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Sample records for staining western blot

  1. 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.

  2. 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.

  3. 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.

  4. 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...

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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...

  12. 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.

  13. 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.

  14. 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.

  15. GAPDH and β-actin protein decreases with aging, making Stain-Free technology a superior loading control in Western blotting of human skeletal muscle

    DEFF Research Database (Denmark)

    Vigelsø Hansen, Andreas; Dybboe, Rie; Hansen, Christina Neigaard

    2015-01-01

    [β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and α-tubulin], as well as TP loaded measured by Stain-Free technology (SF) as normalization tool were tested. This was done using skeletal muscle samples from men subjected to physiological conditions often investigated in applied...... and differences in muscle fiber type. The novel SF technology adds lower variation to the results compared with the existing methods for correcting for loading inaccuracy in Western blotting of human skeletal muscle in applied physiology....

  16. 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.

  17. 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.

  18. GAPDH and β-actin protein decreases with aging, making Stain-Free technology a superior loading control in Western blotting of human skeletal muscle.

    Science.gov (United States)

    Vigelsø, Andreas; Dybboe, Rie; Hansen, Christina Neigaard; Dela, Flemming; Helge, Jørn W; Guadalupe Grau, Amelia

    2015-02-01

    Reference proteins (RP) or the total protein (TP) loaded is used to correct for uneven loading and/or transfer in Western blotting. However, the signal sensitivity and the influence of physiological conditions may question the normalization methods. Therefore, three widely used reference proteins [β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and α-tubulin], as well as TP loaded measured by Stain-Free technology (SF) as normalization tool were tested. This was done using skeletal muscle samples from men subjected to physiological conditions often investigated in applied physiology where the intervention has been suggested to impede normalization (ageing, muscle atrophy, and different muscle fiber type composition). The linearity of signal and the methodological variation coefficient was obtained. Furthermore, the inter- and intraindividual variation in signals obtained from SF and RP was measured in relation to ageing, muscle atrophy, and different muscle fiber type composition, respectively. A stronger linearity of SF and β-actin compared with GAPDH and α-tubulin was observed. The methodological variation was relatively low in all four methods (4-11%). Protein level of β-actin and GAPDH was lower in older men compared with young men. In conclusion, β-actin, GAPDH, and α-tubulin may not be used for normalization in studies that include subjects with a large age difference. In contrast, the RPs may not be affected in studies that include muscle wasting and differences in muscle fiber type. The novel SF technology adds lower variation to the results compared with the existing methods for correcting for loading inaccuracy in Western blotting of human skeletal muscle in applied physiology. Copyright © 2015 the American Physiological Society.

  19. 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.

  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. 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. 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...

  3. 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.

  4. Use of the REVERT®total protein stain as a loading control demonstrates significant benefits over the use of housekeeping proteins when analyzing brain homogenates by Western blot: An analysis of samples representing different gonadal hormone states.

    Science.gov (United States)

    Kirshner, Z Z; Gibbs, R B

    2018-01-30

    Western blot is routinely used to quantify differences in the levels of target proteins in tissues. Standard methods typically use measurements of housekeeping proteins to control for variations in loading and protein transfer. This is problematic, however, when housekeeping proteins also are affected by experimental conditions such as injury, disease, and/or gonadal hormone manipulations. Our goal was to evaluate an alternative and perhaps superior method for conducting Western blot analysis of brain tissue homogenates from rats with distinct physiologically relevant gonadal hormone states. Tissues were collected from the hippocampus, frontal cortex, and striatum of young adult female rats that either were ovariectomized to model surgical menopause, or were treated with the ovatotoxin 4-vinylcyclohexene diepoxide (VCD) to model transitional menopause. Tissues also were collected from rats with a normal estrous cycle killed at proestrus when estradiol levels are high, and at diestrus when estradiol levels are low. Western blot detection of α-tubulin, β-actin, and GAPDH was performed and were compared for sensitivity and reliability with a fluorescent total protein stain (REVERT ® ). Results show that the total protein stain was much less variable across samples and had a greater linear range than α-tubulin, β-actin, or GAPDH. The stain was stable and easy to use, and did not interfere with the immunodetection or multiplexed detection of the housekeeping proteins. In addition, we show that normalization of our data to total protein, but not to GAPDH, revealed significant differences in α-tubulin expression in the hippocampus as a function of treatment, and that gel-to-gel consistency in measuring differences between paired samples run on multiple gels was significantly better when data were normalized to total protein than when normalized to GAPDH. These results demonstrate that the REVERT ® total protein stain can be used in Western blot analysis of brain

  5. 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...

