cellular membrane microviscosity. This study also demonstrates the Rameb ability to extract cholesterol and phospholipids in time- and dose-dependent ways. In addition, ESR spectra enabled to establish that cholesterol is extracted from lipid rafts to form stable aggregates. The present work supports that ESR is an easy, reproducible and noninvasive technique to study the effect of cyclodextrins on cell membranes.

  4. [Sizes of bacterial cells in soils determined by cascade filtration technique].

    Science.gov (United States)

    Polianskaia, L M; Gorodnichev, R B; Zviagintsev, D G

    2013-01-01

    This paper studies the number of bacteria in typical chernozem and mountain-meadow soil by the traditional method and the cascade filtration technique. The total number of bacteria in these soils, which was obtained in filters of different diameters during filtering the suspension of a certain amount, is 1.5-5 times higher than that obtained by the traditional method. In the structure of the bacterial biomass in both soils, the biomass of bacterial cells with a diameter of 0.38-0.43 microm was dominating by 8-90%. In the typical chernozem, the biomass of cells with a diameter of 0.17 microm was slightly more than 1%; in the mountain-meadow soil, the percentage of the biomass of cells with a diameter of 0.17 microm increased by 5%. The average volume and diameter of the bacteria in the studied soils were calculated. In typical chernozem, the average volume of bacterial cells was equal to 0.0046 microm3 and the diameter was 0.206 microm. In the mountain-meadow soils, these values were slightly lower, 0.0038 microm3 and 0.194 microm, respectively. The biomass of the bacterial cells, which is usually calculated based on the cell volume of 0.1 microm3, is overestimated by about five times when counting the number on the filters. The percentage of the real biomass of soil bacteria is traditionally much lower than that estimated.

  5. MS Western, a Method of Multiplexed Absolute Protein Quantification is a Practical Alternative to Western Blotting.

    Science.gov (United States)

    Kumar, Mukesh; Joseph, Shai R; Augsburg, Martina; Bogdanova, Aliona; Drechsel, David; Vastenhouw, Nadine L; Buchholz, Frank; Gentzel, Marc; Shevchenko, Andrej

    2018-02-01

    Absolute quantification of proteins elucidates the molecular composition, regulation and dynamics of multiprotein assemblies and networks. Here we report on a method termed MS Western that accurately determines the molar abundance of dozens of user-selected proteins at the subfemtomole level in whole cell or tissue lysates without metabolic or chemical labeling and without using specific antibodies. MS Western relies on GeLC-MS/MS and quantifies proteins by in-gel codigestion with an isotopically labeled QconCAT protein chimera composed of concatenated proteotypic peptides. It requires no purification of the chimera and relates the molar abundance of all proteotypic peptides to a single reference protein. In comparative experiments, MS Western outperformed immunofluorescence Western blotting by the protein detection specificity, linear dynamic range and sensitivity of protein quantification. To validate MS Western in an in vivo experiment, we quantified the molar content of zebrafish core histones H2A, H2B, H3 and H4 during ten stages of early embryogenesis. Accurate quantification (CV<10%) corroborated the anticipated histones equimolar stoichiometry and revealed an unexpected trend in their total abundance. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Large-Grain Tin-Rich Perovskite Films for Efficient Solar Cells via Metal Alloying Technique.

    Science.gov (United States)

    Tavakoli, Mohammad Mahdi; Zakeeruddin, Shaik Mohammed; Grätzel, Michael; Fan, Zhiyong

    2018-03-01

    Fast research progress on lead halide perovskite solar cells has been achieved in the past a few years. However, the presence of lead (Pb) in perovskite composition as a toxic element still remains a major issue for large-scale deployment. In this work, a novel and facile technique is presented to fabricate tin (Sn)-rich perovskite film using metal precursors and an alloying technique. Herein, the perovskite films are formed as a result of the reaction between Sn/Pb binary alloy metal precursors and methylammonium iodide (MAI) vapor in a chemical vapor deposition process carried out at 185 °C. It is found that in this approach the Pb/Sn precursors are first converted to (Pb/Sn)I 2 and further reaction with MAI vapor leads to the formation of perovskite films. By using Pb-Sn eutectic alloy, perovskite films with large grain sizes up to 5 µm can be grown directly from liquid phase metal. Consequently, using an alloying technique and this unique growth mechanism, a less-toxic and efficient perovskite solar cell with a power conversion efficiency (PCE) of 14.04% is demonstrated, while pure Sn and Pb perovskite solar cells prepared in this manner yield PCEs of 4.62% and 14.21%, respectively. It is found that this alloying technique can open up a new direction to further explore different alloy systems (binary or ternary alloys) with even lower melting point. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Discrepancies between a new highly sensitive Toxoplasma gondii ELISA assay and other reagents: interest of Toxo IgG Western blot.

    Science.gov (United States)

    Leslé, F; Touafek, F; Fekkar, A; Mazier, D; Paris, L

    2011-10-01

    Immunodiagnostic assays are commonly used to screen for maternal toxoplasmic seroconversion during pregnancy. The introduction to the market of a new highly sensitive IgG assay, the Elecsys Toxo IgG test, has resulted in discrepancy issues with other immunoassays because of a lack of standardisation. Western blot appears to be a good alternative gold standard to the dye test, as the latter is not routinely available. For the present prospective study, we compared the analytical performances of two immunoassays, Elecsys Toxo IgG (Roche Diagnostics) and Platelia Toxo IgG (Bio-Rad, Marnes la Coquette, France), to Toxo II IgG Western blot (LDBio, Lyon, France) using 231 consecutive sera with low or equivocal IgG titres. Of these 231 sera, 213 presented discrepancies, which showed the importance of a confirmation test. Of the Elecsys Toxo IgG-positive results, 100% were confirmed by the Western blot with a positive threshold of 30 IU/ml for Elecsys; in the equivocal area (1-30 IU/ml), Western blot is negative in 54% of cases. Our results suggest that the lower diagnostic cut-off of Platelia Toxo IgG should be further reduced. Our study indirectly confirms that monitoring, especially for pregnant women, must be done in the same laboratory using the same technique. The ability to diagnose very early seroconversion using Western blot merits further study.

  8. Assessment of dye distribution in sensitized solar cells by microprobe techniques

    Energy Technology Data Exchange (ETDEWEB)

    Barreiros, M.A., E-mail: alexandra.barreiros@lneg.pt [Laboratório Nacional de Energia e Geologia, LEN/UES, Estrada do Paço do Lumiar, 22, 1649-038 Lisboa (Portugal); Corregidor, V. [IPFN, Instituto Superior Técnico, Universidade de Lisboa, E.N. 10, 2686-953 Sacavém (Portugal); Alves, L.C. [C2TN, Campus Tecnológico e Nuclear, Instituto Superior Técnico, Universidade de Lisboa, E.N. 10, 2686-953 Sacavém (Portugal); Guimarães, F. [Laboratório Nacional de Energia e Geologia, LGM/UCTM, Rua da Amieira, Apartado 1089, 4466-901 S. Mamede de Infesta (Portugal); Mascarenhas, J.; Torres, E.; Brites, M.J. [Laboratório Nacional de Energia e Geologia, LEN/UES, Estrada do Paço do Lumiar, 22, 1649-038 Lisboa (Portugal)

    2015-04-01

    Dye sensitized solar cells (DSCs) have received considerable attention once this technology offers economic and environmental advantages over conventional photovoltaic (PV) devices. The PV performance of a DSC relies on the characteristics of its photoanode, which typically consists of a nanocrystalline porous TiO{sub 2} film, enabled with a large adsorptive surface area. Dye molecules that capture photons from light during device operation are attached to the film nanoparticles. The effective loading of the dye in the TiO{sub 2} electrode is of paramount relevance for controlling and optimizing solar cell parameters. Relatively few methods are known today for quantitative evaluation of the total dye adsorbed on the film. In this context, microprobe techniques come out as suitable tools to evaluate the dye surface distribution and depth profile in sensitized films. Electron Probe Microanalysis (EPMA) and Ion Beam Analytical (IBA) techniques using a micro-ion beam were used to quantify and to study the distribution of the Ru organometallic dye in TiO{sub 2} films, making use of the different penetration depth and beam sizes of each technique. Different 1D nanostructured TiO{sub 2} films were prepared, morphologically characterized by SEM, sensitized and analyzed by the referred techniques. Dye load evaluation in different TiO{sub 2} films by three different techniques (PIXE, RBS and EPMA/WDS) provided similar results of Ru/Ti mass fraction ratio. Moreover, it was possible to assess dye surface distribution and its depth profile, by means of Ru signal, and to visualize the dye distribution in sample cross-section through X-ray mapping by EPMA/EDS. PIXE maps of Ru and Ti indicated an homogeneous surface distribution. The assessment of Ru depth profile by RBS showed that some films have homogeneous Ru depth distribution while others present different Ru concentration in the top layer (2 μm thickness). These results are consistent with the EPMA/EDS maps obtained.

  9. Assessment of dye distribution in sensitized solar cells by microprobe techniques

    International Nuclear Information System (INIS)

    Barreiros, M.A.; Corregidor, V.; Alves, L.C.; Guimarães, F.; Mascarenhas, J.; Torres, E.; Brites, M.J.

    2015-01-01

    Dye sensitized solar cells (DSCs) have received considerable attention once this technology offers economic and environmental advantages over conventional photovoltaic (PV) devices. The PV performance of a DSC relies on the characteristics of its photoanode, which typically consists of a nanocrystalline porous TiO 2 film, enabled with a large adsorptive surface area. Dye molecules that capture photons from light during device operation are attached to the film nanoparticles. The effective loading of the dye in the TiO 2 electrode is of paramount relevance for controlling and optimizing solar cell parameters. Relatively few methods are known today for quantitative evaluation of the total dye adsorbed on the film. In this context, microprobe techniques come out as suitable tools to evaluate the dye surface distribution and depth profile in sensitized films. Electron Probe Microanalysis (EPMA) and Ion Beam Analytical (IBA) techniques using a micro-ion beam were used to quantify and to study the distribution of the Ru organometallic dye in TiO 2 films, making use of the different penetration depth and beam sizes of each technique. Different 1D nanostructured TiO 2 films were prepared, morphologically characterized by SEM, sensitized and analyzed by the referred techniques. Dye load evaluation in different TiO 2 films by three different techniques (PIXE, RBS and EPMA/WDS) provided similar results of Ru/Ti mass fraction ratio. Moreover, it was possible to assess dye surface distribution and its depth profile, by means of Ru signal, and to visualize the dye distribution in sample cross-section through X-ray mapping by EPMA/EDS. PIXE maps of Ru and Ti indicated an homogeneous surface distribution. The assessment of Ru depth profile by RBS showed that some films have homogeneous Ru depth distribution while others present different Ru concentration in the top layer (2 μm thickness). These results are consistent with the EPMA/EDS maps obtained

  10. Techniques for the induction of human pluripotent stem cell differentiation towards cardiomyocytes.

    Science.gov (United States)

    Lewandowski, Jarosław; Kolanowski, Tomasz J; Kurpisz, Maciej

    2017-05-01

    The derivation of pluripotent stem cells from human embryos and the generation of induced pluripotent stem cells (iPSCs) from somatic cells opened a new chapter in studies on the regeneration of the post-infarction heart and regenerative medicine as a whole. Thus, protocols for obtaining iPSCs were enthusiastically adopted and widely used for further experiments on cardiac differentiation. iPSC-mediated cardiomyocytes (iPSC-CMs) under in vitro culture conditions are generated by simulating natural cardiomyogenesis and involve the wingless-type mouse mammary tumour virus integration site family (WNT), transforming growth factor beta (TGF-β) and fibroblast growth factor (FGF) signalling pathways. New strategies have been proposed to take advantage of small chemical molecules, organic compounds and even electric or mechanical stimulation. There are three main approaches to support cardiac commitment in vitro: embryoid bodis (EBs), monolayer in vitro cultures and inductive co-cultures with visceral endoderm-like (END2) cells. In EB technique initial uniform size of pluripotent stem cell (PSC) colonies has a pivotal significance. Hence, some methods were designed to support cells aggregation. Another well-suited procedure is based on culturing cells in monolayer conditions in order to improve accessibility of growth factors and nutrients. Other distinct tactics are using visceral endoderm-like cells to culture them with PSCs due to secretion of procardiac cytokines. Finally, the appropriate purification of the obtained cardiomyocytes is required prior to their administration to a patient under the prospective cellular therapy strategy. This goal can be achieved using non-genetic methods, such as the application of surface markers and fluorescent dyes. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  11. Detection of Infectious Bovine Rhinotracheitis and Bovine Viral Diarrhea Viruses in the Nasal Epithelial Cells by the Direct Immunofluorescence Technique

    Science.gov (United States)

    Silim, A.; Elazhary, M.A.S.Y.

    1983-01-01

    Nasal epithelial cells were collected by cotton swabs for the diagnosis in experimental and field cases of infectious bovine rhinotracheitis and field cases of bovine viral diarrhea in calves. A portion of the cells was washed twice in phosphate buffered saline and a 25 µL drop was placed on microscope slides. The cells were dried, fixed and stained according to the direct fluorescent antibody technique. Another portion of the same specimen was inoculated onto primary bovine skin cell cultures for virus isolation. In the experimental studies for infectious bovine rhinotracheitis, 29/35 specimens were positive by fluorescent antibody technique and 32/35 by cell culture and in the field cases, 22/119 were positive by fluorescent antibody technique and 19/119 by cell culture. In the field cases of bovine viral diarrhea, 28/69 samples were positive by fluorescent antibody technique and 14/69 by cell culture. When fluorescent antibody technique was performed on inoculated cell cultures a total of 24/69 specimens were positive for bovine viral diarrhea. The sensitivity of fluorescent antibody technique was thus comparable to that of cell culture method for infectious bovine rhinotracheitis and bovine viral diarrhea. ImagesFig. 1.Fig. 2.Fig. 3. PMID:6299484

  12. Empirical gradient threshold technique for automated segmentation across image modalities and cell lines.

    Science.gov (United States)

    Chalfoun, J; Majurski, M; Peskin, A; Breen, C; Bajcsy, P; Brady, M

    2015-10-01

    New microscopy technologies are enabling image acquisition of terabyte-sized data sets consisting of hundreds of thousands of images. In order to retrieve and analyze the biological information in these large data sets, segmentation is needed to detect the regions containing cells or cell colonies. Our work with hundreds of large images (each 21,000×21,000 pixels) requires a segmentation method that: (1) yields high segmentation accuracy, (2) is applicable to multiple cell lines with various densities of cells and cell colonies, and several imaging modalities, (3) can process large data sets in a timely manner, (4) has a low memory footprint and (5) has a small number of user-set parameters that do not require adjustment during the segmentation of large image sets. None of the currently available segmentation methods meet all these requirements. Segmentation based on image gradient thresholding is fast and has a low memory footprint. However, existing techniques that automate the selection of the gradient image threshold do not work across image modalities, multiple cell lines, and a wide range of foreground/background densities (requirement 2) and all failed the requirement for robust parameters that do not require re-adjustment with time (requirement 5). We present a novel and empirically derived image gradient threshold selection method for separating foreground and background pixels in an image that meets all the requirements listed above. We quantify the difference between our approach and existing ones in terms of accuracy, execution speed, memory usage and number of adjustable parameters on a reference data set. This reference data set consists of 501 validation images with manually determined segmentations and image sizes ranging from 0.36 Megapixels to 850 Megapixels. It includes four different cell lines and two image modalities: phase contrast and fluorescent. Our new technique, called Empirical Gradient Threshold (EGT), is derived from this reference

  13. The potential of cell sheet technique on the development of hepatocellular carcinoma in rat models.

    Directory of Open Access Journals (Sweden)

    Alaa T Alshareeda

    Full Text Available Hepatocellular carcinoma (HCC is considered the 3rd leading cause of death by cancer worldwide with the majority of patients were diagnosed in the late stages. Currently, there is no effective therapy. The selection of an animal model that mimics human cancer is essential for the identification of prognostic/predictive markers, candidate genes underlying cancer induction and the examination of factors that may influence the response of cancers to therapeutic agents and regimens. In this study, we developed a HCC nude rat models using cell sheet and examined the effect of human stromal cells (SCs on the development of the HCC model and on different liver parameters such as albumin and urea.Transplanted cell sheet for HCC rat models was fabricated using thermo-responsive culture dishes. The effect of human umbilical cord mesenchymal stromal cells (UC-MSCs and human bone marrow mesenchymal stromal cells (BM-MSCs on the developed tumour was tested. Furthermore, development of tumour and detection of the liver parameter was studied. Additionally, angiogenesis assay was performed using Matrigel.HepG2 cells requires five days to form a complete cell sheet while HepG2 co-cultured with UC-MSCs or BM-MSCs took only three days. The tumour developed within 4 weeks after transplantation of the HCC sheet on the liver of nude rats. Both UC-MSCs and BM-MSCs improved the secretion of liver parameters by increasing the secretion of albumin and urea. Comparatively, the UC-MSCs were more effective than BM-MSCs, but unlike BM-MSCs, UC-MSCs prevented liver tumour formation and the tube formation of HCC.Since this is a novel study to induce liver tumour in rats using hepatocellular carcinoma sheet and stromal cells, the data obtained suggest that cell sheet is a fast and easy technique to develop HCC models as well as UC-MSCs have therapeutic potential for liver diseases. Additionally, the data procured indicates that stromal cells enhanced the fabrication of HepG2

  14. Detection and quantification of protein-protein interactions by far-western blotting.

    Science.gov (United States)

    Jadwin, Joshua A; Mayer, Bruce J; Machida, Kazuya

    2015-01-01

    Far-western blotting is a convenient method to characterize protein-protein interactions, in which protein samples of interest are immobilized on a membrane and then probed with a non-antibody protein. In contrast to western blotting, which uses specific antibodies to detect target proteins, far-western blotting detects proteins on the basis of the presence or absence of binding sites for the protein probe. When specific modular protein binding domains are used as probes, this approach allows characterization of protein-protein interactions involved in biological processes such as signal transduction, including interactions regulated by posttranslational modification. We here describe a rapid and simple protocol for far-western blotting, in which GST-tagged Src homology 2 (SH2) domains are used to probe cellular proteins in a phosphorylation-dependent manner. We also present a batch quantification method that allows for the direct comparison of probe binding patterns.

  15. An overview of Western blotting for determining antibody specificities for immunohistochemistry.

    Science.gov (United States)

    Kurien, Biji T; Dorri, Yaser; Dillon, Skyler; Dsouza, Anil; Scofield, R Hal

    2011-01-01

    Despite its overall simplicity, protein blotting or Western blotting has been proven to be a powerful procedure for the immunodetection of proteins, especially those that are of low abundance, following electrophoresis. The usefulness of this procedure stems from its ability to provide simultaneous resolution of multiple immunogenic antigens within a sample for detection by specific antibodies. Protein blotting has evolved greatly since its inception and researchers have a variety of ways and means to carry out this transfer. This procedure is used in combination with other important antibody-based detection methods such as enzyme-linked immunosorbant assay and immunohistochemistry to provide confirmation of results both in research and diagnostic testing. Specificity of antibodies used for immunohistochemistry is of critical importance and therefore Western blot is a "must" to address antibodies' specificity.

  16. Development and evaluation of a Western blot kit for diagnosis of schistosomiasis

    NARCIS (Netherlands)

    Sulahian, Annie; Garin, Yves Jean François; Izri, Arezki; Verret, Caroline; Delaunay, Pascal; van Gool, Tom; Derouin, Francis

    2005-01-01

    We evaluated the performance of Western blot (WB) analysis using commercially available antigen strips and compared the results with those of indirect hemagglutination (IHA) and indirect immunofluorescence (IFAT) for the serodiagnosis of human schistosomiasis. The antigen preparation was a crude

  17. Techniques for early diagnosis of oral squamous cell carcinoma: Systematic review.

    Science.gov (United States)

    Carreras-Torras, Clàudia; Gay-Escoda, Cosme

    2015-05-01

    The diagnosis of early oral potentially malignant disorders (OPMD) and oral squamous cell carcinoma (OSCC) is of paramount clinical importance given the mortality rate of late stage disease. The aim of this study is to review the literature to assess the current situation and progress in this area. A search in Cochrane and PubMed (January 2006 to December 2013) has been used with the key words "squamous cell carcinoma", "early diagnosis" "oral cavity", "Potentially Malignant Disorders" y "premalignant lesions". The inclusion criteria were the use of techniques for early diagnosis of OSCC and OPMD, 7 years aged articles and publications written in English, French or Spanish. The exclusion criteria were case reports and studies in other languages. Out of the 89 studies obtained initially from the search 60 articles were selected to be included in the systematic review: 1 metaanalysis, 17 systematic reviews, 35 prospective studies, 5 retrospective studies, 1 consensus and 1 semi-structured interviews. The best diagnostic technique is that which we have sufficient experience and training. Definitely tissue biopsy and histopathological examination should remain the gold standard for oral cancer diagnose. In this systematic review it has not been found sufficient scientific evidence on the majority of proposed techniques for early diagnosis of OSCC, therefore more extensive and exhaustive studies are needed.

  18. A Flexible Ascorbic Acid Fuel Cell with a Microchannel Fabricated using MEMS Techniques

    Science.gov (United States)

    Mogi, Hiroshi; Fukushi, Yudai; Koide, Syohei; Sano, Ryohei; Sasaki, Tsubasa; Nishioka, Yasushiro

    2013-12-01

    We fabricated a miniature ascorbic acid fuel cells equipped with a microchannel for the circulation of ascorbic acid (AA) solution using micro electronic mechanical system techniques. The fuel cell was fabricated on a flexible polyimide substrate, and its porous carbon-coated aluminium (Al) electrodes of 2.8 mm in width and 11 mm in length were formed using photolithography and screen-printing techniques. The porous carbon was deposited by screen-printing of carbon-black ink on the Al electrode surfaces in order to increase the effective electrode surface area and to absorb more enzymes on the cathode surface. The microchannel with a depth of 200 μm was fabricated using a hot-embossing technique. A maximum power of 0.60 μW at 0.58 V that corresponds to a power density of 1.83 μW/cm2 was realized by introducing a 200 mM concentrated AA solution at room temperature.

  19. Prediction of the optimum hybridization conditions of dot-blot-SNP analysis using estimated melting temperature of oligonucleotide probes.

    Science.gov (United States)

    Shiokai, Sachiko; Kitashiba, Hiroyasu; Nishio, Takeshi

    2010-08-01

    Although the dot-blot-SNP technique is a simple cost-saving technique suitable for genotyping of many plant individuals, optimization of hybridization and washing conditions for each SNP marker requires much time and labor. For prediction of the optimum hybridization conditions for each probe, we compared T (m) values estimated from nucleotide sequences using the DINAMelt web server, measured T (m) values, and hybridization conditions yielding allele-specific signals. The estimated T (m) values were comparable to the measured T (m) values with small differences of less than 3 degrees C for most of the probes. There were differences of approximately 14 degrees C between the specific signal detection conditions and estimated T (m) values. Change of one level of SSC concentrations of 0.1, 0.2, 0.5, and 1.0x SSC corresponded to a difference of approximately 5 degrees C in optimum signal detection temperature. Increasing the sensitivity of signal detection by shortening the exposure time to X-ray film changed the optimum hybridization condition for specific signal detection. Addition of competitive oligonucleotides to the hybridization mixture increased the suitable hybridization conditions by 1.8. Based on these results, optimum hybridization conditions for newly produced dot-blot-SNP markers will become predictable.

  20. Ratiometric analysis of fura red by flow cytometry: a technique for monitoring intracellular calcium flux in primary cell subsets.

    Directory of Open Access Journals (Sweden)

    Emily R Wendt

    Full Text Available Calcium flux is a rapid and sensitive measure of cell activation whose utility could be enhanced with better techniques for data extraction. We describe a technique to monitor calcium flux by flow cytometry, measuring Fura Red calcium dye by ratiometric analysis. This technique has several advantages: 1 using a single calcium dye provides an additional channel for surface marker characterization, 2 allows robust detection of calcium flux by minority cell populations within a heterogeneous population of primary T cells and monocytes 3 can measure total calcium flux and additionally, the proportion of responding cells, 4 can be applied to studying the effects of drug treatment, simultaneously stimulating and monitoring untreated and drug treated cells. Using chemokine receptor activation as an example, we highlight the utility of this assay, demonstrating that only cells expressing a specific chemokine receptor are activated by cognate chemokine ligand. Furthermore, we describe a technique for simultaneously stimulating and monitoring calcium flux in vehicle and drug treated cells, demonstrating the effects of the Gαi inhibitor, pertussis toxin (PTX, on chemokine stimulated calcium flux. The described real time calcium flux assay provides a robust platform for characterizing cell activation within primary cells, and offers a more accurate technique for studying the effect of drug treatment on receptor activation in a heterogeneous population of primary cells.

  1. Western blotting is useful in the salivary diagnosis of Helicobacter pylori infection

    OpenAIRE

    Ballam, L; Mendall, M; Asante, M; Morris, J; Strachan, D; Whincup, P; Cook, D

    2000-01-01

    Background—The salivary diagnosis of Helicobacter pylori infection offers attractive possibilities for the epidemiological study of infection in children. Salivary enzyme linked immunosorbent assay (ELISA) is less reliable then serum ELISA, owing to variable transudation of immunoglobulin. In addition, children are more difficult to study because of lower specific serum antibody concentrations to H pylori. The performance of salivary western blotting in comparison with serum western blotting ...

  2. Modeling and simulation of PEM fuel cell's flow channels using CFD techniques

    Energy Technology Data Exchange (ETDEWEB)

    Cunha, Edgar F.; Andrade, Alexandre B.; Robalinho, Eric; Bejarano, Martha L.M.; Linardi, Marcelo [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)]. E-mails: efcunha@ipen.br; abodart@ipen.br; eric@ipen.br; mmora@ipen.br; mlinardi@ipen.br; Cekinski, Efraim [Instituto de Pesquisas Tecnologicas (IPT-SP), Sao Paulo, SP (Brazil)]. E-mail: cekinski@ipt.br

    2007-07-01

    Fuel cells are one of the most important devices to obtain electrical energy from hydrogen. The Proton Exchange Membrane Fuel Cell (PEMFC) consists of two important parts: the Membrane Electrode Assembly (MEA), where the reactions occur, and the flow field plates. The plates have many functions in a fuel cell: distribute reactant gases (hydrogen and air or oxygen), conduct electrical current, remove heat and water from the electrodes and make the cell robust. The cost of the bipolar plates corresponds up to 45% of the total stack costs. The Computational Fluid Dynamic (CFD) is a very useful tool to simulate hydrogen and oxygen gases flow channels, to reduce the costs of bipolar plates production and to optimize mass transport. Two types of flow channels were studied. The first type was a commercial plate by ELECTROCELL and the other was entirely projected at Programa de Celula a Combustivel (IPEN/CNEN-SP) and the experimental data were compared with modelling results. Optimum values for each set of variables were obtained and the models verification was carried out in order to show the feasibility of this technique to improve fuel cell efficiency. (author)

  3. Identifying plant cell-surface receptors: combining 'classical' techniques with novel methods.

    Science.gov (United States)

    Uebler, Susanne; Dresselhaus, Thomas

    2014-04-01

    Cell-cell communication during development and reproduction in plants depends largely on a few phytohormones and many diverse classes of polymorphic secreted peptides. The peptide ligands are bound at the cell surface of target cells by their membranous interaction partners representing, in most cases, either receptor-like kinases or ion channels. Although knowledge of both the extracellular ligand and its corresponding receptor(s) is necessary to describe the downstream signalling pathway(s), to date only a few ligand-receptor pairs have been identified. Several methods, such as affinity purification and yeast two-hybrid screens, have been used very successfully to elucidate interactions between soluble proteins, but most of these methods cannot be applied to membranous proteins. Experimental obstacles such as low concentration and poor solubility of membrane receptors, as well as instable transient interactions, often hamper the use of these 'classical' approaches. However, over the last few years, a lot of progress has been made to overcome these problems by combining classical techniques with new methodologies. In the present article, we review the most promising recent methods in identifying cell-surface receptor interactions, with an emphasis on success stories outside the field of plant research.

  4. Phenotypic and genetic characterization of circulating tumor cells by combining immunomagnetic selection and FICTION techniques.

    Science.gov (United States)

    Campos, María; Prior, Celia; Warleta, Fernando; Zudaire, Isabel; Ruíz-Mora, Jesús; Catena, Raúl; Calvo, Alfonso; Gaforio, José J

    2008-07-01

    The presence of circulating tumor cells (CTCs) in breast cancer patients has been proven to have clinical relevance. Cytogenetic characterization of these cells could have crucial relevance for targeted cancer therapies. We developed a method that combines an immunomagnetic selection of CTCs from peripheral blood with the fluorescence immunophenotyping and interphase cytogenetics as a tool for investigation of neoplasm (FICTION) technique. Briefly, peripheral blood (10 ml) from healthy donors was spiked with a predetermined number of human breast cancer cells. Nucleated cells were separated by double density gradient centrifugation of blood samples. Tumor cells (TCs) were immunomagnetically isolated with an anti-cytokeratin antibody and placed onto slides for FICTION analysis. For immunophenotyping and genetic characterization of TCs, a mixture of primary monoclonal anti-pancytokeratin antibodies was used, followed by fluorescent secondary antibodies, and finally hybridized with a TOP2A/HER-2/CEP17 multicolor probe. Our results show that TCs can be efficiently isolated from peripheral blood and characterized by FICTION. Because genetic amplification of TOP2A and ErbB2 (HER-2) in breast cancer correlates with response to anthracyclines and herceptin therapies, respectively, this novel methodology could be useful for a better classification of patients according to the genetic alterations of CTCs and for the application of targeted therapies.

  5. Assessing Adipogenic Potential of Mesenchymal Stem Cells: A Rapid Three-Dimensional Culture Screening Technique

    Directory of Open Access Journals (Sweden)

    Jean F. Welter

    2013-01-01

    Full Text Available Bone-marrow-derived mesenchymal stem cells (MSCs have the potential to differentiate into a number of phenotypes, including adipocytes. Adipogenic differentiation has traditionally been performed in monolayer culture, and, while the expression of a fat-cell phenotype can be achieved, this culture method is labor and material intensive and results in only small numbers of fragile adherent cells, which are not very useful for further applications. Aggregate culture is a cell-culture technique in which cells are induced to form three-dimensional aggregates; this method has previously been used successfully, among others, to induce and study chondrogenic differentiation of MSCs. We have previously published an adaptation of the chondrogenic aggregate culture method to a 96-well plate format. Based on the success of this method, we have used the same format for the preparation of three-dimensional adipogenic cultures. The MSCs differentiate rapidly, the aggregates can be handled and processed for histologic and biochemical assays with ease, and the format offers significant savings in supplies and labor. As a differentiation assay, this method can distinguish between degrees of senescence and appears suitable for testing medium or drug formulations in a high-volume, high-throughput fashion.

  6. Assessing adipogenic potential of mesenchymal stem cells: a rapid three-dimensional culture screening technique.

    Science.gov (United States)

    Welter, Jean F; Penick, Kitsie J; Solchaga, Luis A

    2013-01-01

    Bone-marrow-derived mesenchymal stem cells (MSCs) have the potential to differentiate into a number of phenotypes, including adipocytes. Adipogenic differentiation has traditionally been performed in monolayer culture, and, while the expression of a fat-cell phenotype can be achieved, this culture method is labor and material intensive and results in only small numbers of fragile adherent cells, which are not very useful for further applications. Aggregate culture is a cell-culture technique in which cells are induced to form three-dimensional aggregates; this method has previously been used successfully, among others, to induce and study chondrogenic differentiation of MSCs. We have previously published an adaptation of the chondrogenic aggregate culture method to a 96-well plate format. Based on the success of this method, we have used the same format for the preparation of three-dimensional adipogenic cultures. The MSCs differentiate rapidly, the aggregates can be handled and processed for histologic and biochemical assays with ease, and the format offers significant savings in supplies and labor. As a differentiation assay, this method can distinguish between degrees of senescence and appears suitable for testing medium or drug formulations in a high-volume, high-throughput fashion.

  7. Validation of commercial Mas receptor antibodies for utilization in Western Blotting, immunofluorescence and immunohistochemistry studies.

    Science.gov (United States)

    Burghi, Valeria; Fernández, Natalia Cristina; Gándola, Yamila Belén; Piazza, Verónica Gabriela; Quiroga, Diego Tomás; Guilhen Mario, Érica; Felix Braga, Janaína; Bader, Michael; Santos, Robson Augusto Souza; Dominici, Fernando Pablo; Muñoz, Marina Cecilia

    2017-01-01

    Mas receptor (MasR) is a G protein-coupled receptor proposed as a candidate for mediating the angiotensin (Ang)-converting enzyme 2-Ang (1-7) protective axis of renin-angiotensin system. Because the role of this receptor is not definitively clarified, determination of MasR tissue distribution and expression levels constitutes a critical knowledge to fully understanding its function. Commercially available antibodies have been widely employed for MasR protein localization and quantification, but they have not been adequately validated. In this study, we carried on an exhaustive evaluation of four commercial MasR antibodies, following previously established criteria. Western Blotting (WB) and immunohistochemistry studies starting from hearts and kidneys from wild type (WT) mice revealed that antibodies raised against different MasR domains yielded different patterns of reactivity. Furthermore, staining patterns appeared identical in samples from MasR knockout (MasR-KO) mice. We verified by polymerase chain reaction analysis that the MasR-KO mice used were truly deficient in this receptor as MAS transcripts were undetectable in either heart or kidney from this animal model. In addition, we evaluated the ability of the antibodies to detect the human c-myc-tagged MasR overexpressed in human embryonic kidney cells. Three antibodies were capable of detecting the MasR either by WB or by immunofluorescence, reproducing the patterns obtained with an anti c-myc antibody. In conclusion, although three of the selected antibodies were able to detect MasR protein at high expression levels observed in a transfected cell line, they failed to detect this receptor in mice tissues at physiological expression levels. As a consequence, validated antibodies that can recognize and detect the MasR at physiological levels are still lacking.

  8. Localization of human immunodeficiency virus antigens in infected cells by scanning/transmission-immunogold techniques

    International Nuclear Information System (INIS)

    Herrera, M.I.; Santa Maria, I.; de Andres, R.; Najera, R.

    1988-01-01

    An application of high resolution scanning/transmission electron microscopy (STEM) and gold-labelling techniques for the rapid detection of human immunodeficiency virus (HIV) in infected cells has been developed. Experimental in vitro studies for detecting two HIV structural proteins, gp41 and p17, were performed following an indirect labeling procedure that uses monoclonal anti-p17 and anti-gp41 antibodies as primary antibodies and 40 nm gold-linked goat antimouse IgG as secondary antibodies. The cells were then studied by STEM in the scanning mode. Unambiguous localization of the viral antigens was possible by combining the three-dimensional image provided by the secondary electron image and the atomic number-dependent backscattered electron image for the identification of the gold marker. This technique combines both the morphological information and the rapid procedures of scanning electron microscopy with the precise and sensitive antigen detection provided by the use of STEM and immunological methods. The preliminary results of its application to the study of peripheral blood mononuclear cells from four anti-HIV-seropositive patients showing the presence of specific labeling in all of them suggest that it might prove useful for early detection of HIV infection before seroconversion, as well as for quantitative studies

  9. CRITERIA OF POSITIVITY FOR Ig ANTIBODIES IN THE METHOD OF IMMUNE BLOTTING OF LYME DISEASE

    Directory of Open Access Journals (Sweden)

    V G Barskova

    2001-01-01

    Full Text Available There are currently no accepted criteria for positive Western blots in Russian patients with Lyme borreliosis. The purpose of the current study was to develop criteria for a positive IgG westem-blot to aid particularly in the diagnosis of patients with joint manifestation of the disorder. Patients: 97 with Lyme disease, 145 - control subjects. IgG antibody responses were determined to 3 species ofB.burgdorferi sensu lato by Western blotting, using blots prepared by manufacturer. The best discriminatory ability of test criteria was chained by requiring any 3 of 11 IgG bands, a definition that could be used with B. burgdorferi sensu stricto, B.garinii and B.afzelii strains. With these 3 antigen preparation, positive IgG blots were found in 0 to 18% of patients with localized erythema migrans of < 4 weeks duration, 23 to 39% of those with disseminated infection < 20 weeks duration, and in 39 to 46% of those with late arthritis/arthralgia of >6 months duration the specificity was 93 to 99%. Thus, IgG Western blotting may bring greater specificity to serologic testing in Lyme borreliosis, but the sensitivity is limited.

  10. Radio Resource Management Techniques for eMBB and mMTC services in 5G Dense Small Cell Scenarios

    DEFF Research Database (Denmark)

    Mahmood, Nurul Huda; Lauridsen, Mads; Berardinelli, Gilberto

    2016-01-01

    requirements. This article provides an overview of key radio resource management techniques for 5G dense small cells and demonstrates how these techniques can contribute to fulfilling some of the important 5G requirements. Preliminary system level simulation results indicate that a mean throughput gain...... design requirements. Dense small cells with multiple antenna nodes are believed to be key elements in meeting these challenging requirements. 5G will thus feature an adaptable air interface with carefully designed radio resource management techniques that can optimize each link according to its service...... of around 65%, and up to 84% in latency reduction can be achieved utilizing the discussed resource management techniques....

  11. Chondrogenesis of human adipose derived stem cells for future microtia repair using co-culture technique.

    Science.gov (United States)

    Goh, Bee See; Che Omar, Siti Nurhadis; Ubaidah, Muhammad Azhan; Saim, Lokman; Sulaiman, Shamsul; Chua, Kien Hui

    2017-04-01

    In conclusion, these result showed HADSCs could differentiate into chondrocytes-like cells, dependent on signaling induced by TGF-β3 and chondrocytes. This is a promising result and showed that HADSCs is a potential source for future microtia repair. The technique of co-culture is a positive way forward to assist the microtia tissue. Reconstructive surgery for the repair of microtia still remains the greatest challenge among the surgeons. Its repair is associated with donor-site morbidity and the degree of infection is inevitable when using alloplastic prosthesis with uncertain long-term durability. Thus, human adipose derived stem cells (HADSCs) can be an alternative cell source for cartilage regeneration. This study aims to evaluate the chondrogenic potential of HADSCs cultured with transforming growth factor-beta (TGF-β) and interaction of auricular chondrocytes with HADSCs for new cartilage generation. Multi-lineages differentiation features of HADSCs were monitored by Alcian Blue, Alizarin Red, and Oil Red O staining for chondrogenic, adipogenic, and osteogenic differentiation capacity, respectively. Further, HADSCs alone were culture in medium added with TGF-β3; and human auricular chondrocytes were interacted indirectly in the culture with and without TGF-βs for up to 21 days, respectively. Cell morphology and chondrogenesis were monitored by inverted microscope. For cell viability, Alamar Blue assay was used to measure the cell viability and the changes in gene expression of auricular chondrocyte markers were determined by real-time polymerase chain reaction analysis. For the induction of chondrogenic differentiation, HADSCs showed a feature of aggregation and formed a dense matrix of proteoglycans. Staining results from Alizirin Red and Oil Red O indicated the HADSCs also successfully differentiated into adipogenic and osteogenic lineages after 21 days. According to a previous study, HADSCs were strongly positive for the mesenchymal markers CD90, CD73

  12. Techniques for cutting irradiated fuel ducts at FFTF/IEM cell

    International Nuclear Information System (INIS)

    Payzant, W.H.

    1990-09-01

    Two remotely controlled mill-type cutters have been used in the Fast Flux Test Facility Interim Examination and Maintenance Cell to assist in the disassembly of 18 fuel assemblies. These cutters slit the outer duct of the fuel assemblies, which allows the ducts to be removed and provides access to the encased fuel pins. The cutters were developed by Westinghouse Hanford Company and thoroughly tested by cutting prototypic ducts. During actual use, however, occasional loss of cutting depth control occurred. A discussion of the control problems and the operation and design techniques developed for their resolution is presented. 3 refs., 7 figs

  13. An Interference-Aware Distributed Transmission Technique for Dense Small Cell Networks

    DEFF Research Database (Denmark)

    Mahmood, Nurul Huda; Berardinelli, Gilberto; Pedersen, Klaus I.

    2015-01-01

    transmission technique that can efficiently manage the interference in an uncoordinated dense small cell network is investigated in this work. The proposed interference aware scheme only requires instantaneous channel state information at the transmitter end towards the desired receiver. Motivated by penalty...... methods in optimization studies, an interference dependent weighting factor is introduced to control the number of parallel transmission streams. The proposed scheme can outperform a more complex benchmark transmission scheme in terms of the sum network throughput in certain scenarios and with realistic...

  14. Kinetics of Cu (II) separation by ion flotation techniques, in cells with flexible spargers

    International Nuclear Information System (INIS)

    Reyes, M.; Tavera, F. J.; Escudero, R.; Patino, F.; Salinas, E.; Rivera, I.

    2010-01-01

    This research studies and experimentally determines the kinetic parameters and effect of modifying the hydrodynamics and chemical conditions of the air-liquid dispersions during the Cu (II) extraction by ion flotation techniques in cells with porous spargers. Results show that the elimination of Cu (II) from solution can be carried out by ion flotation in one stage, obtaining efficiencies of 68% and 56% for the flat and cylindrical sparger respectively with a xanthate concentration of 0,02 g/l. In multistage systems five cells, recoveries over 92 % were achieved for both sparger geometries. The behavior of the flotation apparent kinetic constant is linear to the parameters that characterize dispersion (Jg, eg y Db), until a point is achieved where the process instability makes the system inoperable. The results show that removing base metal ions by ion flotation is strongly affected by the following factors: collector concentration [C], Jg, eg, Db, Jl and Sb. (Author) 20 refs

  15. Essential Data and Techniques for Conducting Microbial Fuel Cell and other Types of Bioelectrochemical System Experiments

    KAUST Repository

    Logan, Bruce E.

    2012-04-19

    Microbial fuel cells (MFCs) and other bioelectrochemical systems are new technologies that require expertise in a variety of technical areas, ranging from electrochemistry to biological wastewater treatment. There are certain data and critical information that should be included in every MFC study, such as specific surface area of the electrodes, solution conductivity, and power densities normalized to electrode surface area and volumes. Electrochemical techniques such as linear sweep voltammetry can be used to understand the performance of the MFC, but extremely slow scans are required for these biological systems compared to more traditional fuel cells. In this Minireview, the critical information needed for MFC studies is provided with examples of how results can be better conveyed through a full description of materials, the use of proper controls, and inclusion of a more complete electrochemical analysis. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Application of Live-Cell RNA Imaging Techniques to the Study of Retroviral RNA Trafficking

    Directory of Open Access Journals (Sweden)

    Darrin V. Bann

    2012-06-01

    Full Text Available Retroviruses produce full-length RNA that serves both as a genomic RNA (gRNA, which is encapsidated into virus particles, and as an mRNA, which directs the synthesis of viral structural proteins. However, we are only beginning to understand the cellular and viral factors that influence trafficking of retroviral RNA and the selection of the RNA for encapsidation or translation. Live cell imaging studies of retroviral RNA trafficking have provided important insight into many aspects of the retrovirus life cycle including transcription dynamics, nuclear export of viral RNA, translational regulation, membrane targeting, and condensation of the gRNA during virion assembly. Here, we review cutting-edge techniques to visualize single RNA molecules in live cells and discuss the application of these systems to studying retroviral RNA trafficking.

  17. Instrumental analysis of bacterial cells using vibrational and emission Moessbauer spectroscopic techniques

    Energy Technology Data Exchange (ETDEWEB)

    Kamnev, Alexander A. [Laboratory of Biochemistry of Plant-Bacterial Symbioses, Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 410049 Saratov (Russian Federation)]. E-mail: aakamnev@ibppm.sgu.ru; Tugarova, Anna V. [Laboratory of Biochemistry of Plant-Bacterial Symbioses, Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 410049 Saratov (Russian Federation); Antonyuk, Lyudmila P. [Laboratory of Biochemistry of Plant-Bacterial Symbioses, Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 410049 Saratov (Russian Federation); Tarantilis, Petros A. [Laboratory of Chemistry, Department of Science, Agricultural University of Athens, 11855 Athens (Greece); Kulikov, Leonid A. [Laboratory of Nuclear Chemistry Techniques, Department of Radiochemistry, Faculty of Chemistry, M.V. Lomonosov Moscow State University, 119992 Moscow (Russian Federation); Perfiliev, Yurii D. [Laboratory of Nuclear Chemistry Techniques, Department of Radiochemistry, Faculty of Chemistry, M.V. Lomonosov Moscow State University, 119992 Moscow (Russian Federation); Polissiou, Moschos G. [Laboratory of Chemistry, Department of Science, Agricultural University of Athens, 11855 Athens (Greece); Gardiner, Philip H.E. [Division of Chemistry, School of Science and Mathematics, Sheffield Hallam University, Sheffield S1 1WB (United Kingdom)

    2006-07-28

    In biosciences and biotechnology, the expanding application of physicochemical approaches using modern instrumental techniques is an efficient strategy to obtain valuable and often unique information at the molecular level. In this work, we applied a combination of vibrational (Fourier transform infrared (FTIR), FT-Raman) spectroscopic techniques, useful in overall structural and compositional analysis of bacterial cells of the rhizobacterium Azospirillum brasilense, with {sup 57}Co emission Moessbauer spectroscopy (EMS) used for sensitive monitoring of metal binding and further transformations in live bacterial cells. The information obtained, together with ICP-MS analyses for metals taken up by the bacteria, is useful in analysing the impact of the environmental conditions (heavy metal stress) on the bacterial metabolism and some differences in the heavy metal stress-induced behaviour of non-endophytic (Sp7) and facultatively endophytic (Sp245) strains. The results show that, while both strains Sp7 and Sp245 take up noticeable and comparable amounts of heavy metals from the medium (0.12 and 0.13 mg Co, 0.48 and 0.44 mg Cu or 4.2 and 2.1 mg Zn per gram of dry biomass, respectively, at a metal concentration of 0.2 mM in the medium), their metabolic responses differ essentially. Whereas for strain Sp7 the FTIR measurements showed significant accumulation of polyhydroxyalkanoates as storage materials involved in stress endurance, strain Sp245 did not show any major changes in cellular composition. Nevertheless, EMS measurements showed rapid binding of cobalt(II) by live bacterial cells (chemically similar to metal binding by dead bacteria) and its further transformation in the live cells within an hour.

  18. Instrumental analysis of bacterial cells using vibrational and emission Moessbauer spectroscopic techniques

    International Nuclear Information System (INIS)

    Kamnev, Alexander A.; Tugarova, Anna V.; Antonyuk, Lyudmila P.; Tarantilis, Petros A.; Kulikov, Leonid A.; Perfiliev, Yurii D.; Polissiou, Moschos G.; Gardiner, Philip H.E.

    2006-01-01

    In biosciences and biotechnology, the expanding application of physicochemical approaches using modern instrumental techniques is an efficient strategy to obtain valuable and often unique information at the molecular level. In this work, we applied a combination of vibrational (Fourier transform infrared (FTIR), FT-Raman) spectroscopic techniques, useful in overall structural and compositional analysis of bacterial cells of the rhizobacterium Azospirillum brasilense, with 57 Co emission Moessbauer spectroscopy (EMS) used for sensitive monitoring of metal binding and further transformations in live bacterial cells. The information obtained, together with ICP-MS analyses for metals taken up by the bacteria, is useful in analysing the impact of the environmental conditions (heavy metal stress) on the bacterial metabolism and some differences in the heavy metal stress-induced behaviour of non-endophytic (Sp7) and facultatively endophytic (Sp245) strains. The results show that, while both strains Sp7 and Sp245 take up noticeable and comparable amounts of heavy metals from the medium (0.12 and 0.13 mg Co, 0.48 and 0.44 mg Cu or 4.2 and 2.1 mg Zn per gram of dry biomass, respectively, at a metal concentration of 0.2 mM in the medium), their metabolic responses differ essentially. Whereas for strain Sp7 the FTIR measurements showed significant accumulation of polyhydroxyalkanoates as storage materials involved in stress endurance, strain Sp245 did not show any major changes in cellular composition. Nevertheless, EMS measurements showed rapid binding of cobalt(II) by live bacterial cells (chemically similar to metal binding by dead bacteria) and its further transformation in the live cells within an hour

  19. The Use of Atomic Force Microscopy as a Technique for the Identification of Cancerous Cells

    International Nuclear Information System (INIS)

    Lekka, M.

    2007-11-01

    The monograph presents the use of atomic force microscopy (AFM) as a tool for the identification of cancerous cells by studies of the expression of different types of molecules directly on the surface of living cells. The full quantitative description (that is not accessible by other techniques) performed for a given type of molecular interactions has been obtained by using the following quantities: an unbinding force, probability, rupture length and the effective spring constant taking into account the stiffness of a single complex. All, these parameters were extracted from AFM measurements The analysis of the interaction forces performed by AFM allows the quantitative determination of: i) the static properties of a single molecular complex where its strength of interaction and stiffness of the studied complex can be obtained, ii) dynamic properties, on the basis of which the kinetic properties of the unbinding process can be delivered, and iii) properties of adhesion clusters, where the interrelation between single complexes can be characterized, in particular the mechanism of the unbinding can be obtained. The presented characterization of the interaction force between single molecules demonstrates that atomic force microscopy can be used as exceptional technique to study the expression of molecules on a cell surface. Such measurements are not limited to a typical interactions occurring between single molecules but also it is possible to study the interactions between parts of molecules. The results presented in this monograph point to a novel approach to identify cancer-related changes in a quantitative way what can be used for describing and confirming the pathological state of a single cell. (author)

  20. Correlation of open cell-attached and excised patch clamp techniques.

    Science.gov (United States)

    Filipovic, D; Hayslett, J P

    1995-11-01

    The excised patch clamp configuration provides a unique technique for some types of single channel analyses, but maintenance of stable, long-lasting preparations may be confounded by rundown and/or rapid loss of seal. Studies were performed on the amiloride-sensitive Na+ channel, located on the apical surface of A6 cells, to determine whether the nystatin-induced open cell-attached patch could serve as an alternative configuration. Compared to excised inside-out patches, stable preparations were achieved more readily with the open cell-attached patch (9% vs. 56% of attempts). In both preparations, the current voltage (I-V) relation was linear, current amplitudes were equal at opposite equivalent clamped voltages, and Erev was zero in symmetrical Na+ solutions, indicating similar Na+ activities on the cytosolic and external surfaces of the patch. Moreover, there was no evidence that nystatin altered channel activity in the patch because slope conductance (3-4pS) and Erev (75 mV), when the bath was perfused with a high K:low Na solution (ENa = 80 mV), were nearly equal in both patch configurations. Our results therefore indicate that the nystatin-induced open cell-attached patch can serve as an alternative approach to the excised inside-out patch when experiments require modulation of univalent ions in the cytosol.

  1. Research Techniques Made Simple: Experimental Methodology for Single-Cell Mass Cytometry.

    Science.gov (United States)

    Matos, Tiago R; Liu, Hongye; Ritz, Jerome

    2017-04-01

    Growing recognition of the complexity of interactions within cellular systems has fueled the development of mass cytometry. The precision of time-of-flight mass spectrometry combined with the labeling of specific ligands with mass tags enables detection and quantification of more than 40 markers at a single-cell resolution. The 135 available detection channels allow simultaneous study of additional characteristics of complex biological systems across millions of cells. Cutting-edge mass cytometry by time-of-flight (CyTOF) can profoundly affect our knowledge of cell population heterogeneity and hierarchy, cellular state, multiplexed signaling pathways, proteolysis products, and mRNA transcripts. Although CyTOF is currently scarcely used within the field of investigative dermatology, we aim to highlight CyTOF's utility and demystify the technique. CyTOF may, for example, uncover the immunological heterogeneity and differentiation of Langerhans cells, delineate the signaling pathways responsible for each phase of the hair cycle, or elucidate which proteolysis products from keratinocytes promote skin inflammation. However, the success of mass cytometry experiments depends on fully understanding the methods and how to control for variations when making comparisons between samples. Here, we review key experimental methods for CyTOF that enable accurate data acquisition by optimizing signal detection and minimizing background noise and sample-to-sample variation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Where in the Cell Are You? Probing HIV-1 Host Interactions through Advanced Imaging Techniques

    Directory of Open Access Journals (Sweden)

    Brennan S. Dirk

    2016-10-01

    Full Text Available Viruses must continuously evolve to hijack the host cell machinery in order to successfully replicate and orchestrate key interactions that support their persistence. The type-1 human immunodeficiency virus (HIV-1 is a prime example of viral persistence within the host, having plagued the human population for decades. In recent years, advances in cellular imaging and molecular biology have aided the elucidation of key steps mediating the HIV-1 lifecycle and viral pathogenesis. Super-resolution imaging techniques such as stimulated emission depletion (STED and photoactivation and localization microscopy (PALM have been instrumental in studying viral assembly and release through both cell–cell transmission and cell–free viral transmission. Moreover, powerful methods such as Forster resonance energy transfer (FRET and bimolecular fluorescence complementation (BiFC have shed light on the protein-protein interactions HIV-1 engages within the host to hijack the cellular machinery. Specific advancements in live cell imaging in combination with the use of multicolor viral particles have become indispensable to unravelling the dynamic nature of these virus-host interactions. In the current review, we outline novel imaging methods that have been used to study the HIV-1 lifecycle and highlight advancements in the cell culture models developed to enhance our understanding of the HIV-1 lifecycle.