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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...

  10. 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.

  11. 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...

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  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. 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.

  9. 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.

  10. 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.

  11. 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

  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.

    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

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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....

  2. 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…

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. TREATMENTS TO MINIMIZE EXTRACTIVES STAIN IN WESTERN RED CEDAR

    OpenAIRE

    Rod Stirling,; Paul I. Morris

    2012-01-01

    Under certain conditions involving uneven exposure to weather, stains related to the extractives can reduce the aesthetic appeal of western red cedar in exterior applications such as fence boards, siding, and sidewall shingles. Selected chemical treatments were evaluated for their ability to inhibit the formation of extractives stain. DDACarbonate, alkyl amine oxide, and combinations thereof delayed extractives stain formation in an accelerated field test, with higher loadings having greater ...

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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 ...

  14. 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.

  15. 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.

  16. 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).

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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).

  2. 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.

  3. 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...

  4. 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.

  5. 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.

  6. 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...

  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. 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.

  9. 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...

  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. 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…

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. [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.

  18. 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.

  19. 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.

  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

    Diagnosis of allergy to wasp venom and decision to perform immunotherapy are based on the patient's history, along with skin and in vitro tests. Given the high prevalence of specific IgE also in non-allergic individuals, we evaluated the sensitivity and specificity of Western blots as a possible alternative to serum analyses of venom-specific IgE. Skin prick and/or intracutaneous tests were performed in 30 patients with allergy to wasp venom (generalized reaction following sting) along with serum analysis of venom-specific IgE (AlaSTAT microplate) and Western blots. Western blots were subsequently scanned and evaluated qualitatively and semiquantitatively by means of densitometry. Bands were scored 'positive' in cases of signal intensities beyond the mean plus 3 standard deviations of control sera. Twenty newborns (age 2-7 days) and 30 adults without systemic or increased local reactions to hymenoptera stings served as controls. Western blot sensitivity reached 100% in the samples studied and was thus superior to the sensitivities of serum analysis of venom-specific IgE using AlaSTAT microplate assay (90%) and skin tests (87%). The sensitivity of detection of a phospholipase A1 and antigen 5-specific band was higher compared with a hyaluronidase-specific band (97%, 97% and 86%, respectively). Twenty-four out of twenty-nine (83%) patients exhibited specific IgE antibodies against at least three distinct allergens. With regard to the specificities, skin tests as well as AlaSTAT microplate assays were comparable (90% and 93%, respectively), whereas the specificity of the Western blots was 70% if the appearance of any single band was regarded as a positive result. However, when analysing the appearance of a specific band for antigen 5 or hyaluronidase the specificity and overall diagnostic value increased markedly, making it the most efficient test (specificity 97% and 100%, efficiency 96.8% and 93.2%, respectively). As allergy to wasp venom is a severe and potentially

  1. 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.

  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. 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...

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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...

  16. 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.

  17. 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

    INTRODUCTION Differentiating between HIV-1 and HIV-2 infection is the first step to understanding HIV transmission, epidemiology and pathogenesis in geographical areas where both viruses circulate. In Cuba, positive results in mixed HIV-1/2 screening assays are confirmed by HIV-1 Western blot. Indeterminate results constitute the main limitation of this test and HIV-2 infection is among their possible causes; hence the importance of second-stage screening and confirmatory tests for HIV-2 infection. OBJECTIVE Investigate the contribution of HIV-2 antibodies to negative or indeterminate HIV-1 Western blot results in serum samples from 2005 through 2008 in Cuba. METHODS HIV-2 reactivity was studied using the ELISA DAVIH-VIH-2 diagnostic kit (Cuba) in 1723 serum samples with negative or indeterminate results for HIV-1 Western blot from January 2005 through December 2008. Duplicate sera reactive by ELISA were confirmed by HIV-2 Western blot, results interpreted according to WHO criteria. The epidemiological interview established by Cuba's National Program for Prevention and Control Sexually-Transmitted Diseases and HIV/AIDS was applied to HIV-2 Western blot-positive patients. RESULTS Among all sera studied, HIV-2 ELISA identified 12 reactive serum samples (0.70%) and 1711 non-reactive (99.30%). Western blot analysis of the 12 ELISA-reactive samples confirmed two positive samples (16.67%), 4 negative (33.33%) and 6 indeterminate (50%). Positive samples reacted against the p16, p26, gp36, p53, p56, p68 and gp105 proteins. All 12 ELISA-reactive samples belonged to the HIV-1 Western blot indeterminate group. The two HIV-2-positive samples showed well defined reactivity to gp160, p53, p55 and p34 of HIV-1. HIV-1 seroconversion was observed in all 10 remaining samples during serological followup. CONCLUSIONS Two new HIV-2 seropositive cases were diagnosed using DAVIH-VIH-2 and HIV-2 Western blot in indeterminate HIV-1 Western blot samples. Results support the recommendation

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. [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.

  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 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.

  5. Application of Western Blotting for the Immunodiagnosis of Fasciola hepatica in Cattle Using Excretory/Secretory Antigens

    OpenAIRE

    SARIMEHMETOĞLU, H. Oğuz

    2002-01-01

    In this study, protein bands of excretory/secretory antigens of Fasciola hepatica were determined using SDS-PAGE and Western blotting. Blood and faecal samples were obtained from cattle brought to Kazan slaughterhouse. After examining the organs and faecal samples of these cattle for Fasciola hepatica and other helminths, serum samples were divided into two groups as positive (10 cattle) and negative (5 cattle) for F. hepatica. The sera of these two groups were tested using Western blotting. ...

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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...

  2. 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.

  3. Enrichment of PrPSc in formalin-fixed, paraffin-embedded tissues prior to analysis by Western blot.

    Science.gov (United States)

    Nicholson, Eric M

    2011-07-01

    Diagnosis of prion disease is primarily through immunodetection of the infectious agent. Typically, 2 distinct procedures are recommended for a definitive diagnosis, with immunohistochemistry and Western blot providing the most information as to the specific isolate in question. In the past, these approaches required formalin-fixed, paraffin-embedded tissue and fresh or frozen tissue, respectively; however, methods have been developed that allow for use of fixed tissue for Western blot. The present study describes a method of enriching PrP(Sc) in formalin-fixed, paraffin-embedded tissues prior to Western blot analysis for the detection of PrP(Sc). With this modified procedure, 5 times the previously reported sample size may be used for analysis, greatly enhancing the sensitivity of this procedure.

  4. Canine pyometra: a study of the urinary proteins by SDS-PAGE and Western blot.

    Science.gov (United States)

    Zaragoza, C; Barrera, R; Centeno, F; Tapia, J A; Mañe, M C

    2004-05-01

    Canine pyometra often causes glomerulonephritis by immune complex deposition in the glomeruli. Proteinuria, ranging from moderate to severe, may be present secondary to renal damage. To determine urinary protein excretion due to pyometra, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted on urine from 15 bitches with pyometra and 10 healthy bitches. To characterize urinary immunoglobin excretion, Western blot analysis of the urine samples using antibodies to canine IgG and IgA was also performed. Nine bands were detected by electrophoresis in bitches with pyometra, while only four were detected in the healthy animals. The urinary proteins from bitches with pyometra were primarily of glomerular origin; 58% were of medium-high molecular weight (MW), and the remainder were low MW. None of the healthy dogs had IgG or IgA in their urine, whereas three bitches with pyometra had IgG in their urine and another bitch with pyometra had both IgG and IgA. The low proportion of bitches with urinary immunoglobins was probably be due to early diagnosis of the disease. Although only a limited number of dogs was used, this study is apparently the first to characterize the electrophoretic pattern of urinary proteins and to quantify urinary excretion of IgG and IgA in bitches with pyometra.