  3. Evaluation of differential gene expression during behavioral development in the honeybee using microarrays and northern blots

    Science.gov (United States)

    Kucharski, Robert; Maleszka, Ryszard

    2002-01-01

    Background The honeybee (Apis mellifera) has been used with great success in a variety of behavioral studies. The lack of genomic tools in this species has, however, hampered efforts to provide genome-based explanations for behavioral data. We have combined the power of DNA arrays and the availability of distinct behavioral stages in honeybees to explore the dynamics of gene expression during adult development in this insect. In addition, we used caffeine treatment, a procedure that accelerates learning abilities in honeybees, to examine changes in gene expression underlying drug-induced behavioral modifications. Results Spotted microarrays containing several thousand cDNAs were interrogated with RNAs extracted from newly emerged worker bees, experienced foragers and caffeine-treated bees. Thirty-six differentially expressed cDNAs were verified by northern blot hybridization and characterized in silico by sequencing and database searches. Experienced foragers overexpressed royal jelly proteins, a putative imaginal disc growth factor, a transcriptional regulator (Stck) and several enzymes, including α-glucosidases, aminopeptidases and glucose dehydrogenase. Naive workers showed increased expression of members of the SPARC and lectin families, heat-shock cognate proteins and several proteins related to RNA translation and mitochondrial function. A number of novel genes overexpressed in both naive and experienced bees, and genes induced by caffeine, have also been identified. Conclusions We have shown the usefulness of this transcriptome-based approach for gene discovery, in particular in the context of the efficacy of drug treatment, in a model organism in which routine genetic techniques cannot be applied easily. PMID:11864369

  4. Evaluation of the Aspergillus Western blot IgG kit for diagnosis of chronic aspergillosis.

    Science.gov (United States)

    Oliva, A; Flori, P; Hennequin, C; Dubus, J-C; Reynaud-Gaubert, M; Charpin, D; Vergnon, J M; Gay, P; Colly, A; Piarroux, R; Pelloux, H; Ranque, S

    2015-01-01

    Immunoprecipitin detection (IPD) is the current reference confirmatory technique for anti-Aspergillus antibody detection; however, the lack of standardization is a critical drawback of this assay. In this study, we evaluated the performance of the Aspergillus Western blot (Asp-WB) IgG kit (LDBio Diagnostics, Lyon, France), a recently commercialized immunoblot assay for the diagnosis of various clinical presentations of chronic aspergillosis. Three hundred eight serum samples from 158 patients with aspergillosis sensu lato (s.l.) were analyzed. More specifically, 267 serum samples were derived from patients with Aspergillus disease, including 89 cases of chronic pulmonary aspergillosis, 10 of aspergilloma, and 32 of allergic bronchopulmonary aspergillosis, while 41 samples were from patients with Aspergillus colonization, including 15 cystic fibrosis (CF) and 12 non-CF patients. For blood donor controls, the Asp-WB specificity was 94%, while the kit displayed a sensitivity for the aspergillosis s.l. diagnosis of 88.6%, with a diagnostic odds ratio (DOR) of 119 (95% confidence interval [CI], 57 to 251). The DOR values were 185.22 (95% CI,78.79 to 435.45) and 43.74 (95% CI, 15.65 to 122.20) for the diagnosis of Aspergillus disease and Aspergillus colonization, respectively. Among the patients, the sensitivities of the Asp-WB in the diagnosis of Aspergillus colonization were 100% and 41.7% in CF and non-CF patients, respectively. The Asp-WB yielded fewer false-negative results than did IPD. In conclusion, the Asp-WB kit performed well for the diagnosis of various clinical presentations of aspergillosis in nonimmunocompromised patients, with an enhanced standardization and a higher sensitivity than with IPD, which is the current reference method. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  5. Western blot patterns of serum autoantibodies against optic nerve antigens in dogs with goniodysgenesis-related glaucoma.

    Science.gov (United States)

    Pumphrey, Stephanie A; Pizzirani, Stefano; Pirie, Christopher G; Anwer, M Sawkat; Logvinenko, Tanya

    2013-04-01

    To investigate whether differences existed between clinically normal dogs and dogs with goniodysgenesis-related glaucoma (GDRG) in serum autoantibodies against optic nerve antigens. 16 dogs with GDRG, 17 healthy dogs with unremarkable pectinate ligament and iridocorneal angle morphology, and 13 euthanized dogs with no major ocular abnormalities or underlying diseases. Western blotting was performed with optic nerve extracts from the euthanized dogs as an antigen source and serum from clinically normal dogs and dogs with GDRG as a primary antibody (autoantibody) source. Blots were evaluated for presence and density of bands. Multiple bands were identified on western blots from all dogs with GDRG and all clinically normal dogs, with a high degree of variability among individual dogs. Dogs with GDRG were significantly more likely than healthy dogs to have bands present at 38, 40, and 68 kDa. Dogs with GDRG had significant increases in autoreactivity at 40 and 53 kDa and a significant decrease in autoreactivity at 48 kDa. Significant differences in serum autoantibodies against optic nerve antigens were found in dogs with versus without GDRG. Although it remains unclear whether these differences were part of the pathogenesis of disease or were sequelae to glaucomatous changes, these findings provide support for the hypothesis that immune-mediated mechanisms play a role in the development or progression of GDRG. However, the high degree of variability among individual dogs and the considerable overlap between groups suggest that the clinical usefulness of this technique for distinguishing dogs with GDRG from clinically normal dogs is likely limited.

  6. Developing new optical imaging techniques for single particle and molecule tracking in live cells

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Wei [Iowa State Univ., Ames, IA (United States)

    2010-01-01

    Differential interference contrast (DIC) microscopy is a far-field as well as wide-field optical imaging technique. Since it is non-invasive and requires no sample staining, DIC microscopy is suitable for tracking the motion of target molecules in live cells without interfering their functions. In addition, high numerical aperture objectives and condensers can be used in DIC microscopy. The depth of focus of DIC is shallow, which gives DIC much better optical sectioning ability than those of phase contrast and dark field microscopies. In this work, DIC was utilized to study dynamic biological processes including endocytosis and intracellular transport in live cells. The suitability of DIC microscopy for single particle tracking in live cells was first demonstrated by using DIC to monitor the entire endocytosis process of one mesoporous silica nanoparticle (MSN) into a live mammalian cell. By taking advantage of the optical sectioning ability of DIC, we recorded the depth profile of the MSN during the endocytosis process. The shape change around the nanoparticle due to the formation of a vesicle was also captured. DIC microscopy was further modified that the sample can be illuminated and imaged at two wavelengths simultaneously. By using the new technique, noble metal nanoparticles with different shapes and sizes were selectively imaged. Among all the examined metal nanoparticles, gold nanoparticles in rod shapes were found to be especially useful. Due to their anisotropic optical properties, gold nanorods showed as diffraction-limited spots with disproportionate bright and dark parts that are strongly dependent on their orientation in the 3D space. Gold nanorods were developed as orientation nanoprobes and were successfully used to report the self-rotation of gliding microtubules on kinesin coated substrates. Gold nanorods were further used to study the rotational motions of cargoes during the endocytosis and intracellular transport processes in live mammalian

  7. An optimized xylene-free protein extraction method adapted to formalin-fixed paraffin embedded tissue sections for western blot analysis.

    Science.gov (United States)

    Mansour, Anthony G; Khalil, Pamela Abou; Bejjani, Noha; Chatila, Rajaa; Dagher-Hamalian, Carole; Faour, Wissam H

    2017-03-01

    Deparaffinization of formalin-fixed paraffin embedded (FFPE) tissues with xylene currently remains a major challenge to the biomedical community. We developed an efficient xylene-free protocol to isolate proteins from archived FFPE human tissue sections. A total of 79 different types of FFPE tissue sections of 8 µm thickness were obtained from various archived FFPE specimens. Deparaffinization was conducted by gently washing each section with around 1 ml of hot distilled water (≈80°C). The deparaffinized tissues were homogenized in lysis buffer, and the isolated proteins were quantified and efficiently resolved using western blot analysis for the presence of Protein kinase B (PKB/AKT) and β-actin. Moreover, a significant amount of proteins was successfully isolated with an average of 2.31 µg/µl. The migration pattern of AKT and β-actin obtained from the specimens was similar to the positive control obtained from protein lysates prepared from in vitro cultured MDA231 cancer cell lines. AKT was successfully identified in all specimens, and β-actin protein was resolved with an efficiency higher than 80%. The entire extraction procedure requires only 20 minutes. This newly developed technique is an efficient, safe, cost-effective, and rapid method to isolate proteins from FFPE tissue sections adequate for molecular analysis.

  8. Rapid detection of defects in fuel-cell electrodes using infrared reactive-flow-through technique

    Science.gov (United States)

    Das, Prodip K.; Weber, Adam Z.; Bender, Guido; Manak, Austin; Bittinat, Daniel; Herring, Andrew M.; Ulsh, Michael

    2014-09-01

    As fuel cells become more prominent, new manufacturing and production methods will need to be developed to deal efficiently and effectively with increased demand. One necessary component of this industrial growth is the accurate measurement of the variability in the manufacturing process. In this study, we present a diagnostic system that combines infrared thermography with a reactive-flow-through technique to detect catalyst-loading defects in fuel-cell gas-diffusion electrodes accurately with high spatial and temporal resolutions. Experimental results are compared with model predictions of thermal response with good agreement. Data analysis, operating-condition impacts, and detection limits are explored using both experiments and simulation. Overall, the results demonstrate the potential of this technique to measure defects on the millimeter length scale with temporal resolutions appropriate for use on a web-line. Thus we present the first development stage of a next-generation non-destructive diagnostic tool, which may be amenable to eventual use on roll-to-roll manufacturing lines.

  9. Western blotting in the diagnosis of duodenal-biliary and pancreaticobiliary refluxes in biliary diseases.

    Science.gov (United States)

    Xian, Guo-Zhe; Wu, Shuo-Dong; Chen, Chun-Chih; Su, Yang

    2009-12-01

    Currently adopted diagnostic methods for duodenal-biliary and pancreaticobiliary refluxes carry many flaws, so the incidence of the two refluxes demands further larger sample size studies. This study aimed to evaluate Western blotting for the diagnosis of refluxes in biliary diseases. An oral radionuclide 99mTc-DTPA test (radionuclide, RN) was conducted for the observation of duodenal-biliary reflux prior to measuring bile radioactivity and Western blotting for detecting bile enterokinase (EK). Pancreaticobiliary reflux was assessed by biochemical and Western blotting tests for biliary amylase activity and trypsin-1, respectively. In accordance with bile sample origin, our samples were classified into ductal bile and gall bile groups; based on each individual biliary disease, we further classified the ductal bile group into five sub-groups, and the gall bile group into four sub-groups. Western blotting was conducted to assess the two refluxes in biliary diseases. Consistencies were noted between EK and RN tests when diagnosing duodenal-biliary reflux (P0.05); in the common bile duct cyst group, the EK positive rate was significantly lower than the trypsin-1 positive rate (PWestern blotting can accurately reflect duodenal-biliary and pancreaticobiliary refluxes. EK has greater sensitivity than RN for duodenal-biliary reflux. The majority of biliary amylase and lipase comes from the pancreas in all biliary diseases; pancreaticobiliary reflux is the predominant source in the common bile duct cyst group and duodenal-biliary reflux is responsible for the ductal pigment stone group.

  10. Appropriate materials and preparation techniques for polycrystalline-thin-film thermophotovoltaic cells

    Science.gov (United States)

    Dhere, Neelkanth G.

    1997-03-01

    techniques have paved the way for obtaining epitaxial Hg1-xCdxTe thin films at substrate temperatures of ~180 °C with the desired crystalline perfection, stoichiometry, and doping without the necessity of further annealing for improving either the crystalline quality or dopant activity. Retaining larger mercury proportions during annealing would require heated enclosures as in isothermal VPE, hot-wall technique, vacuum evaporation, hot-wall MOCVD, or close-space sublimation. Pb1-xCdxTe thin films can be prepared by magnetron sputtering from cooled Pb1-xCdxTe targets on heated substrates. Hot-wall technique is suitable for the deposition of Pb1-xCdxTe thin films. Hg1-xCdxTe and Pb1-xCdxTe TPV cells will benefit from the substantial work on CdTe thin film solar cells. The paper reviews work on thin films of ternary and pseudoternary compounds of interest for TPV conversion and methods of their preparation with a view to choosing the appropriate materials and fabrication techniques for polycrystalline-thin-film TPV cells.

  11. Electrostatic extrusion as a dispersion technique for encapsulation of cells and bioactive compounds

    Directory of Open Access Journals (Sweden)

    Kostić Ivana T.

    2012-01-01

    Full Text Available Significant development of cells and bioactive compound encapsulation technologies is taking place due to an exceptional possibility of their application in various scientific disciplines, including biomedicine, pharmacy, cosmetology, food and agricultural sciences, beverage production, industrial waste treatment. Despite the broad application of microencapsulation, the literature reviews on dispersion techniques for microcapsule/microbead production, their advantages, restrictions and drawbacks are scarce. The purpose of this paper is to assess the possibilities of electrostatic extrusion for encapsulation of biological material, including living cells in hydrogel microbeads. The paper presents an overview of the mechanisms of droplet formation and controlling experimental parameters for producing microbeads by means of electrostatic extrusion. Electrostatic droplet formation utilizes a special type of physical process taking advantage of electrostatic effects occurring in flowing conductive liquids after introduction of an electric field.When an electrostatic field is applied to the metal needle and an electric charge is induced in the liquid flowing out of the needle, the size of droplet detaching from the needle tip decreases as a funcion of applied electrostatic field. It has been shown that few parameters affect microbead size: applied voltage, electrode geometry, needle size, polarity arrangement and polymer concentration. The electrostatic droplet formation is one of the most precise methods, which enables one to produce spherical and uniform particles ranging from 100 μm up to 1000 μm. Most of the authors report that the encapsulated compounds (drugs, enzymes and living cells remain unaltered after electrostatic extrusion. This technique seems to be particularly promising in biotechnology, pharmaceutical and cosmetics industries, where a low-temperature process, preserving heat-sensitive material is a prerequisite. Future efforts in

  12. A Comparative Study of Non-Viral Gene Delivery Techniques to Human Adipose-Derived Mesenchymal Stem Cell

    Directory of Open Access Journals (Sweden)

    Nur Shuhaidatul Sarmiza Abdul Halim

    2014-08-01

    Full Text Available Mesenchymal stem cells (MSCs hold tremendous potential for therapeutic use in stem cell-based gene therapy. Ex vivo genetic modification of MSCs with beneficial genes of interest is a prerequisite for successful use of stem cell-based therapeutic applications. However, genetic manipulation of MSCs is challenging because they are resistant to commonly used methods to introduce exogenous DNA or RNA. Herein we compared the effectiveness of several techniques (classic calcium phosphate precipitation, cationic polymer, and standard electroporation with that of microporation technology to introduce the plasmid encoding for angiopoietin-1 (ANGPT-1 and enhanced green fluorescent protein (eGFP into human adipose-derived MSCs (hAD-MSCs. The microporation technique had a higher transfection efficiency, with up to 50% of the viable hAD-MSCs being transfected, compared to the other transfection techniques, for which less than 1% of cells were positive for eGFP expression following transfection. The capability of cells to proliferate and differentiate into three major lineages (chondrocytes, adipocytes, and osteocytes was found to be independent of the technique used for transfection. These results show that the microporation technique is superior to the others in terms of its ability to transfect hAD-MSCs without affecting their proliferation and differentiation capabilities. Therefore, this study provides a foundation for the selection of techniques when using ex vivo gene manipulation for cell-based gene therapy with MSCs as the vehicle for gene delivery.

  13. Assessment of IgE Reactivity of β-Casein by Western Blotting After Digestion with Simulated Gastric Fluid.

    Science.gov (United States)

    Benedé, Sara; López-Fandiño, Rosina; Molina, Elena

    2017-01-01

    Cow's milk allergy is defined as an immunologically mediated adverse reaction to cow's milk proteins and it is usually, along with hen's egg allergy, the first food allergy identified in childhood.One of the main aspects to consider when evaluating the allergenic potential of food proteins is the effect of gastric digestion. It is known that allergens are usually able to survive the harsh acidic environment of the stomach, tolerate the presence of surfactants, and resist digestion by pepsin. They might also be digested into high molecular weight peptide fragments, which retain the same, or sometimes increased, IgE-binding. In this respect, western blotting is a highly sensitive and efficient technique that we have used to detect IgE-binding to the digests of milk and egg proteins. Given the importance of the resistance of food proteins to gastric digestion in their capacity to modulate the immune response, we describe in this chapter the assessment of IgE reactivity of a relevant cow's milk allergen, β-casein, by western blotting after simulated digestion under relevant physiological conditions.

  14. [Western Blot diagnostic yield for simultaneous antibody-detection in patients with human cysticercosis, hydatidosis, and human fascioliasis].

    Science.gov (United States)

    Davelois, Kelly; Escalante, Hermes; Jara, César

    2016-01-01

    . To determine the diagnostic yield using western blotting to simultaneously detect antibodies in patients with human cysticercosis, hydatidosis, and human fascioliasis. Materials and methods . Cross-sectional study of diagnostic yield assessment. Excretory/secretory antigens were obtained from Taenia solium larvae, Echinococcus granulosus cysts, and the adult flukes of Fasciola hepática, which were then separated using the polyacrylamide gel electrophoresis technique, transferred, and attached to a nitrocellulose membrane to be probed with sera from the patient infected with the three parasites. The sensitivity of the technique was assessed using 300 individual serum samples, 60 pools of two parasites, and 20 pools of three parasites with 75 sera from patients with other parasites, 10 from patients with other diseases, and 15 from patients without parasites. Results . The technique revealed 13 glycoproteins (GP): GP 35, 31, 24, 23, 18, 17, 14, and 13 kDa for cysticercosis; GP 8, 16, and 21 kDa for hydatidosis; and GP 17 and 23 kDa for fascioliasis. The test detected the presence of antibodies with a sensitivity of 96% (95% confidence interval [CI] = 94.62-98.54%) in the detection of one or the thirteen bands, a specificity of 100% (95% CI = 99.50-100.00%); individually, there was a sensitivity for cysticercosis of 97% (95% CI = 93.16-100.00%), for hydatidosis of 94% (95% CI = 88.85-99.15%) and for fascioliasis of 96% (95% CI = 91.66-100.00%). Conclusions . Western blotting is effective in the simultaneous detection of antibodies in patients with human cysticercosis, hydatidosis, and fascioliasis, and it can be used as a diagnostic test to either rule out or confirm the presence of antibodies in endemic areas.

  15. New designs and characterization techniques for thin-film solar cells

    Science.gov (United States)

    Pang, Yutong

    This thesis presents a fundamentally new thin-film photovoltaic design and develops several novel characterization techniques that improve the accuracy of thin-film solar cell computational models by improving the accuracy of the input data. We first demonstrate a novel organic photovoltaic (OPV) design, termed a "Slot OPV", in which the active layer is less than 50 nm; We apply the principles of slot waveguides to confine light within the active layer. According to our calculation, the guided-mode absorption for a 10nm thick active layer equal to the absorption of normal incidence on an OPV with a 100nm thick active layer. These results, together with the expected improvement in charge extraction for ultrathin layers, suggest that slot OPVs can be designed with greater power conversion efficiency than today's state-of-art OPV architectures if practical challenges, such as the efficient coupling of light into these modes, can be overcome. The charge collection probability, i.e. the probability that charges generated by absorption of a photon are successfully collected as current, is a critical feature for all kinds of solar cells. While the electron-beam-induced current (EBIC) method has been used in the past to successfully reconstruct the charge collection probability, this approach is destructive and requires time-consuming sample preparation. We demonstrate a new nondestructive optoelectronic method to reconstruct the charge collection probability by analyzing the internal quantum efficiency (IQE) data that are measured on copper indium gallium diselenide (CIGS) thin-film solar cells. We further improve the method with a parameter-independent regularization approach. Then we introduce the Self-Constrained Ill-Posed Inverse Problem (SCIIP) method, which improves the signal-to-noise of the solution by using the regularization method with system constraints and optimization via an evolutionary algorithm. For a thin-film solar cell optical model to be an accurate

  16. Joint salvage using sandwich technique for giant cell tumors around knee.

    Science.gov (United States)

    Kundu, Zile Singh; Gogna, Paritosh; Singla, Rohit; Sangwan, Sukhbir Singh; Kamboj, Pradeep; Goyal, Shobit

    2015-04-01

    The most common site for giant cell tumors (GCT) is knee, where the tumor characteristically extends right up to the subarticular bone plate. Extensive curettage with preservation of the joint should be done wherever possible. The alternatives for filling the void left after curettage are either bone graft or bone cement. Sandwich technique uses the advantages of both, taking care to prevent damage to articular cartilage. This study was done to evaluate the results of sandwich technique in tumors around the knee joint. It was a prospective study of 26 consecutive patients (15 females and 11 males) with Campanacci grade II and grade III GCT around the knee, which qualified the inclusion criterion and underwent knee reconstruction with sandwich technique, after extended curettage of the tumor. The mean age of the patients at the time of surgery was 32.73 ± 11.30 years (range, 18-62 years), and the mean follow-up was 3.87 ± 1.26 years (range, 6.5-2 years). At final follow-up, the functional evaluation was done using Musculoskeletal Tumor Society (MSTS) score and measuring range of motion around the knee. Three patients had recurrence of tumor; in one case, we were able to salvage the joint and repeat sandwich surgery was performed, and in the other two cases, the joint was breached; therefore, we resorted to resection arthrodesis. At final follow-up, the mean functional arc of motion around the knee and the mean MSTS score in patients without arthrodesis was 123.52 ± 10.21 degrees (range, 100-130 degrees) and 27.04/30, respectively; all patients were able to do their activities of daily living with ease. Sandwich technique is a good reconstruction procedure in GCT around knee joint with good survival rate, minimal complications, and good functional outcome. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

  17. Artificial Intelligence Techniques for the Estimation of Direct Methanol Fuel Cell Performance

    Science.gov (United States)

    Hasiloglu, Abdulsamet; Aras, Ömür; Bayramoglu, Mahmut

    2016-04-01

    Artificial neural networks and neuro-fuzzy inference systems are well known artificial intelligence techniques used for black-box modelling of complex systems. In this study, Feed-forward artificial neural networks (ANN) and adaptive neuro-fuzzy inference system (ANFIS) are used for modelling the performance of direct methanol fuel cell (DMFC). Current density (I), fuel cell temperature (T), methanol concentration (C), liquid flow-rate (q) and air flow-rate (Q) are selected as input variables to predict the cell voltage. Polarization curves are obtained for 35 different operating conditions according to a statistically designed experimental plan. In modelling study, various subsets of input variables and various types of membership function are considered. A feed -forward architecture with one hidden layer is used in ANN modelling. The optimum performance is obtained with the input set (I, T, C, q) using twelve hidden neurons and sigmoidal activation function. On the other hand, first order Sugeno inference system is applied in ANFIS modelling and the optimum performance is obtained with the input set (I, T, C, q) using sixteen fuzzy rules and triangular membership function. The test results show that ANN model estimates the polarization curve of DMFC more accurately than ANFIS model.

  18. A microculture technique for the evaluation of corneal cell metabolism in vitro.

    Science.gov (United States)

    BenEzra, D

    1977-10-01

    A microculture technique for the evaluation of the metabolic activity of corneal cells is described and analyzed. The extent of DNA synthesis in microcultures with 10(3) to 2.5 X 10(3) cells per well was initially low during day 1, increasing steadily thereafter. Higher initial concentration of 10(4) to 2 X 10(4) cells per microculture demonstrated a high metabolic activity during days 1 and 2 in culture, followed by a rapid and marked decrease on days 3 and 4. The origin and concentration of serum in the system have been found to be crucial. Xenogeneic serum (fetal calf serum--FCS) had the most potent stimulatory effect on DNA and protein synthesis. Syngeneic serum (guinea pig serum, strain 13--SGpS) or allogeneic serum (guinea pig serum strain 2--AGpS) had a generally less stimulatory effect on the metabolic activity. However, both sera had a relatively much stronger effect on the protein synthesis.

  19. The application of next-generation sequencing techniques in studying transcriptional regulation in embryonic stem cells.

    Science.gov (United States)

    Liu, Ya-Jun; Zhang, Feng; Liu, Hong-de; Sun, Xiao

    2017-08-20

    The mechanism of transcriptional regulation has been the focus of many studies in the post-genomic era. The development of sequencing-based technologies for chromatin profiling enables current researchers to experimentally measure chromatin properties. Moreover, many studies aim at annotating the state of the chromatin into broad categories based on observed chromatin features and/or DNA sequences, then associating the resultant distal regulatory regions with the correct target genes based on DNA sequences, and predicting the dependence of epigenetic features on genetic variation. Stem cell biology has many applications in the area of regenerative medicine and tumorigenesis. In this review, we summarize recent research progresses on the application of next-generation sequencing techniques in studying transcriptional regulation in embryonic stem cells. This review mainly focuses on four areas: (1) microarray or RNA-seq; (2) chromatin immunoprecipitation (ChIP); (3) Dnase I hypersensitive sites (DHSs); (4) high-throughput chromosome conformation capture (Hi-C). These technologies have been utilized in studying chromatin on three levels, i.e., gene expression, transcription factor binding and genome three-dimensional structure. We especially emphasize three master transcription factors of pluripotency: Oct4, Sox2 and Nanog. We aim to track the frontier of stem cell transcriptional regulation research and share important progresses in this field.

  20. Inertial focusing and passive micro-mixing techniques for rare cells capturing microfluidic platform

    Science.gov (United States)

    Phadke, Manisha; Shaner, Sebastian; Shah, Shreyas; Rodriguez, Ygnacio; Wibowo, Denni; Whulanza, Yudan; Teriete, Peter; Allen, Jeff; Kassegne, Sam

    2018-02-01

    Isolation and capture of rare cells continues to be a daunting task that is still looking for an innovative and efficient method. While a variety of approaches have been suggested over the past several years, immunocapturing in a microfluidic platform carries a substantial promise as shown by recent published works. In this paper, we introduced a combination of inertial focusing and passive micro-mixing through 3D chevron-type features in a microchannel to induce chaotic mixing within antibody-coated microchannels and, ultimately, promote rare cell capture. The device introduced in this work contains curved microchannels that consist of a series of staggered chevron grooves. The curved channels enable inertial focusing while the chevron grooves allow for chaotic mixing. The microfluidics platform microfabricated through soft lithography has a polydimethylsiloxane (PDMS) foundation and was thinly coated with an alginate hydrogel derivatized with streptavidin. We submitted that our qualitative and quantitative results demonstrated the potentials in advancements in rare cell isolation through this integration of two techniques.

  1. Shift-Western Blotting: Separate Analysis of Protein and DNA from Protein-DNA Complexes.

    Science.gov (United States)

    Harbers, Matthias

    2015-01-01

    The electrophoretic mobility shift assay (EMSA) is the most frequently used experiment for studying protein-DNA interactions and to identify DNA-binding proteins. Protein-DNA complexes formed during EMSA experiments can be further analyzed by shift-western blotting, where the protein and DNA components contained in a polyacrylamide gel are transferred to stacked membranes: First a nitrocellulose membrane retains the proteins while double-stranded DNA passes through the nitrocellulose membrane and binds only to a charged membrane placed below. Immobilized proteins can then be stained with specific antibodies while the DNA can be detected by a radioactive label or a nonradioactive detection system. Shift-western blotting can overcome many limitations of supershift experiments and allows for the analysis of complex protein-DNA complexes containing multiple protein factors. Moreover, proteins and/or DNA may be recovered from membranes after the blotting step for further analysis by other means.

  2. Should we ignore western blots when selecting antibodies for other applications?

    DEFF Research Database (Denmark)

    Uhlén, Mathias

    2017-01-01

    and then heated to very high temperatures (normally >100 °C) in a procedure that is sometimes termed 'epitope retrieval'. Obviously, this procedure might influence the target protein differently than the procedure used to prepare proteins for a western blot, in which the sample is instead treated with a detergent...... (SDS) before the electrophoresis step. Thus, as concluded by the members of the IWGAV1, the results obtained for a given antibody in western blot applications cannot be used to predict the specificity of the antibody in another assay based on an entirely different epitope-retrieval method, such as IHC...

  3. Immunohistochemical and Western Blotting Analyses of Ganoine in the Ganoid Scales of Lepisosteus oculatus: an Actinopterygian Fish.

    Science.gov (United States)

    Sasagawa, Ichiro; Oka, Shunya; Mikami, Masato; Yokosuka, Hiroyuki; Ishiyama, Mikio; Imai, Akane; Shimokawa, Hitoyata; Uchida, Takashi

    2016-05-01

    In order to compare its characteristics with those of jaw tooth collar enamel, normally developing and experimentally regenerating ganoine from ganoid scales of Lepisosteus oculatus (spotted gar), an actinopterygian fish species, was examined by Western blotting and immunohistochemistry. Amelogenin, a major enamel matrix protein (EMP), is widely found from sarcopterygian fish to mammals. Therefore, we used antimammalian amelogenin antibodies and antisera: an antibody against bovine amelogenin; antiserum against porcine amelogenin; and region-specific antibodies or antiserum against the C-terminus, middle region, or N-terminus of porcine amelogenin in this study. Positive immunoreactivity with the antibody against bovine amelogenin, antiserum against porcine amelogenin, and the middle and C-terminal region-specific antibodies was detected in both normally developing and regenerating ganoine matrix, as well as in granules found within inner ganoine epithelial cells. These immunohistochemical analyses indicated that the Lepisosteus ganoine matrix contains EMP-like proteins with epitopes similar to mammalian amelogenins. In Western blotting analyses of regenerating ganoid scales with the antibovine amelogenin antibody, two protein bands with molecular weights of approximately 78 and 65 kDa were detected, which were similar to those found in Lepisosteus tooth enamel. Our study suggests that in Lepisosteus, EMP-like proteins in the ganoine matrix corresponded to those in tooth enamel. However, it was revealed that the 78 and 65 kDa EMP-like proteins were different from 27 kDa bovine amelogenin. © 2016 Wiley Periodicals, Inc.

  4. Identification of Glioblastoma Phosphotyrosine-Containing Proteins with Two-Dimensional Western Blotting and Tandem Mass Spectrometry.

    Science.gov (United States)

    Guo, Tianyao; Wang, Xiaowei; Li, Maoyu; Yang, Haiyan; Li, Ling; Peng, Fang; Zhan, Xianquan

    2015-01-01

    To investigate the presence of, and the potential biological roles of, protein tyrosine phosphorylation in the glioblastoma pathogenesis, two-dimensional gel electrophoresis- (2DGE-) based Western blotting coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis was used to detect and identify the phosphotyrosine immunoreaction-positive proteins in a glioblastoma tissue. MS/MS and Mascot analyses were used to determine the phosphotyrosine sites of each phosphopeptide. Protein domain and motif analysis and systems pathway analysis were used to determine the protein domains/motifs that contained phosphotyrosine residue and signal pathway networks to clarify the potential biological functions of protein tyrosine phosphorylation. A total of 24 phosphotyrosine-containing proteins were identified. Each phosphotyrosine-containing protein contained at least one tyrosine kinase phosphorylation motif and a certain structural and functional domains. Those phosphotyrosine-containing proteins were involved in the multiple signal pathway systems such as oxidative stress, stress response, and cell migration. Those data show 2DGE-based Western blotting, MS/MS, and bioinformatics are a set of effective approaches to detect and identify glioblastoma tyrosine-phosphorylated proteome and to effectively rationalize the biological roles of tyrosine phosphorylation in the glioblastoma biological systems. It provides novel insights regarding tyrosine phosphorylation and its potential role in the molecular mechanism of a glioblastoma.

  5. Properties of graphite-composite bipolar plate prepared by compression molding technique for PEM fuel cell

    Energy Technology Data Exchange (ETDEWEB)

    Dhakate, S.R.; Mathur, R.B.; Dhami, T.L. [National Physical Laboratory, New Delhi (India). Engineering Material Division, Carbon Technology Unit; Kakati, B.K. [Tezpur University, Assam (India). Department of Energy

    2007-12-15

    Bipolar plate is an important key component of fuel cell on the basis of its manifold function. In this direction a lot of effort is going on worldwide to make light-weight and cost-effective bipolar plate for fuel cell application. In the present investigation effort was made to develop graphite-composites bipolar plate by compression molding technique to achieve the requisite goal. The composites plates were prepared by using different reinforcing fillers such as natural graphite, synthetic graphite, carbon black, carbon fibers with phenolic resin as polymer matrix precursor in its liquid and powder form. The composition of different filler constituent adjusted in between 5 and 40 vol%. The composite plates prepared with appropriate proportion of filler components were characterized for physical and mechanical properties. It is found that no single reinforcing filler constituent composites plate gives the requisite properties for being used as bipolar plate in the PEM fuel cell. The judicious combination of reinforcing constituents gives the properties which are required for bipolar plate to use in fuel cell. By controlling the ratio of reinforcing constituents, one can able to achieve properties such as bulk density {proportional_to}1.85gcm{sup -3}, electrical conductivity >150Scm{sup -1}, shore hardness >65, bending strength >60MPa, modulus >10GPa and compressive >70MPa by applying the pressure (100kgcm{sup -2}) during compression molding. I-V characteristic of the composite bipolar plate, with optimum combination of reinforcing constituent, is found to be adequate as per the US-DOE target for composite bipolar plate. (author)

  6. MANAGEMENT OF A RARE RECURRENCE OF DISTAL TIBIAL GIANT CELL TUMOUR BY SANDWICH TECHNIQUE

    Directory of Open Access Journals (Sweden)

    Rajesh Kishanrao

    2016-02-01

    Full Text Available INTRODUCTION First described by Jaffe et al in 1940, giant cell tumour (GCT constitutes 20% of all the skeletal neoplasms with a higher rate of recurrence after excision. Most common sites for the involvement are distal femur and proximal tibia followed by the distal end of radius. Ankle and foot involvement is rare <4%. Usually benign, they are locally aggressive and may occasionally undergo malignant transformation. The surgeon needs to strike a balance during treatment between being aggressive in order to reduce the incidence of local recurrence and being conservative in removing the normal bone to attain maximal function. Current literature suggests that intralesional curettage strikes the best balance between controlling disease and preserving optimum function in the majority of the cases though there may be occasions where the extent of the disease mandates resection to ensure adequate disease clearance. We report a case of Giant Cell Tumour of distal end of left Tibia in a 32-year-old female patient. Initially the condition was treated by curettage and bone grafting. But, due to recurrence of the condition within 9 months, she was treated with extended curettage using hydrogen peroxide, burr and bone cement as adjuvants and reconstruction using the “SANDWICH” Technique. At One year follow up there is no recurrence and reasonably good function around the ankle joint is maintained. Primary Giant Cell Tumours have been traditionally treated with curettage of the lesion followed by bone grafts/bone cement. Recurrent cases often require aggressive management. The adjuvant treatment used in our case offered good stability and allowed early mobilization of the ankle joint. This case substantiates the use of bone cement in the treatment of recurrent Giant Cell Tumour of distal tibia whenever the articular integrity is intact with reasonably good functional outcomes. However, a periodic follow-up is still recommended to watch-out for late re-recurrences

  7. The feasibility of using particle-in-cell technique for modeling liquid crystal devices

    Science.gov (United States)

    Leung, Wing Ching

    1997-12-01

    Liquid crystal materials are used in a variety of electronic devices, but the dynamical behavior of such devices is still not clearly understood. The primary reason for this is that there is a lack of rigorous models for a theoretical treatment for the devices. For the first time we demonstrate here the feasibility of treating such liquid crystal devices using the particle- in-cell (PIC) simulation technique. In a PIC simulation model, the liquid crystal medium is represented by a certain number of particles, each particle representing a fairly large number of real molecules in the medium. The dynamical behavior of the medium is obtained by solving the equations of motions of the particles in self- consistent appropriate fields. The motions may include both translation (flow) and rotation. By neglecting the translational motion and the conductive effects of the materials in this first innovative effort, 1- and 2- dimensional PIC codes, including viscous, electric, and elastic torques affecting the rotation of molecules, are developed for modeling a parallel-plate capacitor cell filled with a nematic liquid crystal material. Using these codes we reproduce the well known phenomenon of Fredericksz transition. Our simulations yield results in excellent agreement with the available analytical results on the threshold voltage for the Fredericksz transition. In addition, we are able to reveal the dynamical behavior of liquid crystal molecules in the device as the material undergoes the Fredericksz transition in response to an applied voltage across the liquid crystal cell. By using the 2-dimensional PIC code to simulate a segmented electrode cell, like those used in liquid crystal devices for optical gratings, the simulation results show the formation and dynamics of the defect walls. The defect wall joins the regions of the material having topologically different orientations. We found that the defect wall is associated with a large gradient in the polarization vector P

  8. Mechanical behavior study of single cell contraction by digital image correlation technique

    Science.gov (United States)

    Huang, Jianyong; Wu, Jia; Qin, Lei; Zhu, Tao; Xiong, Chunyang; Zhang, Youyi; Fang, Jing

    2008-11-01

    Precise determination of cellular traction forces has important significance in assessing cellular mechanical characteristic on micro/nano scale. Elastic substrate method is a useful way to study cellular traction forces, in which the cells are cultured on elastic gel substrate marked by randomly embedding fluorescent microbeads and they exert mechanical forces on the film to cause deformation of the substrate material. In this paper, we investigate the acquisition of deformation field induced by single cardiac myocyte by using digital image correlation (DIC) technique. In view of the fact that cellular force restoration is essentially an ill-posed inverse problem, which implies that the force reconstruction is susceptible to the input displacement noise, we develop a novel optimal filtering scheme in two-dimensional Fourier space to restrain displacement noise amplification. Experiments of traction force recovery for a real cardiac myocyte indicate the optimal scheme in combination with the DIC method enables us to reconstruct cellular traction fields with high accuracy.

  9. Human nail thermal diffusivity obtained using the open photoacoustic cell technique

    Science.gov (United States)

    Dias, D. T.; Nuglish, L. E. R.; Sehn, E.; Baesso, M. L.; Medina, A. N.; Bento, A. C.

    2005-06-01

    In this work the open photoacoustic cell technique (OPC) is applied for measuring the thermal diffusivity (α) of human nail tips. Human nails are natural polymers that receive less attention in clinical analysis than other human body parts, although they are very interesting in giving information about some external diseases like dystrophies. Diagnosis and therapy with topic application of anti-fungal creams could be monitored since thermal properties are known. The OPC experiments in the low frequency range were done and through photoacoustic signal decay, the OPC model were used for fitting data in order to obtain the thermal diffusivity of the human nail in vitro. The average value for the nail tips used was found to be α ˜ (8.9 ± 1.3) × 10-4 cm^2/s, when different light source is used for photothermal heating. This average is of the order of that evaluated for the human skin.

  10. Monitoring programmed cell death of living plant tissues in microfluidics using electrochemical and optical techniques

    DEFF Research Database (Denmark)

    Mark, Christina; Zor, Kinga; Heiskanen, Arto

    This project focuses on developing and applying a tissue culture system with electrochemical and optical detection techniques for tissue culture of barley aleurone layer to increase understanding of the underlying mechanisms of programmed cell death (PCD) in plants. The major advantage...... an optical double-fluorescent probe-system[4]. Currently, we are working on integrating both detection methods into a tissue culture system for immobilised plant tissues....... of electrochemical detection systems is that they can be miniaturized, multiplexed and automated without losing their performance[1,2]. Combining tissue culture with electrochemical and optical detection allows implementation of a wide range of assays for online, real-time, parallel analysis of important parameters...

  11. Photoelectrode Fabrication of Dye-Sensitized Nanosolar Cells Using Multiple Spray Coating Technique

    Directory of Open Access Journals (Sweden)

    Chien-Chih Chen

    2013-01-01

    Full Text Available This paper presents a spray coating technique for fabricating nanoporous film of photoelectrode in dye-sensitized nanosolar cells (DSSCs. Spray coating can quickly fabricate nanoporous film of the photoelectrode with lower cost, which can further help the DSSCs to be commercialized in the future. This paper analyzed photoelectric conversion efficiency of the DSSCs using spray coated photoelectrode in comparison with the photoelectrode made with the doctor blade method. Spray coating can easily control transmittance of the photoelectrode through the multiple spray coating process. This work mainly used a dispersant with help of ultrasonic oscillation to prepare the required nano-TiO2 solution and then sprayed it on the ITO glasses. In this work, a motor-operated conveyor belt was built to transport the ITO glasses automatically for multiple spray coating and drying alternately. Experiments used transmittance of the photoelectrode as a fabrication parameter to analyze photoelectric conversion efficiency of the DSSCs. The influencing factors of the photoelectrode transmittance during fabrication are the spray flow rate, the spray distance, and the moving speed of the conveyor belt. The results show that DSSC with the photoelectrode transmittance of ca. 68.0 ± 1.5% and coated by the spray coating technique has the best photoelectric conversion efficiency in this work.

  12. Remote-welding technique for assembling in-pile IASCC capsule in hot cell

    International Nuclear Information System (INIS)

    Kawamata, Kazuo; Ishii, Toshimitsu; Kanazawa, Yoshiharu; Iwamatsu, Shigemi; Ohmi, Masao; Shimizu, Michio; Matsui, Yoshinori; Saito, Jun-ichi; Ugachi, Hirokazu; Kaji, Yoshiyuki; Tsukada, Takashi

    2006-01-01

    In order to investigate behavior of the irradiation assisted stress corrosion cracking (IASCC) caused by the simultaneous effects of neutron irradiation and high temperature water environment in such a light water reactor (LWR), it is necessary to perform crack growth tests in an in-pile IASCC capsule irradiated in the Japan Materials Testing Reactor (JMTR). The development of the remote-welding technique is essential for remotely assembling the in-pile IASCC capsule installing the pre-irradiated CT specimens. This report describes a new remote-welding machine developed for assembling the in-pile IASCC capsule. The remote-welding technique that the capsule tube is rotated light under the fixed torch was applied to the machine for the welding of thick and large-diameter tubes. The assembly work of four in-pile IASCC capsules having pre-irradiated CT specimens in the hot cell was succeeded for performing the crack growth test under the neutron irradiation in JMTR. The irradiation test of two capsules has been already finished in JMTR without problems. (author)

  13. Flicking technique for microencapsulation of cells in calcium alginate leading to the microtissue formation.

    Science.gov (United States)

    Wong, Soon Chuan; Soon, Chin Fhong; Leong, Wai Yean; Tee, Kian Sek

    2016-01-01

    Microbeads have wide applications in biomedical engineering field that include drug delivery, encapsulation of biomolecules, tissue padding and tissue regeneration. In this paper, we report a simple, yet efficient, flicking technique to produce microcapsules of calcium alginate at a narrow distribution of size. The system consists of an infusion pump and a customised flicker that taps the syringe needle for dispersing microcapsules of sodium alginate that polymerised in the calcium chloride solution. The flow rate of the syringe pump and the velocity of the flicker were studied to achieve a well controlled and tunable size distribution of microbeads ranging from 200 to 400 μm. At a flow rate of 4 μl/min and flicking rate of 80 rpm, a narrow size distribution of microbeads were produced. Via this technique, HaCaT cells were encapsulated in calcium alginate microbeads that grown into microtissues with a size ranging from 100 to 300 μm after two weeks of culture. These microtissues could be potentially useful for pharmacological application.

  14. Imaging and high-sensitivity quantification of chemiluminescent labeled DNA-blots

    International Nuclear Information System (INIS)

    Dorner, G.

    1997-01-01

    The present thesis has for objective the development of both, methods of DNA labeling by chemiluminescence (via the catalytic activity of the enzyme alkaline phosphatase - AP) and an appropriate imaging system. Offering a competitive alternative to the detection of classical radio-labels in molecular-biological experiments of the blotting type, this technique should permit the realization of quantitative studies of gene expression at ultra-high sensitivity necessary in particular for differential-screening experiments. To reach our aim. we separated the project into three different parts. In a first step an imager based on a liquid-nitrogen-cooled CCD coupled to a standard optics (50 mm/fl.2) has been installed and characterized. This system offers a sensitive area of up to 625 cm 2 , a spatial resolution of 0.3-1 mm (depending on the field of view) and a sensitivity sufficient to detect 10 fg/mm 2 labeled DNA. In a second part, the chemiluminescent light-generation process in solution has been investigated to optimize the parameters temperature. pH and concentration of the substrate as well as the enzyme. The substrate offering the highest light yield (CDP-Star in addition with the enhancer EMERALD II) allows quantification of AP down to 10 -15 M within a dynamic range of 10 4 in solution. Finally. preparation, immobilization and detection of AP-labeled DNA probes (via a biotin-streptavidin-biotin-AP bridge) on nylon membranes has been optimized. A linear relation between the light intensities and the amount of DNA was observed in a range of 10 fg/mm 2 - 100 pg/mm 2 . Hybridization of the probes to bacterial cloned target-DNA has been addressed after examination of the best hybridization conditions. Our protocol includes the treatment of a proteinase, which resulted in a significantly lower background on the filter. The results of our investigations suggest that the main conditions for a reliable differential-screening experiment are fulfilled when using

  15. Ferulic acid enhances IgE binding to peanut allergens in western blots.

    Science.gov (United States)

    Phenolic compounds at high concentrations are known to form insoluble complexes with proteins. We hypothesized that this complex formation could interfere with Western blot and ELISA assays for peanut allergens. To verify this, three simple phenolic compounds (ferulic, caffeic, and chlorogenic acids...

  16. Northern and Southern blot analysis of human RNA and DNA in autopsy material

    DEFF Research Database (Denmark)

    Larsen, S; Rygaard, K; Asnaes, S

    1992-01-01

    Fresh biopsy material for molecular biological investigations is not obtainable from all relevant normal human tissues. We studied the feasibility of using RNA and DNA from autopsies for Northern and Southern blot analysis. Tissue samples from seven organs were obtained from 10 autopsies performed...

  17. Northern and Southern blot analysis of human RNA and DNA in autopsy material

    DEFF Research Database (Denmark)

    Larsen, S; Rygaard, K; Asnaes, S

    1992-01-01

    was obtained less than two days postmortem. Histological examination showing slight or no autolysis and the presence of ribosomal bands after gel electrophoresis were both indicative parameters of RNA preservation. DNA was appropriate for Southern blotting when the tissue was obtained less than three to five...

  18. A western blot protocol for detection of proteins heterologously expressed in Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Jørgensen, Morten Egevang; Nour-Eldin, Hussam Hassan; Halkier, Barbara Ann

    2016-01-01

    at the individual oocyte level is often desirable when comparing properties of wild type and mutant transporters. However, a large content of yolk platelets in the oocyte cytoplasm makes this a challenging task. Here we report a method for fast and easy, semiquantitative Western blot analysis of proteins...

  19. Indeterminate human immunodeficiency virus Western blot profiles in ethiopians with discordant screening-assay results

    NARCIS (Netherlands)

    Meles, Hailu; Wolday, Dawit; Fontanet, Arnaud; Tsegaye, Aster; Tilahun, Tesfaye; Aklilu, Mathias; Sanders, Eduard; Rinke de Wit, Tobias F.

    2002-01-01

    The Western blot (WB) assay is the most widely accepted confirmatory assay for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1). However, indeterminate WB reactivity to HIV-1 proteins may occur in individuals who do not appear to be infected with HIV. The profiles of WB

  20. Two-dimensional gel-based protein standardization verified by western blot analysis.

    Science.gov (United States)

    Haniu, Hisao; Watanabe, Daisuke; Kawashima, Yusuke; Matsumoto, Hiroyuki

    2015-01-01

    In data presentation of biochemical investigation the amount of a target protein is shown in the y-axis against the x-axis representing time, concentrations of various agents, or other parameters. Western blot is a versatile and convenient tool in such an analysis to quantify and display the amount of proteins. In western blot, so-called housekeeping gene product(s), or "housekeeping proteins," are widely used as internal standards. The rationale of using housekeeping proteins for standardization of western blot is based on the assumption that the expression of chosen housekeeping gene is always constant, which could be false under certain physiological or pathological conditions. We have devised a two-dimensional gel-based standardization method in which the protein content of each sample is determined by scanning the total protein density of two-dimensional gels and the expression of each protein is quantified as the density ratio of each protein divided by the density of the total proteins on the two-dimensional gel. The advantage of this standardization method is that it is not based on any presumed "housekeeping proteins" that are supposed to be being expressed constantly under all physiological conditions. We will show that the total density of a two-dimensional gel can render a reliable protein standardization parameter by running western blot analysis on one of the proteins analyzed by two-dimensional gels.

  1. Raman Spectroscopy of Solid Oxide Fuel Cells: Technique Overview and Application to Carbon Deposition Analysis

    KAUST Repository

    Maher, R. C.

    2013-07-30

    Raman spectroscopy is a powerful characterization tool for improving the understanding of solid oxide fuel cells (SOFCs), capable of providing direct, molecularly specific information regarding the physical and chemical processes occurring within functional SOFCs in real time. In this paper we give a summary of the technique itself and highlight ex situ and in situ studies that are particularly relevant for SOFCs. This is followed by a case study of carbon formation on SOFC Ni-based anodes exposed to carbon monoxide (CO) using both ex situ and in situ Raman spectroscopy combined with computational simulations. In situ measurements clearly show that carbon formation is significantly reduced for polarized SOFCs compared to those held at open circuit potential (OCP). Ex situ Raman mapping of the surfaces showed clear variations in the rate of carbon formation across the surface of polarized anodes. Computational simulations describing the geometry of the cell showed that this is due to variations in gas access. These results demonstrate the ability of Raman spectroscopy in combination with traditional characterization tools, to provide detailed understanding of critical processes occurring within functional SOFCs. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Development of tensile test techniques for irradiated fuel cladding in hot cell

    International Nuclear Information System (INIS)

    Kim, D. S.; Hong, G. P.; Joo, Y. S.; Ahn, S. B.; Jeong, Y. H.; Oh, W. H.; Baek, S. J.

    2003-01-01

    To estimate the longitudinal and transverse tensile properties of fuel cladding in hot cell, existing tensile test techniques are reviewed. The specimen geometries have been optimized to determine the constitutive stress-strain properties of the cladding in both the longitudinal and transverse directions. The dogbone tube specimen for the longitudinal tensile test is designed to have a uniform strain distribution at the gage section. The ring specimen design for the transverse tensile test is conducted to maximize uniformity of strain distribution in the uniaxial ring specimen and to assure plane strain conditions in the plane-strain ring specimen. Fuel pellets in the cladding are removed by using the mechanical(grinding or drilling) or chemical(dissolution) method. The specimens are machined by a traveling-wire electrical discharge machine in hot cell. The pin-loaded grip is used for the longitudinal tensile test of an irradiated specimen. The grip for the transverse tensile test is designed such that a constant specimen curvature is maintained during deformation, and the interface was lubricated to minimize the friction between the outer surface of the die insert and the inner surface of the cladding specimen. In order to determine the constitutive stress-strain response of the cladding specimens, the machine compliance should be considered. The essential data for fuel damage criteria used in regulation and the material properties used in safety analyses could be obtained

  3. Detection of KatG Gen Mutation on Mycobacterium Tuberculosis by Means of PCR-Dot Blot Hybridization with 32P Labeled Oligonucleotide Probe Methods

    International Nuclear Information System (INIS)

    Maria Lina R; Budiman Bela; Andi Yasmon

    2009-01-01

    Handling and controlling of tuberculosis, a disease caused by Mycobacterium tuberculosis (MTB), is now complicated since there are many MTBs that are resistant against anti-tuberculosis drugs such as isoniazid. The drug resistance could occurred due to the inadequate and un-regular drug utilization that cause gene mutation of the drug target such as katG gene for isoniazid. The molecular biology techniques such as the PCR- dot blot hybridization with radioisotope ( 32 P) labeled oligonucleotide probe, has been reported as a technique that is more sensitive and rapid for detection of gene mutations related with drug resistances. Hence, the aim of this study was to apply the PCR- dot blot hybridization technique using 32 P labeled oligonucleotide probe for detection of single mutation at codon 315 of katG gene of MTBs that rise the isoniazid resistance. In this study, we used 89 sputum specimens and a standard MTB (MTB H 37 RV) as a control. DNA extractions were performed by the BOOM method and the phenol chloroform for sputum samples and standard MTB, respectively. Primers used for PCR technique were Pt8 and Pt9 and RTB59 and RTB36 for detecting tuberculosis causing Mycobacterium and the existence of katG gene, respectively. Both of the primers are specific for IS6110 region and katG gene, respectively. PCR products were detected by an agarose gel electrophoresis technique. Dot blot hybridization with 32 P-oligonucleotide probe 315mu was performed to detect mutation at codon 315 of tested samples. Results of the PCR using primer Pt8 and Pt9 showed that all sputum specimens had positive results. Mutation detection by PCR- dot blot hybridization with 32 P-oligonucleotide probe 315mu, revealed that 11 of 89 tested samples had a mutation at their codon 315 of katG gene. Based upon these results, it is concluded that PCR-dot blot hybridization with 32 P-oligonucleotide probe is a technique that is rapid and highly specific and sensitive for detection of mutation at codon

  4. Fabrication of CdS/CdTe-Based Thin Film Solar Cells Using an Electrochemical Technique

    Directory of Open Access Journals (Sweden)

    I. M. Dharmadasa

    2014-06-01

    Full Text Available Thin film solar cells based on cadmium telluride (CdTe are complex devices which have great potential for achieving high conversion efficiencies. Lack of understanding in materials issues and device physics slows down the rapid progress of these devices. This paper combines relevant results from the literature with new results from a research programme based on electro-plated CdS and CdTe. A wide range of analytical techniques was used to investigate the materials and device structures. It has been experimentally found that n-, i- and p-type CdTe can be grown easily by electroplating. These material layers consist of nano- and micro-rod type or columnar type grains, growing normal to the substrate. Stoichiometric materials exhibit the highest crystallinity and resistivity, and layers grown closer to these conditions show n → p or p → n conversion upon heat treatment. The general trend of CdCl2 treatment is to gradually change the CdTe material’s n-type electrical property towards i-type or p-type conduction. This work also identifies a rapid structural transition of CdTe layer at 385 ± 5 °C and a slow structural transition at higher temperatures when annealed or grown at high temperature. The second transition occurs after 430 °C and requires more work to understand this gradual transition. This work also identifies the existence of two different solar cell configurations for CdS/CdTe which creates a complex situation. Finally, the paper presents the way forward with next generation CdTe-based solar cells utilising low-cost materials in their columnar nature in graded bandgap structures. These devices could absorb UV, visible and IR radiation from the solar spectrum and combine impact ionisation and impurity photovoltaic (PV effect as well as making use of IR photons from the surroundings when fully optimised.