  5. Serological diagnosis of North American Paragonimiasis by Western blot using Paragonimus kellicotti adult worm antigen.

    Science.gov (United States)

    Fischer, Peter U; Curtis, Kurt C; Folk, Scott M; Wilkins, Patricia P; Marcos, Luis A; Weil, Gary J

    2013-06-01

    Abstract. We studied the value of an IgG Western blot (WB) with Paragonimus kellicotti (Pk) antigen for diagnosis of North American paragonimiasis. The test was evaluated with sera from patients with Pk and Paragonimus westermani infections, with control sera from patients with other helminth infections, and sera from healthy Americans. All 11 proven Pk infection sera and two samples from suspected cases that were negative by P. westermani WB at the Centers for Disease Control and Prevention (CDC) contained antibodies to antigens at 34 kDa and at 21/23 kDa. Seven of 7 P. westermani sera contained antibodies to the 34 kDa antigen, but only 2 recognized the 21/23 kDa doublet. No control samples were reactive with these antigens. Antibody reactivity declined after praziquantel treatment. Thus, the P. kellicotti WB appears to be superior to P. westermani WB for diagnosing Pk infections, and it may be useful for assessing responses to treatment.

  6. 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.

  7. 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.

  8. Clinical usefulness of Western blotting and ELISA avidity for the diagnosis of human toxocariasis.

    Science.gov (United States)

    Rudzińska, M; Kowalewska, B; Sikorska, K

    2017-01-01

    The serodiagnosis of human toxocariasis is difficult. Specific IgGs detected routinely with ELISA based on Toxocara excretory-secretory (TES) antigens often persist for years at an elevated level, which does not allow either the differentiation between an active and persistent infection or monitoring of the effect of treatment. Additionally, false-positive results may occur in co-infections with other helminths due to cross-reactions. We evaluated the usefulness of an IgG avidity index (AI) and a Western blotting (WB) IgG in the diagnosis of patients suspected of Toxocara infection. We studied 138 subjects who were submitted to serological testing two or more times. Confirmation of an infection by WB was achieved in 73.2% of patients. A high AI was obtained in 89.1% of patients, and low AI and borderline AI were found in only 10.9%. Low and borderline values of AI remained at similar levels in subsequent studies over 2-3 years. The results showed the necessity of obligatory verification of all ELISA IgG positive and questionable results by WB. The index of IgG avidity may be helpful in excluding recent infection, but its usefulness in detecting an active phase of invasion requires further research. © 2016 John Wiley & Sons Ltd.

  9. 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.

  10. 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.

  11. Detection of IgA and IgM antibodies to HIV-1 in neonates by radioimmune western blotting.

    OpenAIRE

    Portincasa, P.; Conti, G.; Re, M. C.; Chezzi, C.

    1992-01-01

    OBJECTIVE--To detect infection with HIV-1 by IgA and IgM response at birth in children born to HIV-1 seropositive mothers. DESIGN--Western blotting and radioimmune western blotting on stored sera from infected and uninfected babies born to HIV-1 seropositive mothers. Sera were pretreated to remove IgG. SETTING--Parma and Bologna, Italy. SUBJECTS--12 infected and five uninfected babies born to HIV-1 seropositive mothers and three babies born to seronegative mothers. MAIN OUTCOME MEASURES--Effe...

  12. Antibody responses to Borrelia burgdorferi detected by western blot vary geographically in Canada.

    Science.gov (United States)

    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, geographic variations in serological responses were also explored. Metrics of relative sensitivity, specificity and the kappa statistic measure of concordance were used to assess the capacity of responses to individual proteins to predict the overall IgG WB result of 2524 EIA (C6)-positive samples from across Canada. Geographic and interannual variations in proportions of samples testing positive were explored by logistic regression. No one protein was highly concordant with the IgG WB result. Significant variations were found amongst years and geographic regions in the prevalence of samples testing positive using the overall IgG WB algorithm, and for individual proteins of the algorithm. In most cases the prevalence of samples testing positive were highest in Nova Scotia, and lower in samples from Manitoba westwards. These findings suggest that the current two tier test may not be simplified and continued use of the current two-tier test method and interpretation is recommended. Geographic and interannual variations in the prevalence of samples testing positive may be consistent with B. burgdorferi strain variation in Canada, and further studies are needed to explore this.