  5. [Clinical manifestation of Lyme borreliosis in children with positive and negatiwe western blot results].

    Science.gov (United States)

    Ołdak, Elzbieta; Rozkiewicz, Doroto; Sulik, Artur

    2008-01-01

    In the afforested area of North-Eastern Poland the risk of Borrelia burgdorferi infection seems to be higher compared to the other regions. Because of unspecific clinical manifestation of Lyme borreliosis in children the positive ELISA IgM results should be confirmed with Western blot IgM tests. Retrospective analysis of clinical signs and symptoms of Lyme borreliosis in children with positive ELISA IgM and positive Western blot IgM results and in children with positive ELISA IgM and negative Western blot IgM results. The study included 20 children reactive with ELISA IgM (Bellco Biomedica, Austria), hospitalized in Pediatric Infectious Diseases Clinic in 2007 due to probable diagnosis of Lyme disease. All children were tested with B. burgdorferi Western blot IgM and/or IgG assay (DRG, Diagnostics, Germany) as a second-step diagnosis. In 10 (50% females, 50% males) out of 20 children the results were positive (borreliosis) and in other 10 (80% females, 20% males) the results were negative (controls). In both groups of patients the retrospective analysis of signs and symptoms was done. The most often clinical manifestation of Lyme borreliosis in children was neuroborreliosis. Children presented Lyme meningitis (30%), facial nerve palsy (10%) and chronic or recurrent headaches (40%), associated with vertigo (20%), weakness (30%), fever (40%), and fatigue syndrome (30%). One patient presented Lyme arthritis. Children of control group presented with unspecific symptoms like isolated headaches (40%), arthralgias (70%), myalgias (10%) and abdomen pain (20%) (1) The most frequent clinical presentation of Lyme borreliosis in analyzed children was neuroborreliosis; (2) Isolated arthralgias in children reactive with B. burgdorferi ELISA IgM need to be confirmed with Western blot assay before implementing the antibiotic therapy.

  6. Diagnosis of Giardia duodenalis Infection using Dot Blot in Comparison with Microscopy.

    Science.gov (United States)

    Yazdani, Hajar; Sharafi, Seyedeh Maryam; Yousefi, Hoseynali; Hadipur, Mahbobeh; Sepahvand, Akram; Darani, Hossein Yousofi

    2016-01-01

    Giardia duodenalis is an intestinal flagellate parasite which spreads all over the world and is considered as a health problem in the most rural and low sanitation areas. Many diagnostic tests have been developed for the detection of Giardia parasite in stool samples but all of them have some disadvantages such as lack of sensitivity and specificity. In search for a simple and accurate test, diagnosis of Giardia infection using dot blot method has been investigated in this work. In this descriptive study, 30 stool samples which their infection with Giardia were confirmed by direct examination and formalin ether considered as case group. Thirty stool samples without Giardia infection according to formalin ether examination were also considered as a control group. Giardia cysts were isolated from the stool samples using sucrose method. In order to raise antiserum against Giardia cysts, the purified cysts were then sonicated and injected to a rabbit. A mono specific antiserum against the 66KDa band of Giardia cyst antigen was also prepared. The two antisera were used in the dot blot test. Finally, the sensitivity and specificity of the dot-blot method were estimated by considering formalin ether as the gold standard. When Poly specific antiserum was used, the sensitivity and specificity of the dot blot for detection of Giardia infection were 77% and 64% respectively. However the sensitivity and specificity of this assay were 97% and 64% respectively when monospecific antiserum was used. It seems that dot blot is an easy method for the diagnosis of Giardia especially in the rural areas. However more work is recommended for further development of this test.

  7. Measurement of the viability of stored red cells by the single-isotope technique using 51Cr. Analysis of validity

    International Nuclear Information System (INIS)

    Beutler, E.; West, C.

    1984-01-01

    A single-isotope 51 Cr method often is used to evaluate the viability of stored red cells. In this technique, the red cell mass is measured by back-extrapolation to time zero (t0) of the radioactivity of the blood between 5 and 20 minutes after infusion of the sample. If there is early destruction of stored cells, this method provides an overestimate of the red cell mass and, hence, of the viability of the stored cells. Freshly drawn red cells from normal donors were labeled with /sup 99m/Tc, and cells from the same donor which had been stored in citrate-phosphate-dextrose-adenine-one (CPDA-1) for periods ranging from 7 to 49 days were labeled with 51 Cr. A comparison of the ''true red cell mass'' as determined with /sup 99m/Tc with the back-extrapolated red cell mass from stored 51 Cr-labeled cells has made it possible to define the magnitude of error introduced by early loss of red cells. The overestimation of red cell mass and viability was diminished if only the 51 Cr radioactivity between 5 and 15 minutes after infusion was used in back-extrapolating to t0. The degree of overestimation of red cell mass was greatest when the red cell viability had declined to very low levels. However, in the entire range of 10 to 80 percent viability, the overestimate of viability was usually less than 4 percent. The overestimate of viability proved to be quite similar for all samples and may be taken into account when using the single-isotope technique for measurement of red cell viability

  8. Detection of human cytomegalovirus by slot-blot hybridization assay employing oligo-primed 32P-labelled probe

    International Nuclear Information System (INIS)

    Agha, S.A.; Coleman, J.C.; Selwyn, S.; Mahmound, L.A.; Abd-Elaal, A.M.; Archard, L.C.

    1988-01-01

    A 32 P-labelled Hind III-0 DNA fragment (nine Kilobases; Kb) from human cytomegalovirus AD-169 (HCMV) was used in slot-blot hybridization assay for the detection of HCMV in clinical samples. The results obtained with DNA hybridization assay (DNA HA) were compared with virus isolation using conventional tube cell culture (CTC) and centrifugation vial culture (CVC), immunofluorescence (IF), and complement fixation test (CFT). Of 15 CTC-positive samples, 13 were positive with DNA HA (sensitivity 86.7%). Also, 14 additional samples were DNA HA-positive but CTC-negative. CVC and/or IF confirmed the diagnosis in nine of 14; the remaining five samples were from three patients who showed fourfold rising antibody titre by CFT. Although DNA HA using 32 P-labelled probes is relatively cumbersome and expensive, it is a valuable test for quantitation of viral shedding in patients with HCMV infections who may benefit from antiviral therapy

  9. Investigation of CPD and HMDS Sample Preparation Techniques for Cervical Cells in Developing Computer-Aided Screening System Based on FE-SEM/EDX

    Science.gov (United States)

    Ng, Siew Cheok; Abu Osman, Noor Azuan

    2014-01-01

    This paper investigated the effects of critical-point drying (CPD) and hexamethyldisilazane (HMDS) sample preparation techniques for cervical cells on field emission scanning electron microscopy and energy dispersive X-ray (FE-SEM/EDX). We investigated the visualization of cervical cell image and elemental distribution on the cervical cell for two techniques of sample preparation. Using FE-SEM/EDX, the cervical cell images are captured and the cell element compositions are extracted for both sample preparation techniques. Cervical cell image quality, elemental composition, and processing time are considered for comparison of performances. Qualitatively, FE-SEM image based on HMDS preparation technique has better image quality than CPD technique in terms of degree of spread cell on the specimen and morphologic signs of cell deteriorations (i.e., existence of plate and pellet drying artifacts and membrane blebs). Quantitatively, with mapping and line scanning EDX analysis, carbon and oxygen element compositions in HMDS technique were higher than the CPD technique in terms of weight percentages. The HMDS technique has shorter processing time than the CPD technique. The results indicate that FE-SEM imaging, elemental composition, and processing time for sample preparation with the HMDS technique were better than CPD technique for cervical cell preparation technique for developing computer-aided screening system. PMID:25610902

  10. In Vitro Growth of Human Keratinocytes and Oral Cancer Cells into Microtissues: An Aerosol-Based Microencapsulation Technique

    Directory of Open Access Journals (Sweden)

    Wai Yean Leong

    2017-05-01

    Full Text Available Cells encapsulation is a micro-technology widely applied in cell and tissue research, tissue transplantation, and regenerative medicine. In this paper, we proposed a growth of microtissue model for the human keratinocytes (HaCaT cell line and an oral squamous cell carcinoma (OSCC cell line (ORL-48 based on a simple aerosol microencapsulation technique. At an extrusion rate of 20 μL/min and air flow rate of 0.3 L/min programmed in the aerosol system, HaCaT and ORL-48 cells in alginate microcapsules were encapsulated in microcapsules with a diameter ranging from 200 to 300 μm. Both cell lines were successfully grown into microtissues in the microcapsules of alginate within 16 days of culture. The microtissues were characterized by using a live/dead cell viability assay, field emission-scanning electron microscopy (FE-SEM, fluorescence staining, and cell re-plating experiments. The microtissues of both cell types were viable after being extracted from the alginate membrane using alginate lyase. However, the microtissues of HaCaT and ORL-48 demonstrated differences in both nucleus size and morphology. The microtissues with re-associated cells in spheroids are potentially useful as a cell model for pharmacological studies.

  11. A double labeling technique for performing immunocytochemistry and in situ hybridization in virus infected cell cultures and tissues

    International Nuclear Information System (INIS)

    Gendelman, H.E.; Moench, T.R.; Narayan, O.; Griffin, D.E.; Clements, J.E.

    1985-01-01

    This report describes a combined immunocytochemical and in situ hybridization procedure which allows visualization of cellular or viral antigens and viral RNA in the same cell. Cultures infected with visna or measles virus were fixed in periodate-lysine-paraformaldehyde-glutaraldehyde, stained by the avidin-biotin-peroxidase technique using antibodies to viral or cellular proteins and then incubated with radiolabeled specific DNA probes (in situ hybridization). This technique provides a new approach to the study of viral pathogenesis by: (1) identifying the types of cells which are infected in the host and (2) identifying points of blockade in the virus life cycle during persistent infections. (Auth.)

  12. [Evaluation of the IgG anti-Toxoplasma response and its avidity by western-blot in HIV-patients].

    Science.gov (United States)

    Cristina Sarmiento, María; Gómez Marín, Jorge Enrique; Castaño Osorio, Jhon Carlos

    2005-01-01

    The IgG anti-Toxoplasma western blot technique was used in 25 HIV-cases and 8 control sera from patients without HIV infection aimed at evaluating the humoral response in these patients. They were divided into 3 groups: 14 HIV positive cases with cerebral toxoplasmosis and IgG anti-Toxoplasma serological titers, 11 HIV positive cases without cerebral toxoplasmosis and with IgG anti-Toxoplasma titers, and 8 HIV negative patients with IgG anti-Toxoplasma titers. It was found that the higher the IgG anti-Toxoplasma serum titers are, the greater the number of bands in the western-blot is. The intensity of the bands measured by densitometry varied significantly for proteins of 66 and 31 kDa. According to the results, these proteins are of interest to evaluate their role in the reactivation of toxoplasmosis in HIV patients.

  13. DISKRIMINASI KELAMIN PADA IKAN TUNA SIRIP KUNING, Yellowfin tuna MENGGUNAKAN ANALISIS DOT BLOT DAN ELISA

    Directory of Open Access Journals (Sweden)

    Gusti Ngurah Permana

    2014-08-01

    Full Text Available Pemahaman tentang penentuan jenis kelamin dalam populasi induk merupakan hal yang sangat penting bagi keberhasilan program pembenihan. Pengukuran reaksi antibodi dan aktivitas hormon testosterone, serta estradiol adalah metode dengan potensi yang secara akurat dapat menentukan jenis kelamin ikan tanpa mematikan ikan. Tujuan penelitian ini adalah untuk mengetahui akurasi metode dot blot dan ELISA dengan 11-ketotestorsterone (11-KT yang tersedia secara komersial EIA-kit untuk membedakan jenis kelamin ikan tuna sirip kuning. Hasil analisis menunjukkan bahwa metode dot blot menghasilkan ekspresi vitelogenin tampak jelas pada individu betina dan efek plasma terlihat transparan, jika dibandingkan dengan individu jantan. Interpretasi dari metode ini memerlukan pengalaman dan keahlian dalam akurasi pembacaan hasil. Aktivitas hormon 11-KT dengan sampel klip sirip dan plasma memberikan hasil yang baik dengan aktivitas hormon terlihat jelas.

  14. Total protein or high-abundance protein: Which offers the best loading control for Western blotting?

    Science.gov (United States)

    Thacker, Jonathan S; Yeung, Derrick H; Staines, W Richard; Mielke, John G

    2016-03-01

    Western blotting routinely involves a control for variability in the amount of protein across immunoblot lanes. Normalizing a target signal to one found for an abundantly expressed protein is widely regarded as a reliable loading control; however, this approach is being increasingly questioned. As a result, we compared blotting for two high-abundance proteins (actin and glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) and two total protein membrane staining methods (Ponceau and Coomassie Brilliant Blue) to determine the best control for loading variability. We found that Ponceau staining optimally balanced accuracy and precision, and we suggest that this approach be considered as an alternative to normalizing with a high-abundance protein. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Nanogold Immunodetection Detection Systems for the Identification of Autoantigens by Western Blotting.

    Science.gov (United States)

    Moore, Jacen S; Scofield, R Hal

    2015-01-01

    Nanogold-conjugated immunodetection systems are now widely and commercially available for use in a number of research applications including electron microscopy, light microscopy, and western blotting. Nanogold clusters are small, uniform in size, and stable, unlike gold colloids historically used in protein detection. Covalent linkage of nanogold particles to secondary antibodies prevents dissociation of the gold particles during the staining process, making protein detection reliable, antigen specific, and highly sensitive. Nanogold labeling is extremely versatile and can be used in conjunction with other staining methodologies including Alexa Fluor immunofluorescence detection to perform coupled staining procedures. Silver enhancement increases the limits of sensitivity for nanogold staining, thus improving detection signals for antigens with reduced expression levels. Herein, we describe the use of nanogold-silver detection as an immunodetection system for standard western blotting of autoantigens.

  16. Western blot detection of brain phosphoproteins after performing Laser Microdissection and Pressure Catapulting (LMPC).

    Science.gov (United States)

    Maitre, Marlène; Roullot-Lacarrière, Valérie; Piazza, Pier Vincenzo; Revest, Jean-Michel

    2011-06-15

    The Central Nervous System (CNS) is constituted of complex and specific anatomical regions that cluster together and interact with each other with the ultimate objective of receiving and delivering information. This information is characterized by selective biochemical changes that happen within specific brain sub-regions. Most of these changes involve a dynamic balance between kinase and phosphatase activities. The fine-tuning of this kinase/phosphatase balance is thus critical for neuronal adaptation, transition to long-term responses and higher brain functions including specific behaviors. Data emerging from several biological systems may suggest that disruption of this dynamic cell signaling balance within specific brain sub-regions leads to behavioral impairments. Therefore, accurate and powerful techniques are required to study global changes in protein expression levels and protein activities in specific groups of cells. Laser-based systems for tissue microdissection represent a method of choice enabling more accurate proteomic profiling. The goal of this study was to develop a methodological approach using Laser Microdissection and Pressure Catapulting (LMPC) technology combined with an immunoblotting technique in order to specifically detect the expression of phosphoproteins in particular small brain areas. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Investigation of anti-Relaxation coatings for alkali-metal vapor cells using surface science techniques

    Energy Technology Data Exchange (ETDEWEB)

    Seltzer, S. J.; Michalak, D. J.; Donaldson, M. H.; Balabas, M. V.; Barber, S. K.; Bernasek, S. L.; Bouchiat, M.-A.; Hexemer, A.; Hibberd, A. M.; Jackson Kimball, D. F.; Jaye, C.; Karaulanov, T.; Narducci, F. A.; Rangwala, S. A.; Robinson, H. G.; Shmakov, A. K.; Voronov, D. L.; Yashchuk, V. V.; Pines, A.; Budker, D.

    2010-10-11

    Many technologies based on cells containing alkali-metal atomic vapor benefit from the use of antirelaxation surface coatings in order to preserve atomic spin polarization. In particular, paraffin has been used for this purpose for several decades and has been demonstrated to allow an atom to experience up to 10?000 collisions with the walls of its container without depolarizing, but the details of its operation remain poorly understood. We apply modern surface and bulk techniques to the study of paraffin coatings in order to characterize the properties that enable the effective preservation of alkali spin polarization. These methods include Fourier transform infrared spectroscopy, differential scanning calorimetry, atomic force microscopy, near-edge x-ray absorption fine structure spectroscopy, and x-ray photoelectron spectroscopy. We also compare the light-induced atomic desorption yields of several different paraffin materials. Experimental results include the determination that crystallinity of the coating material is unnecessary, and the detection of C=C double bonds present within a particular class of effective paraffin coatings. Further study should lead to the development of more robust paraffin antirelaxation coatings, as well as the design and synthesis of new classes of coating materials.

  18. The observation report of red blood cell morphology in Thailand teenager by using data mining technique.

    Science.gov (United States)

    Saichanma, Sarawut; Chulsomlee, Sucha; Thangrua, Nonthaya; Pongsuchart, Pornsuri; Sanmun, Duangmanee

    2014-01-01

    It is undeniable that laboratory information is important in healthcare in many ways such as management, planning, and quality improvement. Laboratory diagnosis and laboratory results from each patient are organized from every treatment. These data are useful for retrospective study exploring a relationship between laboratory results and diseases. By doing so, it increases efficiency in diagnosis and quality in laboratory report. Our study will utilize J48 algorithm, a data mining technique to predict abnormality in peripheral blood smear from 1,362 students by using 13 data set of hematological parameters gathered from automated blood cell counter. We found that the decision tree which is created from the algorithm can be used as a practical guideline for RBC morphology prediction by using 4 hematological parameters (MCV, MCH, Hct, and RBC). The average prediction of RBC morphology has true positive, false positive, precision, recall, and accuracy of 0.940, 0.050, 0.945, 0.940, and 0.943, respectively. A newly found paradigm in managing medical laboratory information will be helpful in organizing, researching, and assisting correlation in multiple disciplinary other than medical science which will eventually lead to an improvement in quality of test results and more accurate diagnosis.

  19. The Observation Report of Red Blood Cell Morphology in Thailand Teenager by Using Data Mining Technique

    Directory of Open Access Journals (Sweden)

    Sarawut Saichanma

    2014-01-01

    Full Text Available It is undeniable that laboratory information is important in healthcare in many ways such as management, planning, and quality improvement. Laboratory diagnosis and laboratory results from each patient are organized from every treatment. These data are useful for retrospective study exploring a relationship between laboratory results and diseases. By doing so, it increases efficiency in diagnosis and quality in laboratory report. Our study will utilize J48 algorithm, a data mining technique to predict abnormality in peripheral blood smear from 1,362 students by using 13 data set of hematological parameters gathered from automated blood cell counter. We found that the decision tree which is created from the algorithm can be used as a practical guideline for RBC morphology prediction by using 4 hematological parameters (MCV, MCH, Hct, and RBC. The average prediction of RBC morphology has true positive, false positive, precision, recall, and accuracy of 0.940, 0.050, 0.945, 0.940, and 0.943, respectively. A newly found paradigm in managing medical laboratory information will be helpful in organizing, researching, and assisting correlation in multiple disciplinary other than medical science which will eventually lead to an improvement in quality of test results and more accurate diagnosis.

  20. Metastatic Renal Cell Cancer to Thyroid Diagnosed by Endoscopic Ultrasound Guided Fine Needle Aspiration Technique

    Directory of Open Access Journals (Sweden)

    Yousef Abdel-Aziz

    2017-01-01

    Full Text Available Medical literature about the role of endoscopic ultrasound (EUS in identifying thyroid lesions is limited. We present a case of secondary thyroid cancer from renal cell carcinoma (RCC metastasis, diagnosed by thyroid EUS-fine needle aspiration (FNA approach that was done for staging of esophageal adenocarcinoma, in a patient with 11-year history of complete right nephrectomy for RCC. An 81-year-old female patient underwent EUS for the evaluation of a newly discovered distal esophageal cancer. A hypoechoic, round, and well-demarcated mass that measured 26.9 mm × 21.9 mm was noticed in the right lobe thyroid gland. Therefore FNA was performed. The cytological results were consistent with metastatic RCC. In conclusion, EUS-FNA of thyroid nodule is a feasible and safe technique that can be used to evaluate any suspicious thyroid nodule. This case emphasizes the importance of carefully examining the thyroid gland during routine upper esophageal EUS examinations in the presence of history of nonthyroidal cancer.

  1. Metastatic Renal Cell Cancer to Thyroid Diagnosed by Endoscopic Ultrasound Guided Fine Needle Aspiration Technique.

    Science.gov (United States)

    Abdel-Aziz, Yousef; Hammad, Tariq; Nawras, Mohamad; Abdulwahid, Hayder; Nawras, Ali

    2017-01-01

    Medical literature about the role of endoscopic ultrasound (EUS) in identifying thyroid lesions is limited. We present a case of secondary thyroid cancer from renal cell carcinoma (RCC) metastasis, diagnosed by thyroid EUS-fine needle aspiration (FNA) approach that was done for staging of esophageal adenocarcinoma, in a patient with 11-year history of complete right nephrectomy for RCC. An 81-year-old female patient underwent EUS for the evaluation of a newly discovered distal esophageal cancer. A hypoechoic, round, and well-demarcated mass that measured 26.9 mm × 21.9 mm was noticed in the right lobe thyroid gland. Therefore FNA was performed. The cytological results were consistent with metastatic RCC. In conclusion, EUS-FNA of thyroid nodule is a feasible and safe technique that can be used to evaluate any suspicious thyroid nodule. This case emphasizes the importance of carefully examining the thyroid gland during routine upper esophageal EUS examinations in the presence of history of nonthyroidal cancer.

  2. Antibody responses to Borrelia burgdorferi detected by western blot vary geographically in Canada

    OpenAIRE

    Ogden, Nicholas H.; Arsenault, Julie; Hatchette, Todd F.; Mechai, Samir; Lindsay, L. Robbin

    2017-01-01

    Lyme disease is emerging in eastern and central Canada, and most cases are diagnosed using the two-tier serological test (Enzyme Immuno Assay [EIA] followed by Western blot [WB]). Simplification of this algorithm would be advantageous unless it impacts test performance. In this study, accuracy of individual proteins of the IgG WB algorithm in predicting the overall test result in samples from Canadians was assessed. Because Borrelia burgdorferi strains vary geographically in Canada, geographi...

  3. Development of a cell culture surface conversion technique using alginate thin film for evaluating effect upon cellular differentiation

    International Nuclear Information System (INIS)

    Nakashima, Y.; Tsusu, K.; Minami, K.; Nakanishi, Y.

    2014-01-01

    Here, we sought to develop a cell culture surface conversion technique that would not damage living cells. An alginate thin film, formed on a glass plate by spin coating of sodium alginate solution and dipping into calcium chloride solution, was used to inhibit adhesion of cells. The film could be removed by ethylenediaminetetraacetate (EDTA) at any time during cell culture, permitting observation of cellular responses to conversion of the culture surface in real time. Additionally, we demonstrated the validity of the alginate thin film coating method and the performance of the film. The thickness of the alginate thin film was controlled by varying the rotation speed during spin coating. Moreover, the alginate thin film completely inhibited the adhesion of cultured cells to the culture surface, irrespective of the thickness of the film. When the alginate thin film was removed from the culture surface by EDTA, the cultured cells adhered to the culture surface, and their morphology changed. Finally, we achieved effective differentiation of C2C12 myoblasts into myotube cells by cell culture on the convertible culture surface, demonstrating the utility of our novel technique

  4. Development of a cell culture surface conversion technique using alginate thin film for evaluating effect upon cellular differentiation.

    Science.gov (United States)

    Nakashima, Y; Tsusu, K; Minami, K; Nakanishi, Y

    2014-06-01

    Here, we sought to develop a cell culture surface conversion technique that would not damage living cells. An alginate thin film, formed on a glass plate by spin coating of sodium alginate solution and dipping into calcium chloride solution, was used to inhibit adhesion of cells. The film could be removed by ethylenediaminetetraacetate (EDTA) at any time during cell culture, permitting observation of cellular responses to conversion of the culture surface in real time. Additionally, we demonstrated the validity of the alginate thin film coating method and the performance of the film. The thickness of the alginate thin film was controlled by varying the rotation speed during spin coating. Moreover, the alginate thin film completely inhibited the adhesion of cultured cells to the culture surface, irrespective of the thickness of the film. When the alginate thin film was removed from the culture surface by EDTA, the cultured cells adhered to the culture surface, and their morphology changed. Finally, we achieved effective differentiation of C2C12 myoblasts into myotube cells by cell culture on the convertible culture surface, demonstrating the utility of our novel technique.

  5. Direct Blue 71 staining as a destaining-free alternative loading control method for Western blotting.

    Science.gov (United States)

    Zeng, Li; Guo, Jing; Xu, Hong-Bo; Huang, Rongzhong; Shao, Weihua; Yang, Liu; Wang, Mingju; Chen, Jianjun; Xie, Peng

    2013-08-01

    In Western blotting, a suitable loading control is indispensable for correcting errors in the total amount of loaded protein. Immunodetection of housekeeping proteins and total protein staining have traditionally been used as loading control methods. Direct Blue 71 (DB71) staining-a novel, sensitive, dye-binding staining method compatible with immunodetection-may offer advantages over these traditional loading control methods. Three common neuroscientific samples (human plasma, human oligodendrocytes, and rat brain) were employed to assess DB71 staining as a loading control method for Western blotting. DB71, CBB, one traditional housekeeping protein, and one protein of interest were comparatively assessed for reliability and repeatability and linear dynamic range over 2.5-40 μg of protein loaded. DB71's effect on the reliability and repeatability and linear dynamic range of immunoreaction were also assessed. Across all three sample types, DB71 was either equivalent or superior to CBB and housekeeping protein-based methods in terms of reliability and repeatability and linear dynamic range. Across all three sample types, DB71 staining did not impair the reliability and repeatability or linear dynamic range of immunoreaction. Our results demonstrate that the DB71 staining can be used as a destaining-free alternative loading control method for Western blotting. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Banding pattern indicative of echinococcosis in a commercial cysticercosis western blot

    Directory of Open Access Journals (Sweden)

    Tappe D

    2009-09-01

    Full Text Available Abstract Objective A commercial cysticercosis Western blot was evaluated for serological cross-reactivity of sera from patients with alveolar (AE and cystic echinococcosis (CE. Methods A total of 161 sera were examined, including 31 sera from AE-patients, 11 sera from CE-patients, 9 sera from patients with other parasitic diseases and 109 sera from patients with unrelated medical conditions. All AE-and CE-sera were also examined by the echinococcosis Western blot. Results More sera from patients with AE than with CE showed cross-reactivity in the form of ladder-like patterns ("Mikado aspect" and untypical bands at 6-8 kDa (71% and 77.4% versus 27.3% and 45.5%, respectively. In contrast, triplets of bands in the area above 50 kDa and between 24 and 39-42 kDa were more frequent in CE than in AE sera. The fuzzy band at 50-55 kDa typical for cysticercosis was absent in all AE and CE sera. Conclusions Atypical banding patterns in the cysticercosis Western blot should raise the suspicion of a metacestode infection different from Taenia solium, i.e. Echinococcus multilocularis or E. granulosus, especially when the Mikado aspect and an altered 6-8 kDa band is visible in the absence of a fuzzy 50-55 kDa band.

  7. Diagnosis of paracoccidioidomycosis by a dot blot assay using a recombinant Paracoccidioides brasiliensis p27 protein.

    Science.gov (United States)

    Correa, M M; Bedoya, A M; Guerrero, M P; Méndez, J; Restrepo, A; McEwen, J G

    2007-01-01

    A variety of immunological methods have proven useful for Paracoccidioidomycosis (PCM) diagnosis; however, they are often time consuming and many lack sensitivity and specificity, partially attributed to the use of crude antigens, which give cross reactivity. Until now, attempts to clone and express Paracoccidioides brasiliensis immunodominant antigens have presented difficulties of process and problems of cost. In an attempt to obtain a more rapid, sensitive, and specific test for PCM diagnosis, we subcloned the P. brasiliensis p27 gene and used the recombinant protein as the antigen in dot blot assays to evaluate its usefulness in paracoccidioidomicosis diagnosis. The development of an optimised procedure for p27 recombinant protein purification and production led to an easier and less expensive process than the one previously used in our laboratory and allowed the availability of enough purified protein for its evaluation as the antigen in the dot blot assays. In these assays, antibodies present in ten serum samples from seven patients with PCM recognised the recombinant protein showing a sensitivity of 100% with a specificity of 98%. These results confirm the value of the 27-kDa recombinant antigen in the serodiagnosis of paracoccidioidomycosis and that the dot blot format is an alternative to the immunoenzymatic assay procedure.

  8. Positive IgG Western Blot for Borrelia burgdorferi in Colombia

    Directory of Open Access Journals (Sweden)

    Palacios Ricardo

    1999-01-01

    Full Text Available In order to evaluate the presence of specific IgG antibodies to Borrelia burgdorferi in patients with clinical manifestations associated with Lyme borreliosis in Cali, Colombia, 20 serum samples from patients with dermatologic signs, one cerebrospinal fluid (CSF sample from a patient with chronic neurologic and arthritic manifestations, and twelve serum samples from individuals without clinical signs associated with Lyme borreliosis were analyzed by IgG Western blot. The results were interpreted following the recommendations of the Centers for Diseases Control and Prevention (CDC for IgG Western blots. Four samples fulfilled the CDC criteria: two serum specimens from patients with morphea (localized scleroderma, the CSF from the patient with neurologic and arthritic manifestations, and one of the controls. Interpretation of positive serology for Lyme disease in non-endemic countries must be cautious. However these results suggest that the putative "Lyme-like" disease may correlate with positivity on Western blots, thus raising the possibility that a spirochete genospecies distinct from B. burgdorferi sensu stricto, or a Borrelia species other than B. burgdorferi sensu lato is the causative agent. Future work will focus on a survey of the local tick and rodent population for evidence of spirochete species that could be incriminated as the etiologic agent.

  9. An electrorotation technique for measuring the dielectric properties of cells with simultaneous use of negative quadrupolar dielectrophoresis and electrorotation.

    Science.gov (United States)

    Han, Song-I; Joo, Young-Don; Han, Ki-Ho

    2013-03-07

    This paper presents an effective electrorotation technique for measuring the dielectric properties of cells using a superposed electrical signal, which can simultaneously generate negative quadrupolar dielectrophoretic (nQDEP) force and electrorotational (ROT) torque. The proposed technique involves a three-dimensional (3D) octode, which includes four electrodes arranged in a crisscross pattern on the top and bottom of a microchannel, respectively. A single cell was trapped in the center of the 3D octode by the nQDEP force and simultaneously rotated by the ROT torque. Using the proposed electrorotation technique, ROT spectra of human leukocyte subpopulations (T and B lymphocytes, granulocytes, and monocytes) and metastatic human breast (SkBr3) and lung (A549) cancer cell lines were accurately measured without any disturbance. Torque on the cells generated by the ROT signal was analyzed theoretically based on the single-shell dielectric model for the cells. Furthermore, the dielectric properties of the cells, such as area-specific membrane capacitance and cytoplasm conductivity, were extracted using the measured ROT spectra and the analyzed torque.

  10. A Historical Perspective on the Identification of Cell Types in Pancreatic Islets of Langerhans by Staining and Histochemical Techniques.

    Science.gov (United States)

    Baskin, Denis G

    2015-08-01

    Before the middle of the previous century, cell types of the pancreatic islets of Langerhans were identified primarily on the basis of their color reactions with histological dyes. At that time, the chemical basis for the staining properties of islet cells in relation to the identity, chemistry and structure of their hormones was not fully understood. Nevertheless, the definitive islet cell types that secrete glucagon, insulin, and somatostatin (A, B, and D cells, respectively) could reliably be differentiated from each other with staining protocols that involved variations of one or more tinctorial techniques, such as the Mallory-Heidenhain azan trichrome, chromium hematoxylin and phloxine, aldehyde fuchsin, and silver impregnation methods, which were popularly used until supplanted by immunohistochemical techniques. Before antibody-based staining methods, the most bona fide histochemical techniques for the identification of islet B cells were based on the detection of sulfhydryl and disulfide groups of insulin. The application of the classical islet tinctorial staining methods for pathophysiological studies and physiological experiments was fundamental to our understanding of islet architecture and the physiological roles of A and B cells in glucose regulation and diabetes. © The Author(s) 2015.

  11. Lorentz boosted frame simulation technique in Particle-in-cell methods

    Science.gov (United States)

    Yu, Peicheng

    In this dissertation, we systematically explore the use of a simulation method for modeling laser wakefield acceleration (LWFA) using the particle-in-cell (PIC) method, called the Lorentz boosted frame technique. In the lab frame the plasma length is typically four orders of magnitude larger than the laser pulse length. Using this technique, simulations are performed in a Lorentz boosted frame in which the plasma length, which is Lorentz contracted, and the laser length, which is Lorentz expanded, are now comparable. This technique has the potential to reduce the computational needs of a LWFA simulation by more than four orders of magnitude, and is useful if there is no or negligible reflection of the laser in the lab frame. To realize the potential of Lorentz boosted frame simulations for LWFA, the first obstacle to overcome is a robust and violent numerical instability, called the Numerical Cerenkov Instability (NCI), that leads to unphysical energy exchange between relativistically drifting particles and their radiation. This leads to unphysical noise that dwarfs the real physical processes. In this dissertation, we first present a theoretical analysis of this instability, and show that the NCI comes from the unphysical coupling of the electromagnetic (EM) modes and Langmuir modes (both main and aliasing) of the relativistically drifting plasma. We then discuss the methods to eliminate them. However, the use of FFTs can lead to parallel scalability issues when there are many more cells along the drifting direction than in the transverse direction(s). We then describe an algorithm that has the potential to address this issue by using a higher order finite difference operator for the derivative in the plasma drifting direction, while using the standard second order operators in the transverse direction(s). The NCI for this algorithm is analyzed, and it is shown that the NCI can be eliminated using the same strategies that were used for the hybrid FFT

  12. Application of laser tweezers Raman spectroscopy techniques to the monitoring of single cell response to stimuli

    Science.gov (United States)

    Chan, James W.; Liu, Rui; Matthews, Dennis L.

    2012-06-01

    Laser tweezers Raman spectroscopy (LTRS) combines optical trapping with micro-Raman spectroscopy to enable label-free biochemical analysis of individual cells and small biological particles in suspension. The integration of the two technologies greatly simplifies the sample preparation and handling of suspension cells for spectroscopic analysis in physiologically meaningful conditions. In our group, LTRS has been used to study the effects of external perturbations, both chemical and mechanical, on the biochemistry of the cell. Single cell dynamics can be studied by performing longitudinal studies to continuously monitor the response of the cell as it interacts with its environment. The ability to carry out these measurements in-vitro makes LTRS an attractive tool for many biomedical applications. Here, we discuss the use of LTRS to study the response of cancer cells to chemotherapeutics and bacteria cells to antibiotics and show that the life cycle and apoptosis of the cells can be detected. These results show the promise of LTRS for drug discovery/screening, antibiotic susceptibility testing, and chemotherapy response monitoring applications. In separate experiments, we study the response of red blood cells to the mechanical forces imposed on the cell by the optical tweezers. A laser power dependent deoxygenation of the red blood cell in the single beam trap is reported. Normal, sickle cell, and fetal red blood cells have a different behavior that enables the discrimination of the cell types based on this mechanochemical response. These results show the potential utility of LTRS for diagnosing and studying red blood cell diseases.

  13. Materials and characterization techniques for high-temperature polymer electrolyte membrane fuel cells.

    Science.gov (United States)

    Zeis, Roswitha

    2015-01-01

    The performance of high-temperature polymer electrolyte membrane fuel cells (HT-PEMFC) is critically dependent on the selection of materials and optimization of individual components. A conventional high-temperature membrane electrode assembly (HT-MEA) primarily consists of a polybenzimidazole (PBI)-type membrane containing phosphoric acid and two gas diffusion electrodes (GDE), the anode and the cathode, attached to the two surfaces of the membrane. This review article provides a survey on the materials implemented in state-of-the-art HT-MEAs. These materials must meet extremely demanding requirements because of the severe operating conditions of HT-PEMFCs. They need to be electrochemically and thermally stable in highly acidic environment. The polymer membranes should exhibit high proton conductivity in low-hydration and even anhydrous states. Of special concern for phosphoric-acid-doped PBI-type membranes is the acid loss and management during operation. The slow oxygen reduction reaction in HT-PEMFCs remains a challenge. Phosphoric acid tends to adsorb onto the surface of the platinum catalyst and therefore hampers the reaction kinetics. Additionally, the binder material plays a key role in regulating the hydrophobicity and hydrophilicity of the catalyst layer. Subsequently, the binder controls the electrode-membrane interface that establishes the triple phase boundary between proton conductive electrolyte, electron conductive catalyst, and reactant gases. Moreover, the elevated operating temperatures promote carbon corrosion and therefore degrade the integrity of the catalyst support. These are only some examples how materials properties affect the stability and performance of HT-PEMFCs. For this reason, materials characterization techniques for HT-PEMFCs, either in situ or ex situ, are highly beneficial. Significant progress has recently been made in this field, which enables us to gain a better understanding of underlying processes occurring during fuel cell

  14. Protein multiplicity can lead to misconduct in western blotting and misinterpretation of immunohistochemical staining results, creating much conflicting data.

    Science.gov (United States)

    Liu, Xingde; Wang, Yiming; Yang, Wenxiu; Guan, Zhizhong; Yu, Wenfeng; Liao, D Joshua

    2016-11-01

    Western blotting (WB) and immunohistochemical staining (IHC) are common techniques for determining tissue protein expression. Both techniques require a primary antibody specific for the protein in question. WB data is band(s) on a membrane while IHC result is a staining on a tissue section. Most human genes are known to produce multiple protein isoforms; in agreement with that, multiple bands are often found on the WB membrane. However, a common but unspoken practice in WB is to cut away the extra band(s) and present for publication only the band of interest, which implies to the readers that only one form of protein is expressed and thus the data interpretation is straightforward. Similarly, few IHC studies discuss whether the antibody used is isoform-specific and whether the positive staining is derived from only one isoform. Currently, there is no reliable technique to determine the isoform-specificity of an antibody, especially for IHC. Therefore, cutting away extra band(s) on the membrane usually is a form of misconduct in WB, and a positive staining in IHC only indicates the presence of protein product(s) of the to-be-interrogated gene, and not necessarily the presence of the isoform of interest. We suggest that data of WB and IHC involving only one antibody should not be published and that relevant reports should discuss whether there may be protein multiplicity and whether the antibody used is isoform-specific. Hopefully, techniques will soon emerge that allow determination of not only the presence of protein products of genes but also the isoforms expressed. Copyright © 2016. Published by Elsevier GmbH.

  15. New views of the human NK cell immunological synapse: recent advances enabled by super- and high- resolution imaging techniques

    Directory of Open Access Journals (Sweden)

    Emily M. Mace

    2013-01-01

    Full Text Available Imaging technology has undergone rapid growth with the development of super resolution microscopy, which enables resolution below the diffraction barrier of light (~200 nm. In addition, new techniques for single molecule imaging are being added to the cell biologist’s arsenal. Immunologists have exploited these techniques to advance understanding of NK biology, particularly that of the immune synapse. The immune synapse’s relatively small size and complex architecture combined with its exquisitely controlled signaling milieu have made it a challenge to visualize. In this review we highlight and discuss new insights into NK cell immune synapse formation and regulation revealed by cutting edge imaging techniques, including super resolution microscopy and high resolution total internal reflection microscopy and Förster resonance energy transfer.

  16. Basal cell carcinoma of the outer nose: Overview on surgical techniques and analysis of 312 patients

    Directory of Open Access Journals (Sweden)

    Uwe Wollina

    2014-01-01

    Full Text Available Background: Basal cell carcinoma of the nose is common, with a potential of local recurrence and high-risk features. Materials and Methods: We provide a review on anatomy of the nose, tumour surgery and defect closure on the nose. We analysed our own patients with nasal BCC of a 24 months period. Results: We identified 321 patients with nasal BCC. There was a predominance of female patients of 1.2 to 1. The mean age was 74.8 years. Slow Mohs technique was employed for all tumours until 3D tumour-free margins were achieved. That resulted on average in 1.8 ± 0.7 Mohs stages. The most common histologic types were solitary (n = 182, morpheic (79, and micronodular (20, Perineural infiltration was evident in 56 tumours. Primary closure after mobilisation of soft tissue was possible in 105 BCCs. Advancement flaps were used in 91 tumours, rotation flaps in 47, transposition flaps in 34 tumours, and combined procedures in 6 cases. In 36 patients full-thickness skin grafting was performed. In two patients healing by second intention was preferred. Partial flap loss was seen in four patients (1.4%. All of them had significant underlying pathologies. None of the tumours treated showed a relapse during the observation time. However, this is a limitation of the present study since follow-up was on average only 10 months. Conclusions: BCCs of the nose are common. Only 3D-controlled micrographic surgery (Mohs or slow Mohs guarantee a high rate of complete tumour removal and a very low risk of recurrence.

  17. Establishment of Hepatocellular Cancer Induced Pluripotent Stem Cells Using a Reprogramming Technique.

    Science.gov (United States)

    Kim, Han Joon; Jeong, Jaemin; Park, Sunhoo; Jin, Young-Woo; Lee, Seung-Sook; Lee, Seung Bum; Choi, Dongho

    2017-03-15

    Cancer is known to be a disease by many factors. However, specific results of reprogramming by pluripotency-related transcription factors remain to be scarcely reported. Here, we verified potential effects of pluripotent-related genes in hepatocellular carcinoma cancer cells. To better understand reprogramming of cancer cells in different genetic backgrounds, we used four liver cancer cell lines representing different states of p53 (HepG2, Hep3B, Huh7 and PLC). Retroviral-mediated introduction of reprogramming related genes (KLF4, Oct4, Sox2, and Myc) was used to induce the expression of proteins related to a pluripotent status in liver cancer cells. Hep3B cells (null p53) exhibited a higher efficiency of reprogramming in comparison to the other liver cancer cell lines. The reprogrammed Hep3B cells acquired similar characteristics to pluripotent stem cells. However, loss of stemness in Hep3B-iPCs was detected during continual passage. We demonstrated that reprogramming was achieved in tumor cells through retroviral induction of genes associated with reprogramming. Interestingly, the reprogrammed pluripotent cancer cells (iPCs) were very different from original cancer cells in terms of colony shape and expressed markers. The induction of pluripotency of liver cancer cells correlated with the status of p53, suggesting that different expression level of p53 in cancer cells may affect their reprogramming.

  18. Evaluation of the Cell Proliferation Process of Ovarian Follicles in Hypothyroid Rats by Proliferation Cell Nuclear Antigen Immunohistochemical Technique

    Directory of Open Access Journals (Sweden)

    M. Moghaddam Dorafshani

    2012-10-01

    Full Text Available Introduction & Objective: The normal females reproductive function , needs hypothalamus-hypophysis-ovarian extensive hormonal messages. Primary hypothyroidism is characterized by reduced production and secretion of thyroid hormones. During follicular growth PCNA (Proliferating Cell Nuclear Antigen and cycklin D complex play an important role in regulating cell proliferation .This study aimed to determine the cell proliferation index and how this process changes induced by thyroid hormone decreased in rat ovarian follicles.Materials & Methods: In this experimental study, 20 Wistar female rats were divided into experimental and control groups. Experimental group was chemically thyroidectomized by administering propylthiouracil (PTU (500 mg per liter of drinking water. The control group received normal drinking water. After three weeks rats were killed and their ovaries dissected and fixed for the histological preparation. Cell proliferation was determined by PCNA and stereological methods were used for counting cells.Results: Cell proliferation index showed a significant decrease in the frequency of follicular growth from prenatal to graafian follicles in hypothyroidism groups(P0.05 . PCNA expression determined that Primary follicle growth begins earlier. Positive PCNA cells were not observed in primordial follicles of the groups.Conclusion: According to the results of our study, this hypothesis is raised that granulosa cells in growing follicles may be increased by follicle adjacent cells in ovarian stroma . Hormonal changes following the reduction of thyroid hormones may greatly affect the cell proliferation index and lead to faster follicle degeneration.(Sci J Hamadan Univ Med Sci 2012; 19 (3:5-15

  19. The liquid overlay technique is the key to formation of co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells.

    Science.gov (United States)

    Metzger, Wolfgang; Sossong, Daniela; Bächle, Annick; Pütz, Norbert; Wennemuth, Gunther; Pohlemann, Tim; Oberringer, Martin

    2011-09-01

    The 3-dimensional (3-D) culture of various cell types reflects the in vivo situation more precisely than 2-dimensional (2-D) cell culture techniques. Spheroids as 3-D cell constructs have been used in tumor research for a long time. They have also been used to study angiogenic mechanisms, which are essential for the success of many tissue-engineering approaches. Several methods of forming spheroids are known, but there is a lack of systematic studies evaluating the performance of these techniques. We evaluated the performance of the hanging drop technique, carboxymethyl cellulose technique and liquid overlay technique to form both mono- and co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells. The performance of the three techniques was evaluated in terms of rate of yield and reproducibility. The size of the generated spheroids was determined systematically. The liquid overlay technique was the most suitable for generating spheroids reproducibly. The rate of yield for this technique was between 60% and 100% for monoculture spheroids and 100% for co-culture spheroids. The size of the spheroids could be adjusted easily and precisely by varying the number of seeded cells organized in one spheroid. The formation of co-culture spheroids consisting of three different cell types was possible. Our results show that the most suitable technique for forming spheroids can vary from the chosen cell type, especially if primary cells are used. Co-culture spheroids consisting of three different cell types will be used to study angiogenic phenomena in further studies.

  20. Gene Profiling Technique to Accelerate Stem Cell Therapies for Eye Diseases

    Science.gov (United States)

    ... can be derived from a patient’s skin or blood cells, coaxed into other cell types (such as neurons or muscle), and in theory, re-implanted without causing immune rejection. To verify the identity of RPE made from ...

  1. An ultrafiltration technique for labeling red blood cells with Tc-99m

    International Nuclear Information System (INIS)

    Hendershott, L.R.; Gatson, R.C.; Ordway, F.S.; Ahmad, M.; Saint Louis Univ., MO; Saint Louis Univ., MO

    1979-01-01

    This method automates the preparation of autologous Tc-99m labeled red blood cells utilizing the Amicon on-line column eluate concentrator to separate the plasma from the red blood cells. The red blood cells were pre-tinned with stannous diphosphonate and continuously recirculated over a 0.6 μ filter until all of the plasma was removed and the red blood cells remained suspended in a solution of 0.9% sodium chloride. Once the plasma has been removed the red blood cells are incubated with Tc-99m pertechnetate. The above Tc-99m red blood cells were compared to Tc-99m red blood cells produced in a similar manner except that centrifugation was used to separate the red blood cells from the plasma. Both preparations had a tagging efficiency of 98% or greater and rat distribution studies demonstrate that both preparations are equally stable as an in vivo intravascular agent. (orig.) [de

  2. Applying a Commercial Atomic Force Microscope for Scanning Near-field Optical Microscopy Techniques and Investigation of Cell-cell Signaling

    Science.gov (United States)

    Lopez Ayon, Gabriela Monserratt

    The field of research of this thesis is Condensed Matter Physics applied to Biology. Specifically it describes the development of different Atomic Force Microscopy techniques and tools towards the study of living cells in physiological solution. Particular interest is put into the understanding of the influence of noise in the determination of ordered liquid layers above a mica surface - as work towards the study of the role of water and ions in biological processes - and the influence of "diving bell" to boost the Q factor and allow stable imaging and force spectroscopy with tips based on Scanning Near-field Optical Microscopy [LeDue, 2010 and LeDue, 2008]. By combining SNOM techniques as a local illumination method (and thus avoiding photo bleaching of individual molecules) and high resolution AFM techniques we will be able to investigate mechano-transduction and associated signaling in living cells and individual proteins.

  3. A no film slot blot for the detection of developing P. falciparum oocysts in mosquitoes.

    Directory of Open Access Journals (Sweden)

    Bryan Grabias

    Full Text Available Non-microscopy-based assays for sensitive and rapid detection of Plasmodium infection in mosquitoes are needed to allow rapid and high throughput measurement of transmission intensity and malaria control program effectiveness. Here, we report on a modified enhanced chemiluminescence-based slot blot assay for detection of Plasmodium falciparum (Pf circumsporozite protein (PfCSP expressed on parasite oocysts developing inside the mosquito midgut. This modified assay has several novel features that include eliminating the need for exposure to autoradiography (AR film, as well as utilizing a novel high affinity anti-CSP antibody, and optimizing assay procedures resulting in significant reduction in the time required to perform the assay. The chemiluminescent signal for the detection of PfCSP in mosquito samples was captured digitally utilizing the C-Digit blot scanner that, allowed the detection of 0.01 pg of recombinant P. falciparum CSP and as few as 0.02 P. falciparum oocysts in a little over two hours. The earlier ECL-SB detected rCSP and oocysts and took approximately 5 h to perform. Whole mosquito lysates from both high and low prevalence-infected mosquito populations were prepared and evaluated for PfCSP detection on the ECL-SB by both AR film and digital data capture and analysis. There was a 100% agreement between the AR film and the C-Digit scanner methods for PfCSP detection in randomly sampled mosquitoes. This novel "No Film" Slot Blot assay obviates the need for AR film exposure and development and significantly reduces the assay time enabling widespread use in field settings.

  4. FANCD2 Western blot as a diagnostic tool for Brazilian patients with Fanconi anemia

    Directory of Open Access Journals (Sweden)

    D.V. Pilonetto

    2009-03-01

    Full Text Available Fanconi anemia is a rare hereditary disease showing genetic heterogeneity due to a variety of mutations in genes involved in DNA repair pathways, which may lead to different clinical manifestations. Phenotypic variability makes diagnosis difficult based only on clinical manifestations, therefore laboratory tests are necessary. New advances in molecular pathogenesis of this disease led researchers to develop a diagnostic test based on Western blot for FANCD2. The objective of the present study was to determine the efficacy of this method for the diagnosis of 84 Brazilian patients with Fanconi anemia, all of whom tested positive for the diepoxybutane test, and 98 healthy controls. The FANCD2 monoubiquitinated isoform (FANCDS+/FANCD2L- was not detected in 77 patients (91.7%. In 2 patients (2.4%, there was an absence of both the monoubiquitinated and the non-ubiquitinated proteins (FANCD2S-/FANCD2L- and 5 patients (5.9% had both isoforms (FANCD2S+/FANCD2L+. This last phenotype suggests downstream subtypes or mosaicism. All controls were diepoxybutane negative and were also negative on the FANCD2 Western blot. The Western blot for FANCD2 presented a sensitivity of 94% (79/84 and specificity of 100% (98/98. This method was confirmed as an efficient approach to screen Brazilian patients with deleterious mutations on FANCD2 (FANCD2S-/FANCD2L- or other upstream genes of the FA/BRCA pathway (FANCDS+/FANCD2L-, to confirm the chromosome breakage test and to classify patients according to the level of FA/BRCA pathway defects. However, patients showing both FANCD2 isoforms (FANCD2S+/FANCD2L+ require additional studies to confirm mutations on downstream Fanconi anemia genes or the presence of mosaicism.

  5. SDS-PAGE and Western blot of urinary proteins in dogs with leishmaniasis.

    Science.gov (United States)

    Zaragoza, Concepción; Barrera, Rafael; Centeno, Francisco; Tapia, Jose A; Durán, Esther; González, Marta; Mañé, M Cinta

    2003-01-01

    Canine leishmaniasis is an endemic disease in the Mediterranean area caused by the protozoan Leishmania infantum, which usually produces renal failure. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blot using antibodies to IgG and IgA from dogs were carried out in the urine of 22 dogs with leishmaniasis diagnosed by ELISA and confirmed by PCR, and 20 healthy dogs. The results were compared to renal function laboratory tests and to those from a histopathological study of the kidneys from sick animals that died naturally or were euthanized. Five different bands with molecular weights ranging from 10 to 110 kDa were obtained from the electrophoresis of the urine of healthy dogs. 33.5% of total proteins corresponded to low molecular weight proteins and the other proteins had middle and high molecular weights. However, in the group with leishmaniasis, a maximum of 11 different bands with molecular weights ranging from 10 kDa to 150 kDa were displayed in the electrophoresis of the urine. The urine electrophoretic pattern in the sick dogs was classified as mixed (proteins with high and low molecular weights) because low molecular weight proteins made up 57.9% and the rest of the proteins had middle and high molecular weights. In Western blot, none of the healthy dogs showed excretion of IgG and/or IgA, whereas IgG and IgA were detected in the Western blot of urine of 68% and 55% respectively of dogs with leishmaniasis. The results obtained in the leishmaniasis group agreed with glomerular and tubular damage, which were confirmed by the histopathological findings.

  6. Western Blotting Is an Efficient Tool for Differential Diagnosis of Paracoccidioidomycosis and Pulmonary Tuberculosis

    Science.gov (United States)

    Bertoni, Thâmara Aline; Perenha-Viana, Maysa Cláudia Zolin; Patussi, Eliana Valéria; Cardoso, Rosilene Fressatti

    2012-01-01

    Sputum and sera from 134 patients screened for tuberculosis (TB) were analyzed to investigate TB and paracoccidioidomycosis (PCM). Of these patients, 11 (8.2%) were confirmed to have TB, but six (4.5%) were positive only for PCM. All patients with PCM presented anti-43-kDa-component antibodies in Western blotting (WB) assays, while in the TB-positive patients these antibodies did not appear. This preliminary study suggests WB as a potential tool for differential laboratory diagnosis between TB and PCM. PMID:22971781

  7. Blotting Assisted by Heating and Solvent Extraction for DESI-MS Imaging

    Science.gov (United States)

    Cabral, Elaine C.; Mirabelli, Mario F.; Perez, Consuelo J.; Ifa, Demian R.

    2013-06-01

    Imprints of potato sprout ( Solanum tuberosum L.), gingko leaves (Gingko biloba L. ) and strawberries (Fragaria x ananassa Duch. ) were successfully imaged by desorption electrospray ionization mass spectrometry (DESI-MS) on TLC plates through blotting assisted by heating and/or solvent extraction. Ion images showing the distribution of significant compounds such as glycoalkaloid toxins in potato sprout, ginkgolic acids and flavonoids in ginkgo leaves, and sugars and anthocyanidin in strawberry were obtained. Practical implications of this work include analysis of a wide range of irregular or soft materials by different imprinting conditions without requiring the addition of matrices or use of specific kinds of surfaces.

  8. Proteínas inmunodominantes de Brucella Melitensis evaluadas por Western Blot

    Directory of Open Access Journals (Sweden)

    Elizabeth Anaya

    1997-01-01

    Full Text Available Se separaron extractos de proteínas totales de Brucella melitensis en gel 15% SDS-PAGE. Su seroreactividad fue analizada por Western Blot con resultados satisfactorios. Para éste propósito sueros controles negativos (n=03, sueros de pacientes con brucelosis (n=34, cólera (n=12, tifoidea (n=02 y tuberculosis (n=02 fueron usados. Esta prueba inmunodiagnóstica detectó bandas seroreactivas altamente específicas (100% correspondientes a 8,14,18, un complejo de 25-48 y 58kDa. La sensibilidad del test fue del 90% usando los sueros antes mencionados.

  9. An overview of techniques for the measurement of calcium distribution, calcium fluxes, and cytosolic free calcium in mammalian cells

    International Nuclear Information System (INIS)

    Borle, A.B.

    1990-01-01

    An array of techniques can be used to study cell calcium metabolism that comprises several calcium compartments and many types of transport systems such as ion channels, ATP-dependent pumps, and antiporters. The measurement of total call calcium brings little information of value since 60 to 80% of total cell calcium is actually bound to the extracellular glycocalyx. Cell fractionation and differential centrifugation have been used to study intracellular Ca 2+ compartmentalization, but the methods suffer from the possibility of Ca 2+ loss or redistribution among cell fractions. Steady-state kinetic analyses of 45 Ca uptake or desaturation curves have been used to study the distribution of Ca 2+ among various kinetic pools in living cells and their rate of Ca 2+ exchange, but the analyses are constrained by many limitations. Nonsteady-state tracer studies can provide information about rapid changes in calcium influx or efflux in and out of the cell. Zero-time kinetics of 45 Ca uptake can detect instantaneous changes in calcium influx, while 45 Ca fractional efflux ratio, can detect rapid stimulations or inhibitions of calcium efflux out of cells. The best strategy to study cell calcium metabolism is to use several different methods that focus on a specific problem from widely different angles

  10. MIMO wireless networks channels, techniques and standards for multi-antenna, multi-user and multi-cell systems

    CERN Document Server

    Clerckx, Bruno

    2013-01-01

    This book is unique in presenting channels, techniques and standards for the next generation of MIMO wireless networks. Through a unified framework, it emphasizes how propagation mechanisms impact the system performance under realistic power constraints. Combining a solid mathematical analysis with a physical and intuitive approach to space-time signal processing, the book progressively derives innovative designs for space-time coding and precoding as well as multi-user and multi-cell techniques, taking into consideration that MIMO channels are often far from ideal. Reflecting developments

  11. Measurement of Relaxation Time of Excess Carriers in Si and CIGS Solar Cells by Modulated Electroluminescence Technique

    Energy Technology Data Exchange (ETDEWEB)

    Khatavkar, Sanchit [Indian Institute of Technology Bombay, Powai Mumbai 400076 India; Sanjivani College of Engineering, Kopargaon 423601 India; Muniappan, Kulasekaran [Indian Institute of Technology Bombay, Powai Mumbai 400076 India; Kannan, Chinna V. [MoserBaer Photovoltaic Pvt. Ltd., U.P. Greater Noida 201306 India; Kumar, Vijay [MoserBaer Photovoltaic Pvt. Ltd., U.P. Greater Noida 201306 India; Narsimhan, Krishnamachari L. [Indian Institute of Technology Bombay, Powai Mumbai 400076 India; Nair, Pradeep R. [Indian Institute of Technology Bombay, Powai Mumbai 400076 India; Vasi, Juzer M. [Indian Institute of Technology Bombay, Powai Mumbai 400076 India; Contreras, Miguel A. [National Renewable Energy Laboratory, Golden CO 80401 USA; van Hest, Maikel F. A. M. [National Renewable Energy Laboratory, Golden CO 80401 USA; Arora, Brij M. [Indian Institute of Technology Bombay, Powai Mumbai 400076 India; Indian Institute of Technology Goa, Farmagudi Ponda 403401 India

    2017-11-10

    Excess carrier lifetime plays a crucial role in determining the efficiency of solar cells. In this paper, we use the frequency dependence of inphase and quadrature components of modulated electroluminescence (MEL) to measure the relaxation time (decay) of excess carriers. The advantage of the MEL technique is that the relaxation time is obtained directly from the angular frequency at which the quadrature component peaks. It does not need knowledge of the material parameters like mobility, etc., and can be used for any finished solar cells which have detectable light emission. The experiment is easy to perform with standard electrical equipment. For silicon solar cells, the relaxation time is dominated by recombination and hence, the relaxation time is indeed the excess carrier lifetime. In contrast, for the CIGS solar cells investigated here, the relaxation time is dominated by trapping and emission from shallow minority carrier traps.

  12. Capillary blotting of glycosaminoglycans on nitrocellulose membranes after agarose-gel electrophoresis separation.

    Science.gov (United States)

    Volpi, Nicola; Maccari, Francesca

    2009-01-01

    A method for the blotting and immobilizing of several nonsulfated and sulfated complex polysaccharides on membranes made hydrophilic and positively charged by cationic detergent after their separation by conventional agarose gel electrophoresis is illustrated. This new approach to the study of glycosaminoglycans (GAGs) utilizes the capacity of agarose gel electrophoresis to separate single species of polysaccharides from mixtures and the membrane technology for further preparative and analytical uses.Nitrocellulose membranes are derivatized with the cationic detergent cetylpyridinium chloride and mixtures of GAGs are capillary blotted after their separation in agarose gel electrophoresis. Single purified species of variously sulfated polysaccharides are transferred on derivatized membranes with an efficiency of 100% and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining). This enables a lower amount limit of detection of 0.1 microg. Nonsulfated polyanions, for example hyaluronic acid, may also be transferred to membranes with a limit of detection of approximately 0.1-0.5 microg after irreversible or reversible staining. The membranes may be stained with reversible staining and the same lanes are used for immunological detection or other applications.

  13. Checking transfer efficiency and equal loading via qualitative optical way in western blotting.

    Science.gov (United States)

    Gong, Jun-Hua; Gong, Jian-Ping; Zheng, Kai-Wen

    2017-11-01

    The ability to determine that successful transfer and equal loading occur prior to using primary antibodies is important. And total protein staining is commonly used to check transfer efficiency and normalization, which play a crucial role in western blotting. Ponceau S and coomassie blue are commonly used, but there are disadvantages reported in recent years. Therefore, we are interested in finding another method, which is cheap, easy and fast. As we know, protein binding region of PVDF membrane is still hydrophilic when carbinol volatilizes, however, the non-protein binding region of PVDF membrane became hydrophobic again. And this different wettability between non-protein binding region and protein binding region of Polyvinylidene difluoride membrane may be used to check transfer efficiency and equal loading in western blotting. Based on the principle above, we describe an optical approach where an experimenter can observe that the proteins have been transferred to the membrane without any staining within minutes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Monitoring programmed cell death of living plant tissues in microfluidics using electrochemical and optical techniques

    DEFF Research Database (Denmark)

    Mark, Christina; Heiskanen, Arto; Svensson, Birte

    or optically detectable events during PCD in barley aleurone layer, a cell model for living plant tissues, for a better understanding of the underlying mechanisms of PCD in plants. Microfluidic cell culture enables in vitro experiments to approach in vivo conditions. The major advantage of electrochemical......Programmed cell death (PCD) in plants can influence the outcome of yield and quality of crops through its important role in seed germination and the defence process against pathogens. The main scope of the project is to apply microfluidic cell culture for the measurement of electrochemically......, since it is known that reactive oxygen species, which are affected by changes in the redox activity of the cells3, are involved in PCD in plants, but the relationship between and mechanisms behind ROS and PCD is only poorly understood in plant cells4. Recently, it has been shown, using optical detection...

  15. Proton Exchange Membrane Fuel Cell Modelling Using Moving Least Squares Technique

    Directory of Open Access Journals (Sweden)

    Radu Tirnovan

    2009-07-01

    Full Text Available Proton exchange membrane fuel cell, with low polluting emissions, is a great alternative to replace the traditional electrical power sources for automotive applications or for small stationary consumers. This paper presents a numerical method, for the fuel cell modelling, based on moving least squares (MLS. Experimental data have been used for developing an approximated model of the PEMFC function of the current density, air inlet pressure and operating temperature of the fuel cell. The method can be applied for modelling others fuel cell sub-systems, such as the compressor. The method can be used for off-line or on-line identification of the PEMFC stack.

  16. A Novel Technique for Accelerated Culture of Murine Mesenchymal Stem Cells that Allows for Sustained Multipotency.

    Science.gov (United States)

    Caroti, Courtney M; Ahn, Hyunhee; Salazar, Hector F; Joseph, Giji; Sankar, Sitara B; Willett, Nick J; Wood, Levi B; Taylor, W Robert; Lyle, Alicia N

    2017-10-17

    Bone marrow derived mesenchymal stem cells (MSCs) are regularly utilized for translational therapeutic strategies including cell therapy, tissue engineering, and regenerative medicine and are frequently used in preclinical mouse models for both mechanistic studies and screening of new cell based therapies. Current methods to culture murine MSCs (mMSCs) select for rapidly dividing colonies and require long-term expansion. These methods thus require months of culture to generate sufficient cell numbers for feasibility studies in a lab setting and the cell populations often have reduced proliferation and differentiation potential, or have become immortalized cells. Here we describe a simple and reproducible method to generate mMSCs by utilizing hypoxia and basic fibroblast growth factor supplementation. Cells produced using these conditions were generated 2.8 times faster than under traditional methods and the mMSCs showed decreased senescence and maintained their multipotency and differentiation potential until passage 11 and beyond. Our method for mMSC isolation and expansion will significantly improve the utility of this critical cell source in pre-clinical studies for the investigation of MSC mechanisms, therapies, and cell manufacturing strategies.

  17. Complementary techniques for solid oxide electrolysis cell characterisation at the micro- and nano-scale

    DEFF Research Database (Denmark)

    Wiedenmann, D.; Hauch, Anne; Grobety, B.

    2010-01-01

    ), material degradation and evaporation can occur, e.g., from the cell-sealing material, leading to poisoning effects and aging mechanisms that decrease the cell efficiency and long-term durability. To investigate such cell degradation processes, thorough examination of SOCs often requires a chemical...... approach for the structural and chemical characterisation of changes in aged cathode-supported electrolysis cells produced at Risø DTU, Denmark. Additionally, we present results from the characterisation of impurities at the electrolyte/hydrogen interface caused by evaporation of sealing material....

  18. A Modified Quantum Dot-Based Dot Blot Assay for Rapid Detection of Fish Pathogen Vibrio anguillarum.

    Science.gov (United States)

    Zhang, Yang; Xiao, Jingfan; Wang, Qiyao; Zhang, Yuanxing

    2016-08-28

    Vibrio anguillarum, a devastating pathogen causing vibriosis among marine fish, is prevailing in worldwide fishery industries and accounts for grievous economic losses. Therefore, a rapid on-site detection and diagnostic technique for this pathogen is in urgent need. In this study, two mouse monoclonal antibodies (MAbs) against V. anguillarum, 6B3-C5 and 8G3-B5, were generated by using hybridoma technology and their isotypes were characterized. MAb 6B3-C5 was chosen as the detector antibody and conjugated with quantum dots. Based on MAb 6B3- C5 labeled with quantum dots, a modified dot blot assay was developed for the on-site determination of V. anguillarum. It was found that the method had no cross-reactivity with other than V. anguillarum bacteria. The detection limit (LOD) for V. anguillarum was 1 × 10(3) CFU/ml in cultured bacterial suspension samples, which was a 100-fold higher sensitivity than the reported colloidal gold immunochromatographic test strip. When V. anguillarum was mixed with turbot tissue homogenates, the LOD was 1 × 10(3) CFU/ml, suggesting that tissue homogenates did not influence the detection capabilities. Preenrichment with the tissue homogenates for 12 h could raise the LOD up to 1 × 10(2) CFU/ml, confirming the reliability of the method.

  19. Profile of anti-Tp47 antibodies in patients with positive serology for syphilis analized by Western Blot.

    Science.gov (United States)

    Miranda, Ana Paula Félix de; Sato, Neuza Satomi

    2008-04-01

    In Brazil, syphilis is still a great problem of public health. Serological test is essential for syphilis diagnosis and the current trend is the use of recombinant antigen in the treponemal tests, due to its confirmed higher sensibility and specificity. The purpose of the present study was to analyze the profile of anti-Tp47 antibodies in patients with positive serology for syphilis. One hundred positive sera samples were analyzed by Western Blot (WB) technique, using the recombinant antigen (rTp47). Ten of them did not present antibodies against the fraction rTp47, the results were confirmed by WB using native T. pallidum antigen. All ten samples had antibodies against the fractions Tp17 and Tp15 and presented low reactivity in VDRL, negative results or title below than 1:4. Considering that VDRL is used for therapeutic monitoring due to seroreversion of nontreponemal antibodies in response to the treatment, and that some studies reported loss of treponemal antibodies after treatment, we could speculate if these ten samples are cases of serological memory from patients previously treated for syphilis. In addition, although several features state the Tp47 fraction as one of the major antigenic components, based on our results we point out to the importance of including other antigenic proteins such as Tp17 and Tp15 in addition to Tp47 in tests for serological screening of syphilis.

  20. Development of enzyme immunoassays (ELISA and Western blot) for the serological diagnosis of dermatophytosis in symptomatic and asymptomatic cats.

    Science.gov (United States)

    Santana, Aline Elisa; Taborda, Carlos Pelleschi; Severo, Julia So; Rittner, Glauce Mary Gomes; Muñoz, Julian Esteban; Larsson, Carlos Eduardo; Larsson, Carlos Eduardo

    2018-01-01

    Dermatophytosis is the most common fungal infection in cats worldwide and plays an important role in both animal and human health due to their high zoonotic potential. Effective screening is a strong preventive measure and the fungal culture is quite useful but requires full laboratorial experience and it takes a long time to obtain the result. A rapid and accurate screening test for dermatophytosis in cats is crucial for the effective control of disease outbreaks. The aim of this study was to develop and evaluate the diagnostic efficacy of enzyme immunoassays (ELISA and Western blot [WB]) for the rapid and precise diagnosis of dermatophytosis in cats. Seventy cats of various ages were divided into three groups: S (symptomatic, n = 20), AS (asymptomatic, n = 30), and N (negative, n = 20). All animals were submitted to fungal culture and blood samples for carrying out the serological tests. A significant difference (P technique detected 13 bands, and the 50 kDa protein was considered the most immunogenic protein, observing reactivity in 83.3% in the symptomatic group and 66.6% in the asymptomatic group. The study concluded that ELISA and WB were useful tools to reliably detect cats that have been exposed to M. canis. © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  1. Detection of Zika virus using reverse-transcription LAMP coupled with reverse dot blot analysis in saliva.

    Directory of Open Access Journals (Sweden)

    Maite Sabalza

    Full Text Available In recent years, there have been increasing numbers of infectious disease outbreaks that spread rapidly to population centers resulting from global travel, population vulnerabilities, environmental factors, and ecological disasters such as floods and earthquakes. Some examples of the recent outbreaks are the Ebola epidemic in West Africa, Middle East respiratory syndrome coronavirus (MERS-Co in the Middle East, and the Zika outbreak through the Americas. We have created a generic protocol for detection of pathogen RNA and/or DNA using loop-mediated isothermal amplification (LAMP and reverse dot-blot for detection (RDB and processed automatically in a microfluidic device. In particular, we describe how a microfluidic assay to detect HIV viral RNA was converted to detect Zika virus (ZIKV RNA. We first optimized the RT-LAMP assay to detect ZIKV RNA using a benchtop isothermal amplification device. Then we implemented the assay in a microfluidic device that will allow analyzing 24 samples simultaneously and automatically from sample introduction to detection by RDB technique. Preliminary data using saliva samples spiked with ZIKV showed that our diagnostic system detects ZIKV RNA in saliva. These results will be validated in further experiments with well-characterized ZIKV human specimens of saliva. The described strategy and methodology to convert the HIV diagnostic assay and platform to a ZIKV RNA detection assay provides a model that can be readily utilized for detection of the next emerging or re-emerging infectious disease.

  2. Scaling-Up Techniques for the Nanofabrication of Cell Culture Substrates via Two-Photon Polymerization for Industrial-Scale Expansion of Stem Cells

    Directory of Open Access Journals (Sweden)

    Davide Ricci

    2017-01-01

    Full Text Available Stem-cell-based therapies require a high number (106–109 of cells, therefore in vitro expansion is needed because of the initially low amount of stem cells obtainable from human tissues. Standard protocols for stem cell expansion are currently based on chemically-defined culture media and animal-derived feeder-cell layers, which expose cells to additives and to xenogeneic compounds, resulting in potential issues when used in clinics. The two-photon laser polymerization technique enables three-dimensional micro-structures to be fabricated, which we named synthetic nichoids. Here we review our activity on the technological improvements in manufacturing biomimetic synthetic nichoids and, in particular on the optimization of the laser-material interaction to increase the patterned area and the percentage of cell culture surface covered by such synthetic nichoids, from a low initial value of 10% up to 88% with an optimized micromachining time. These results establish two-photon laser polymerization as a promising tool to fabricate substrates for stem cell expansion, without any chemical supplement and in feeder-free conditions for potential therapeutic uses.

  3. Sulphate reduction and vertical distribution of sulphate-reducing bacteria quantified by rRNA slot-blot hybridization in a coastal marine sediment

    DEFF Research Database (Denmark)

    Sahm, K.; MacGregor, BJ; Jørgensen, BB

    1999-01-01

    In the past, enumeration of sulphate-reducing bacteria (SRB) by cultivation-based methods generally contradicted measurements of sulphate reduction, suggesting unrealistically high respiration rates per cell. Here, we report evidence that quantification of SRB rRNA by slot-blot hybridization...... between 18% and 25% to the prokaryotic rRNA pool. The dominant SRB were related to complete oxidizing genera (Desulphococcus, Desulphosarcina and Desulphobacterium), while Desulpho-bacter could not be detected. The vertical profile and quantity of rRNA from SRB was compared with sulphate reduction rates......, directly above the sulphate reduction maximum. Cell numbers calculated by converting the relative contribution of SRB rRNA to the percentage of DAPI-stained cells indicated a population size for SRB of 2.4-6.1 x 10(8) cells cm(-3) wet sediment. Cellular sulphate reduction rates calculated on the basis...

  4. Intensity modulated radiation therapy for squamous cell carcinoma of the vulva: Treatment technique and outcomes

    Directory of Open Access Journals (Sweden)

    Yuan James Rao, MD

    2017-04-01

    Conclusions: IMRT for vulvar cancer is associated with high rates of LRC in the postoperative setting and limited radiation-related toxicity. Durable LRC of disease after definitive IMRT remains challenging, and several refinements to our treatment technique are suggested.

  5. A new asymmetric 6T SRAM cell with a write assist technique in 65nm CMOS technology

    DEFF Research Database (Denmark)

    Farkhani, Hooman; Peiravi, Ali; Moradi, Farshad

    2014-01-01

    A new asymmetric 6T-SRAM cell design is presented for low-voltage low-power operation under process variations. The write margin of the proposed cell is improved by the use of a new write-assist technique. Simulation results in 65nm technology show that the proposed cell achieves the same RSNM...... as the asymmetric 5T-SRAM cell and 77% higher RSNM than the standard 6T-SRAM cell while it is able to perform write operation without any write assist at VDD=1V. Monte Carlo simulations for an 8Kb SRAM (256×32) array indicate that the scalability of operating supply voltage of the proposed cell can be improved...... overhead for the write assist, replica column and the replica column driver of 2.6%, the overall area reduction in die area is 6.3% and 16.3% as compared with array designs with asymmetric 5T- and standard 6T-SRAM cells....

  6. Single-channel data-validation technique for ΔP cells in turbulent flow

    International Nuclear Information System (INIS)

    Goodrich, L.D.; Brower, R.W.

    1983-01-01

    This paper discusses a single-channel-analysis, data-validation technique which is applicable to all flow-measuring devices in turbulent conditions, can be applied in either real time or batch mode, and allows online correction of zero offset and slope coefficients. This technique of validating flow measurements eliminates the need for multiple measuring devices, thus reducing the complexity of the overall instrumentation system

  7. Comparative evaluation of optical methods and conventional isotope techniques for the detection of insulin receptors in heterogenous cell systems

    International Nuclear Information System (INIS)

    Thun, C.

    1984-01-01

    The findings of studies using radioactively labelled (I-125) insulin to characterise its binding to various heterogenous cell systems had led to a classification of the relevant receptors with those of high affinity and low capacity or vice versa. This, in turn, raised questions as to the binding properties of each individual cell or cell material of a heterogenous nature. Apparently homogenous (lymphocytes) and heterogenous (blood and islet cells) cell populations were investigated on the basis of various techniques for the separate evaluation of individual cells, which were cytofluorometry using FITC insulin and the analysis of gold insulin under the electron microscope. For the association kinetics and equilibration analysis or affinity and receptor quantity a radioactive tracer and light microscope were used. Insulin was shown to bind to erythrocytes, reticulocytes, monocytes and lymphocytes and this result finds confirmation in the relevant literature. Furthermore, binding parameters could be determined for isolated islet cells. Cytofluorometry pointed to the fact that the insulin receptors of an apparently homogenous cell system differed in affinity and number and permitted the use of a multiple parameter procedure. Thus, it holds out promise as a method to be routinely used in the clinical diagnosis of binding parameters, without requiring previous separation procedures that are complicated or involve a loss of material. Transmission electron microscopy permitted conclusions to be drawn as to the type of cell to which insulin is attached. Owing to the use of gold insulin it was possible to throw some light on the factors determining the fate of membrane-bound insulin during its uptake into the cell. (TRV) [de

  8. Value of window technique in diagnosis of the ground glass opacities in patients with non-small cell pulmonary cancer

    OpenAIRE

    Yao, Gang

    2016-01-01

    The aim of the present study was to examine the value of window technique in qualitative diagnosis of the ground glass opacities (GGO) in patients with non-small cell pulmonary cancer. A total of 124 clinically suspected pulmonary cancer patients were analyzed retrospectively. The lesions were affirmed by puncture biopsy, and were GGO on pulmonary window while were invisible on mediastinal window. Sixty-four multi-detector spiral computed tomography with the window width and window level of 1...

  9. Intracellular microelectrode measurements in small cells evaluated with the patch clamp technique

    NARCIS (Netherlands)

    Ince, C.; van Bavel, E.; van Duijn, B.; Donkersloot, K.; Coremans, A.; Ypey, D. L.; Verveen, A. A.

    1986-01-01

    Microelectrode penetration of small cells leads to a sustained depolarization of the resting membrane potential due to a transmembrane shunt resistance (Rs) introduced by the microelectrode. This has led to underestimation of the resting membrane potential of various cell types. However, measurement

  10. Predicting bacteriophage proteins located in host cell with feature selection technique.

    Science.gov (United States)

    Ding, Hui; Liang, Zhi-Yong; Guo, Feng-Biao; Huang, Jian; Chen, Wei; Lin, Hao

    2016-04-01

    A bacteriophage is a virus that can infect a bacterium. The fate of an infected bacterium is determined by the bacteriophage proteins located in the host cell. Thus, reliably identifying bacteriophage proteins located in the host cell is extremely important to understand their functions and discover potential anti-bacterial drugs. Thus, in this paper, a computational method was developed to recognize bacteriophage proteins located in host cells based only on their amino acid sequences. The analysis of variance (ANOVA) combined with incremental feature selection (IFS) was proposed to optimize the feature set. Using a jackknife cross-validation, our method can discriminate between bacteriophage proteins located in a host cell and the bacteriophage proteins not located in a host cell with a maximum overall accuracy of 84.2%, and can further classify bacteriophage proteins located in host cell cytoplasm and in host cell membranes with a maximum overall accuracy of 92.4%. To enhance the value of the practical applications of the method, we built a web server called PHPred (〈http://lin.uestc.edu.cn/server/PHPred〉). We believe that the PHPred will become a powerful tool to study bacteriophage proteins located in host cells and to guide related drug discovery. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. [Study on the setup of a new technique of tissue and cell culture for vitreous fluid of vitrectomy].

    Science.gov (United States)

    Li, Gen-lin; Liu, Yue-yue; Zhang, Xiang; Zhang, Cheng-yue; Wang, Liang-hai

    2008-12-01

    To setup a new technique of tissue and cell culture for vitreous aspirates. Experiment study. Specimens used for supporting new culture technique were selected based on random digit table. Thirty cases with rhegmatogenous retinal detachment (RRD) and forty-eight with proliferative diabetic retinopathy (PDR), which undergoing primary pars plana vitrectomy, were selected randomly and included in the study. After being antiphase stained with fluorescein-natrium (0.5%) and digested with hyaluronidase (10(5) U/L) combined with collagenase I (10(6) U/L) for removing vitreous gel, sediment of vitreous fluid after centrifugation were inoculated into standard culturing bottle with which polylysine (0.01%) was pre-set. The bottle which contained F12 medium with 30% fetal bovine serum was placed upside down for 24 hours and consecutively upside for 6 days. During which, F12 medium was replaced once in half volume, and cell growth along the edge of sedimentary membrane was observed at time of the 3rd and the 6th day after upside culture. Under condition of pre-setting by polylysine (0.01%) and being placed upside down for 24 hours, pieces from vitreous fluids could adhere to the bottom of bottle in a way of semi-xerosis with adherence rate of 100% (78/78). No bacteria, fungus and mycoplasma contamination was found within 7 days. Antiphase stained with fluorescein-natrium (0.5%) and digested with hyaluronidase (10(5) U/L) combined with collagenase I (10(6) U/L) for 30 minutes, vitreous gel in 78 specimens could be digested (78/78). Cell emigration could be found in edge area of some pieces of vitreous fluid and cell growth as well as proliferation was shown. In 30 specimens of RRD, cell growth rate were 43.33% (13/30). In 48 specimens of PDR, cell growth rate were 37.50% (18/48). Concerning PDR phase V (PDR-V), cell growth rate reach 41.67% (10/24). Enzymolysis with upside down and semi-xerosis could ensure good adherence of membrane, moreover, no contamination and obvious cell

  12. Potential for broad applications of flow cytometry and fluorescence techniques in microbiological and somatic cell analyses of milk.

    Science.gov (United States)

    Gunasekera, T S; Veal, D A; Attfield, P V

    2003-08-25

    Monitoring the quality and safety of milk requires careful analysis of microbial and somatic cell loading. Our aim was to demonstrate proof of the principle that flow cytometry (FCM), coupled with fluorescence techniques for distinguishing between cell types, could potentially be employed in a wide variety of biological assays relevant to the dairy industry. To this end, we studied raw milk samples and ultraheat-treated milk, into which known numbers of bacteria or mouse cells were inoculated. For bacterial analyses, protein and lipids were removed, whereas only centrifugal lipid clearing was needed for somatic cell analyses. Cleared samples were stained with fluorescent dyes or with bacterial-specific fluorescent-labeled oligonucleotides and analyzed by FCM. A fluoresceinated peptide nucleic acid probe enabled efficient enumeration of bacteria in milk. Dual staining of samples with fluorescent dyes that indicate live (5-cyanol-2,3-ditolyl tetrazolium chloride, CTC or SYTO 9) or damaged cells (oxonol or propidium iodide, PI) enabled determination of viable bacteria in milk. Gram-positive and -negative bacteria were distinguished using hexidium iodide and SYTO 13 in dual staining of cleared milk samples. An FCM-based method gave a good correlation (r=0.88) with total microscopic counts of somatic cells in raw milk. The FCM method also correlated strongly (r=0.98) with the standard Fossomatic method for somatic cell detection. We conclude that FCM, coupled with fluorescence staining techniques, offers potentially diverse and rapid approaches to biological safety and quality testing in the dairy industry. Potential application of flow cytometers to a broad range of assays for milk biological quality should make this instrumentation more attractive and cost effective to the dairy industry and indeed the broader food industry.

  13. Safe genetic modification of cardiac stem cells using a site-specific integration technique.

    Science.gov (United States)

    Lan, Feng; Liu, Junwei; Narsinh, Kazim H; Hu, Shijun; Han, Leng; Lee, Andrew S; Karow, Marisa; Nguyen, Patricia K; Nag, Divya; Calos, Michele P; Robbins, Robert C; Wu, Joseph C

    2012-09-11

    Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts. We used the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells. Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared with unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging, and positron emission tomography. Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function 2 weeks after cell delivery, as assessed by echocardiography (P=0.002) and MRI (P=0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated human endothelial cells, which enhanced hind limb perfusion (P<0.05 at day 7 and 14 after transplantation) on laser Doppler imaging. The phiC31 integrase genomic modification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types.

  14. Survival of irradiated glia and glioma cells studied with a new cloning technique

    International Nuclear Information System (INIS)

    Nilsson, S.; Carlsson, J.; Larsson, B.; Ponten, J.

    1980-01-01

    A method allowing cloning of monolayer cultured cells with a low plating efficiency was developed. Cells were grown in several small palladium squares to obtain a high cell density. These squares were surrounded by non-adhesive agarose to prevent large distance migration and thereby mixing of the clones. By using easily-cloned hamster cells for comparison it was found that the survival curves were similar to the curves obtained with conventional cloning. The new method was used to compare the radiosensitivity of cultured human glia and glioma cells which both have a low plating efficiency ( 0 -values (1.5 to 2.5 Gy) and large shoulders (extrapolation numbers around 5) indicating that they were rather resistant and had a high capacity for accumulation of sublethal damage. The survival curves for glia cells had lower D 0 -values (1.3 to 1.5 Gy) and no shoulders at all, indicating that they were more sensitive than the glioma cells. (author)

  15. A noninvasive technique for the measurement of the energetic state of free-suspension mammalian cells.

    Science.gov (United States)

    Ben-Tchavtchavadze, M; Chen, J; Perrier, Michel; Jolicoeur, Mario

    2010-01-01

    A perfusion small-scale bioreactor allowing on-line monitoring of the cell energetic state was developed for free-suspension mammalian cells. The bioreactor was designed to perform in vivo nuclear magnetic resonance (NMR) spectroscopy, which is a noninvasive and nondestructive method that permits the monitoring of intracellular nutrient concentrations, metabolic precursors and intermediates, as well as metabolites and energy shuttles, such as ATP, ADP, and NADPH. The bioreactor was made of a 10-mm NMR tube following a fluidized bed design. Perfusion flow rate allowing for adequate oxygen supply was found to be above 0.79 mL min(-1) for high-density cell suspensions (10(8) cells). Chinese hamster ovary (CHO) cells were studied here as model system. Hydrodynamic studies using coloration/decoloration and residence time distribution measurements were realized to perfect bioreactor design as well as to determine operating conditions bestowing adequate homogeneous mixing and cell retention in the NMR reading zone. In vivo (31)P NMR was performed and demonstrated the small-scale bioreactor platform ability to monitor the cell physiological behavior for 30-min experiments.

  16. Examining the impact of cell phone conversations on driving using meta-analytic techniques.

    Science.gov (United States)

    Horrey, William J; Wickens, Christopher D

    2006-01-01

    The performance costs associated with cell phone use while driving were assessed meta-analytically using standardized measures of effect size along five dimensions. There have been many studies on the impact of cell phone use on driving, showing some mixed findings. Twenty-three studies (contributing 47 analysis entries) met the appropriate conditions for the meta-analysis. The statistical results from each of these studies were converted into effect sizes and combined in the meta-analysis. Overall, there were clear costs to driving performance when drivers were engaged in cell phone conversations. However, subsequent analyses indicated that these costs were borne primarily by reaction time tasks, with far smaller costs associated with tracking (lane-keeping) performance. Hands-free and handheld phones revealed similar patterns of results for both measures of performance. Conversation tasks tended to show greater costs than did information-processing tasks (e.g., word games). There was a similar pattern of results for passenger and remote (cell phone) conversations. Finally, there were some small differences between simulator and field studies, though both exhibited costs in performance for cell phone use. We suggest that (a) there are significant costs to driver reactions to external hazards or events associated with cell phone use, (b) hands-free cell phones do not eliminate or substantially reduce these costs, and (c) different research methodologies or performance measures may underestimate these costs. Potential applications of this research include the assessment of performance costs attributable to different types of cell phones, cell phone conversations, experimental measures, or methodologies.

  17. Assessment of environmental insults on lymphoid cells as detected by computer assisted morphometric techniques

    International Nuclear Information System (INIS)

    Olson, G.B.; Bartels, P.H.

    1984-01-01

    Ability to detect and monitor specific environmental insults with great sensitivity prompted the following study - the use of splenocytes and PBL as indicator cells to detect, monitor and differentiate between physically, chemically and biologically induced environmental insults. To be tested in this study is the hypothesis that different types of chemical, physical and biologic insults cause distinctive changes in the nuclear chromatin of the exposed cells. Should this assumption be warranted, one could monitor water, soil, air and food samples for undesirable chemicals and biological vectors (viruses), monitor people living near nuclear energy sites and people in radiation-related professions, and monitor cell samples obtained from people exposed to viral injections

  18. Combination of Small Molecule Microarray and Confocal Microscopy Techniques for Live Cell Staining Fluorescent Dye Discovery

    Directory of Open Access Journals (Sweden)

    Attila Bokros

    2013-08-01

    Full Text Available Discovering new fluorochromes is significantly advanced by high-throughput screening (HTS methods. In the present study a combination of small molecule microarray (SMM prescreening and confocal laser scanning microscopy (CLSM was developed in order to discover novel cell staining fluorescent dyes. Compounds with high native fluorescence were selected from a 14,585-member library and further tested on living cells under the microscope. Eleven compartment-specific, cell-permeable (or plasma membrane-targeted fluorochromes were identified. Their cytotoxicity was tested and found that between 1–10 micromolar range, they were non-toxic even during long-term incubations.

  19. Technique Comparison of the Fracture Toughness Tests for Irradiated Fuel Claddings in a Hot Cell

    International Nuclear Information System (INIS)

    Ahn, Sangbok; Kim, Dosik; Jung, Yanghong; Choo, Yongsun; Ryu, Wooseog

    2007-01-01

    The degradation of a fracture toughness in a fuel cladding is a important factor to restrict the operation safety in nuclear power plants. The fracture properties of claddings were traditionally measured through a rubber bung test, a burst test, etc. Those results were the qualitative fracture characteristics, and could not be used as design or operation safety evaluation data. We need to evaluate the quantitative characteristics of claddings under normal operation and in accidents. The application of a fracture mechanics concept in testing a fuel cladding is restricted by the cladding geometry and creating the correct stress-state conditions. The geometry of claddings does not meet the requirement of the ASTM Standards for a specimen configuration and an applied load. The specimen may be produced from previously flattened claddings, but the flattening causes some uncertainties in the results due to changes in the microstructure of the material and a new distribution of the internal stresses. Therefore many efforts have been devoted to developing new test techniques, to quantify the fracture characteristics of claddings. Researchers from JAEA and NFI in Japan, Studsvik Company Ltd in Sweden, IAEA in Australia, and KAERI in Korea have independently developed fracture test techniques. This study is designed to review the independently developed techniques and to compare of their merits. Finally we shall apply the other techniques to upgrade our developing techniques

  20. Cell mediated lympholysis: CML. A microplate technique requiring few target cells and employing a new method of supernatant collection

    International Nuclear Information System (INIS)

    Hirschberg, A.; Thorsby, E.

    1977-01-01

    A micromethod for the 51 Cr release assay is described. Allogeneically induced cytotoxic lymphocytes are generated in mixed lymphocyte microcultures in the wells of microplates. Their cytotoxic capacity is assayed by adding 51 Cr-labelled PHA derived lymphoblasts directly into the microcultures with no pooling or transfer of the cytotoxic effector cells being required. The 51 Cr isotope released into the cell supernatants is collected by inserting a cellulose acetate absorption cartridge into each well. A glass fiber filter attached to the cartridge effectively separates the supernatant from the cellular elemets. This system allows the simultaneous collection of the supernatant from 96 wells, and can be used with either adherent or non-adherent target cells

  1. Experimental models of brain ischemia: a review of techniques, magnetic resonance imaging and investigational cell-based therapies

    Directory of Open Access Journals (Sweden)

    Alessandra eCanazza

    2014-02-01

    Full Text Available Stroke continues to be a significant cause of death and disability worldwide. Although major advances have been made in the past decades in prevention, treatment and rehabilitation, enormous challenges remain in the way of translating new therapeutic approaches from bench to bedside. Thrombolysis, while routinely used for ischemic stroke, is only a viable option within a narrow time window. Recently, progress in stem cell biology has opened up avenues to therapeutic strategies aimed at supporting and replacing neural cells in infarcted areas. Realistic experimental animal models are crucial to understand the mechanisms of neuronal survival following ischemic brain injury and to develop therapeutic interventions. Current studies on experimental stroke therapies evaluate the efficiency of neuroprotective agents and cell-based approaches using primarily rodent models of permanent or transient focal cerebral ischemia. In parallel, advancements in imaging techniques permit better mapping of the spatial-temporal evolution of the lesioned cortex and its functional responses. This review provides a condensed conceptual review of the state of the art of this field, from models and magnetic resonance imaging techniques through to stem cell therapies.

  2. Quantitative detection for plant virus's RNA-loading by dot-blot

    International Nuclear Information System (INIS)

    Chai Lihong; Xu Bujin; Chen Jishuang

    2003-01-01

    A new method, RNA dot blot combined with direct determination of the radioactivity by BIO-Imaging Analyzer (dRH-dBIA) was used for detecting RNA of plant virus in infected plant tissue. This method was used for the influence of RNA-loading level of tobacco mosaic virus (TMV) in tobacco leave tissues after treatment of a plant hormone relatives (n-Propyl dihydro-jasmonate, PDJ) in the concentration range of 0.001-10 ppm. The results indicate that after PDJ application onto tobacco leaves for 3 days all PDJ treatments cause increase of TMV RNA-loading level except 0.001 ppm treatment, and the higher the concentration, the more obvious increase was observed. This phenomenon was confirmed with semi-leaf lesion spot on Nicotiana glutinosa as a local lesion host. The dRH-dBIA method is applicable in quantitative determination of RNA without obvious artificial influence

  3. A simple DNA recombination screening method by RT-PCR as an alternative to Southern blot

    DEFF Research Database (Denmark)

    Albers, Eliene; Sbroggiò, Mauro; Martin Gonzalez, Javier

    2017-01-01

    genomic locus, but limits the detection of its correct genomic integration by standard PCR methods. Genomic Long Range PCR (LR-PCR), using primers adjacent to the homology arms, has been used as an alternative to radioactive-based Southern blot screenings. However, LR-PCRs are often difficult and render...... many false positive and false negative results. Here, we propose an alternative screening method based on the detection of a genetic modification at the mRNA level, which we successfully optimized in two mouse models. This screening method consists of a reverse-transcription PCR (RT-PCR) using primers...... that match exons flanking the targeting construct. The detection of the expected modification in this PCR product confirms the integration at the correct genomic location and shows that the mutant mRNA is expressed. This is a simple and sensitive strategy to screen locus-specific recombination of targeting...

  4. Post-stained Western blotting, a useful approach in immunoproteomic studies.

    Science.gov (United States)

    Reguera-Brito, Mercedes; Fernández-Garayzábal, José F; Blanco, M Mar; Aguado-Urda, Mónica; Gibello, Alicia

    2014-12-15

    The precise localisation of immunogenic proteins on stained two-dimensional electrophoresis (2DE) gels is occasionally difficult, contributing to the erroneous identification of unrelated non-immunogenic proteins, which is expensive and time consuming. This inconvenience can be solved by performing immunoblotting using previously stained polyacrylamide gels. This approach was proposed nearly 20 years ago but is now almost forgotten. We have evaluated the suitability of this approach to identify immunogenic proteins from Lactococcus garvieae. Some of the immunogenic proteins identified in L. garvieae, such as Gls24, have been considered important as immunotarget in different bacterial species. Post-staining western blotting facilitated the correct selection of immunogenic proteins of interest in 2D gels before their identification. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Using Phos-Tag in Western Blotting Analysis to Evaluate Protein Phosphorylation.

    Science.gov (United States)

    Horinouchi, Takahiro; Terada, Koji; Higashi, Tsunehito; Miwa, Soichi

    2016-01-01

    Protein phosphorylation has traditionally been detected by radioisotope phosphate labeling of proteins with radioactive ATP. Several nonradioactive assays with phosphorylation site-specific antibodies are now available for the analysis of phosphorylation status at target sites. However, due to their high specificity, these antibodies they cannot be used to detect unidentified phosphorylation sites. Recently, Phos-tag technology has been developed to overcome the disadvantages and limitations of phosphospecific antibodies. Phos-tag and its derivatives conjugated to biotin, acrylamide, or agarose, form alkoxide-bridged dinuclear metal complexes, which can capture phosphate monoester dianions bound to serine, threonine, and tyrosine residues, in an amino acid sequence-independent manner. Here, we describe our method, which is based on in vitro kinase assay and Western blotting analysis using biotinylated Phos-tag and horseradish peroxidase-conjugated streptavidin, to determine the sites of TRPC6 (transient receptor potential canonical 6) channel phosphorylated by protein kinase A.

  6. Electrospun nitrocellulose and nylon: Design and fabrication of novel high performance platforms for protein blotting applications

    Directory of Open Access Journals (Sweden)

    Bowlin Gary L

    2007-10-01

    Full Text Available Abstract Background Electrospinning is a non-mechanical processing strategy that can be used to process a variety of native and synthetic polymers into highly porous materials composed of nano-scale to micron-scale diameter fibers. By nature, electrospun materials exhibit an extensive surface area and highly interconnected pore spaces. In this study we adopted a biological engineering approach to ask how the specific unique advantages of the electrospinning process might be exploited to produce a new class of research/diagnostic tools. Methods The electrospinning properties of nitrocellulose, charged nylon and blends of these materials are characterized. Results Nitrocellulose electrospun from a starting concentration of Conclusion The flexibility afforded by electrospinning process makes it possible to tailor blotting membranes to specific applications. Electrospinning has a variety of potential applications in the clinical diagnostic field of use.

  7. Evaluation of fluorescence in situ hybridization techniques to study long non-coding RNA expression in cultured cells

    DEFF Research Database (Denmark)

    Soares, Ricardo J; Maglieri, Giulia; Gutschner, Tony

    2018-01-01

    approach with or without enzymatic signal amplification, a branched-DNA (bDNA) probe and an LNA-modified probe with enzymatic signal amplification. All four methods adequately stained MALAT1 in the nucleus in all of three cell lines investigated, HeLa, NHDF and T47D, and three of the methods detected......Deciphering the functions of long non-coding RNAs (lncRNAs) is facilitated by visualization of their subcellular localization using in situ hybridization (ISH) techniques. We evaluated four different ISH methods for detection of MALAT1 and CYTOR in cultured cells: a multiple probe detection...... the less expressed CYTOR. The sensitivity of the four ISH methods was evaluated by image analysis. In all three cell lines, the two methods involving enzymatic amplification gave the most intense MALAT1 signal, but the signal-to-background ratios were not different. CYTOR was best detected using the b...

  8. Comparison of CdS films deposited by different techniques: Effects on CdTe solar cell

    International Nuclear Information System (INIS)

    Lee, Jaehyeong

    2005-01-01

    Polycrystalline cadmium sulfide (CdS) thin-films were deposited on glass substrate by chemical bath deposition (CBD) and vacuum evaporation (VE) techniques. VE-CdS films consisted primarily of hexagonal phase, whereas CBD CdS films containing primarily the cubic form. VE-grown films were shown to have better crystallinity than CBD-grown films. The grain size of the CBD films is smaller than the ones of VE films. VE-CdS films exhibited relatively high transmittance in the above-gap region and band gap compared with CBD films. However, CdTe solar cells with these low quality CBD-CdS layers yield higher and more stable characteristics. Current-voltage-temperature measurements showed that the current transport for both cells was controlled by both tunneling and interface recombination but the cells with CBD-CdS displayed less tunneling

  9. Evaluation of the Western blotting method for the diagnosis of congenital toxoplasmosis,

    Directory of Open Access Journals (Sweden)

    Jaqueline Dario Capobiango

    Full Text Available Abstract Objective: To evaluate the Western blotting method for the detection of IgG anti-Toxoplasma gondii (T. gondii (IgG-WB in the serum of children with suspected congenital toxoplasmosis. Methods: We accompanied 47 mothers with acquired toxoplasmosis in pregnancy and their children, between June of 2011 and June of 2014. The IgG-WB was done in house and the test was considered positive if the child had antibodies that recognized at least one band on IgG blots different from the mother's or with greater intensity than the corresponding maternal band, during the first three months of life. Results: 15 children (15.1% met the criteria for congenital toxoplasmosis and 32 (32.3% had the diagnosis excluded. The symptoms were observed in 12 (80.0% children and the most frequent were cerebral calcification in 9 (60.0%, chorioretinitis in 8 (53.3%, and hydrocephalus in 4 (26.6%. IgM antibodies anti-T. gondii detected by chemiluminescence (CL were found in 6 (40.0% children and the polymerase chain reaction (PCR for detection of T. gondii DNA was positive in 5 of 7 performed (71.4%. The sensitivity of IgG-WB was of 60.0% [95% confidence interval (CI 32.3-83.7%] and specificity 43.7% (95% CI 26.7-62.3%. The sensitivity of IgG-WB increased to 76.0 and 89.1% when associated to the research of IgM anti-T. gondii or PCR, respectively. Conclusions: The IgG-WB showed greater sensitivity than the detection of IgM anti-T. gondii; therefore, it can be used for the diagnosis of congenital toxoplasmosis in association with other congenital infection markers.

  10. Detection of specific antigens of Newcastle disease virus using an absorbed Western blotting method.

    Science.gov (United States)

    Hemmatzadeh, F; Kazemimanesh, M

    2017-01-01

    Newcastle disease virus (NDV) is an economically important poultry pathogen with a worldwide distribution that may infect a wide range of domestic and wild avian species. The identification of different pathotypes of NDVs plays an important role in the diagnosis and development of vaccines to control and eradicate NDV infections. In our previous study, we showed that mono-specific antibodies can differentiate velogenic and lentogenic strains of NDV in Agar Gel Immuno-Diffusion tests. To evaluate the ability of the specific antibodies to detect NDV specific antigens, this study was conducted with a range of NDV isolates. The samples included 9 NDV neuropathogenic/velogenic isolates from diseased chickens collected from poultry farms in central and northern parts of Iran plus La-Sota and B1 vaccine strains. All samples were propagated in embryonated chicken eggs and concentrated and purified by ultra-centrifugation. All samples were subjected to 12.5% SDS-PAGE and Western blotting using the specific antibodies mentioned previously. In SDS-PAGE all velogenic and vaccine strains showed the same electrophoretic pattern. The detected bands included 15, 38, 46, 48, 53, 55, 68, 74 and 220 kDa proteins. In Western blotting analysis, the mono-specific antibodies reacted specifically to the viral proteins with 15, 38, 48, 55, 74 and 220 kDa and non-specifically to the viral protein with 53 kDa. The results suggest that specific anti-NDV antibodies can react specifically to glycoproteins (haemagglutin-neuraminidase and fusion proteins) but not to internal proteins (nucleoprotein or matrix protein) of NDV strains.

  11. Evaluation of the Western blotting method for the diagnosis of congenital toxoplasmosis.

    Science.gov (United States)

    Capobiango, Jaqueline Dario; Monica, Thaís Cabral; Ferreira, Fernanda Pinto; Mitsuka-Breganó, Regina; Navarro, Italmar Teodorico; Garcia, João Luis; Reiche, Edna Maria Vissoci

    To evaluate the Western blotting method for the detection of IgG anti-Toxoplasma gondii (T. gondii) (IgG-WB) in the serum of children with suspected congenital toxoplasmosis. We accompanied 47 mothers with acquired toxoplasmosis in pregnancy and their children, between June of 2011 and June of 2014. The IgG-WB was done in house and the test was considered positive if the child had antibodies that recognized at least one band on IgG blots different from the mother's or with greater intensity than the corresponding maternal band, during the first three months of life. 15 children (15.1%) met the criteria for congenital toxoplasmosis and 32 (32.3%) had the diagnosis excluded. The symptoms were observed in 12 (80.0%) children and the most frequent were cerebral calcification in 9 (60.0%), chorioretinitis in 8 (53.3%), and hydrocephalus in 4 (26.6%). IgM antibodies anti-T. gondii detected by chemiluminescence (CL) were found in 6 (40.0%) children and the polymerase chain reaction (PCR) for detection of T. gondii DNA was positive in 5 of 7 performed (71.4%). The sensitivity of IgG-WB was of 60.0% [95% confidence interval (CI) 32.3-83.7%] and specificity 43.7% (95% CI 26.7-62.3%). The sensitivity of IgG-WB increased to 76.0 and 89.1% when associated to the research of IgM anti-T. gondii or PCR, respectively. The IgG-WB showed greater sensitivity than the detection of IgM anti-T. gondii; therefore, it can be used for the diagnosis of congenital toxoplasmosis in association with other congenital infection markers. Copyright © 2016 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

  12. Monitoring apoptosis of TK-GFP-expressing ACC-M cells induced by ACV using FRET technique

    Science.gov (United States)

    Xiong, Tao; Zhang, Zhihong; Lin, Juqiang; Yang, Jie; Zeng, Shaoqun; Luo, Qingming

    2006-09-01

    Apoptosis is an evolutionary conserved cellular process that plays an important role during development, but it is also involved in tissue homeostasis and in many diseases. To study the characteristics of suicide gene system of the herpes simplex virus thymidine kinase (HSV-tk) gene in tumor cells and explore the apoptosis phenomena in this system and its effect on the human adenoid cystic carcinoma line ACC-M cell, we detected apoptosis of CD3- (ECFP-CRS-DsRed) and TK-GFP-expressing ACC-M (ACC-M-TK-GFP-CD3) cells induced by acyclovir (ACV) using fluorescence resonance energy transfer (FRET) technique. CD3 is a FRET-based indicator for activity of caspase-3, which is composed of an enhanced cyan fluorescent protein, a caspase-3 sensitive linker, and a red fluorescent protein from Discosoma with efficient maturation property. FRET from ECFP to DsRed could be detected in normal ACC-M-TK-GFP-CD3 cells, and the FRET efficient was remarkably decreased and then disappeared during the cells apoptosis induced by ACV. It was due to the activated caspase-3 cleaved the CD3 fusion protein. In this study, the results suggested that the AVC-induced apoptosis of ACC-M-TK-GFP-CD3 cells was through caspase-3 pathway.

  13. Investigation of HIV-1 infected and uninfected cells using the optical trapping technique

    CSIR Research Space (South Africa)

    Ombinda-Lemboumba, Saturnin

    2017-02-01

    Full Text Available pathological information as possible, we use a home-build optical trapping and spectroscopy system for real time probing human immunodeficiency virus (HIV-1) infected and uninfected single cells. Briefly, our experimental rig comprises an infrared continuous...

  14. Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale.

    Science.gov (United States)

    Wachtmeister, Jochen; Rother, Dörte

    2016-12-01

    Recent advances in biocatalysis have strongly boosted its recognition as a valuable addition to traditional chemical synthesis routes. As for any catalytic process, catalyst's costs and stabilities are of highest relevance for the economic application in chemical manufacturing. Employing biocatalysts as whole cells circumvents the need of cell lysis and enzyme purification and hence strongly cuts on cost. At the same time, residual cell wall components can shield the entrapped enzyme from potentially harmful surroundings and aid to enable applications far from natural enzymatic environments. Further advantages are the close proximity of reactants and catalysts as well as the inherent presence of expensive cofactors. Here, we review and comment on benefits and recent advances in whole cell biocatalysis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Magnetic techniques for the detection and determination of xenobiotics and cells in water.

    Science.gov (United States)

    Safarik, Ivo; Horska, Katerina; Pospiskova, Kristyna; Safarikova, Mirka

    2012-09-01

    Magnetic techniques based on the application of magnetic nanoparticles and microparticles and films have been successfully used for the determination and detection of different types of xenobiotics (e.g. herbicides, insecticides, fungicides, aromatic and polyaromatic hydrocarbons, pentachlorophenol and heavy metal ions) as well as viruses, microbial pathogens and protozoan parasites in water samples. Preconcentration of xenobiotics from large volumes of samples can be performed using magnetic solid-phase extraction, stir-bar sorptive extraction and related procedures. This review provides basic information about these techniques. Published examples of successful applications document the importance of these simple and efficient procedures employing magnetic materials.

  16. Perovskite solar cells with a planar heterojunction structure prepared using room-temperature solutio