  13. Antibody responses to Borrelia burgdorferi detected by western blot vary geographically in Canada.

    Directory of Open Access Journals (Sweden)

    Nicholas H Ogden

    Full Text Available 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, geographic variations in serological responses were also explored. Metrics of relative sensitivity, specificity and the kappa statistic measure of concordance were used to assess the capacity of responses to individual proteins to predict the overall IgG WB result of 2524 EIA (C6-positive samples from across Canada. Geographic and interannual variations in proportions of samples testing positive were explored by logistic regression. No one protein was highly concordant with the IgG WB result. Significant variations were found amongst years and geographic regions in the prevalence of samples testing positive using the overall IgG WB algorithm, and for individual proteins of the algorithm. In most cases the prevalence of samples testing positive were highest in Nova Scotia, and lower in samples from Manitoba westwards. These findings suggest that the current two tier test may not be simplified and continued use of the current two-tier test method and interpretation is recommended. Geographic and interannual variations in the prevalence of samples testing positive may be consistent with B. burgdorferi strain variation in Canada, and further studies are needed to explore this.

  14. Electrostatic protein immobilization using charged polyacrylamide gels and cationic detergent microfluidic Western blotting.

    Science.gov (United States)

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

    2012-03-06

    We report a novel protein immobilization matrix for fully integrated microfluidic Western blotting (WB). The electrostatic immobilization gel (EIG) enables immobilization of all proteins sized using cetyl trimethylammonium bromide polyacrylamide gel electrophoresis (CTAB-PAGE), for subsequent electrophoretic probing with detection affinity reagents (e.g., labeled antibodies). The "pan-analyte" capture strategy introduced here uses polyacrylamide gel grafted with concentrated point charges (zwitterionic macromolecules), in contrast to existing microfluidic WB strategies that rely on a sandwich immunoassay format for analyte immobilization and detection. Sandwich approaches limit analyte immobilization to capture of only a priori known targets. A charge interaction mechanism study supports the hypothesis that electrostatic interaction plays a major role in analyte immobilization on the EIG. We note that protein capture efficiency depends on both the concentration of copolymerized charges and ionic strength of the gel buffer. We demonstrate pan-analyte immobilization of sized CTAB-laden model proteins (protein G, ovalbumin, bovine serum albumin, β-galactosidase, lactoferrin) on the EIG with initial capture efficiencies ranging from 21 to 100%. Target proteins fixed on the EIG (protein G, lactoferrin) are detected using antibody probes with signal-to-noise ratios of 34 to 275. The approach advances protein immunoblotting performance through 200× reduction on sample consumption, 12× reduction in assay duration, and automated assay operation, compared to slab-gel WB. Using the microfluidic WB assay, assessment of lactoferrin in human tear fluid is demonstrated with a goal of advancing toward nonbiopsy-based diagnosis of Sjögren's Syndrome, an autoimmune disease.

  15. Western blot analysis of sera from dogs with suspected food allergy.

    Science.gov (United States)

    Favrot, Claude; Linek, Monika; Fontaine, Jacques; Beco, Luc; Rostaher, Ana; Fischer, Nina; Couturier, Nicolas; Jacquenet, Sandrine; Bihain, Bernard E

    2017-04-01

    Food allergy is often suspected in dogs with clinical signs of atopic dermatitis. This diagnosis is confirmed with an elimination diet and a subsequent challenge with regular food. Laboratory tests for the diagnosis of food allergy in dogs are unreliable and/or technically difficult. Cyno-DIAL ® is a Western blot method that might assist with the selection of an appropriate elimination diet. To evaluate the performance of Cyno-DIAL ® for the selection of an elimination diet and diagnosis of food allergy. Thirty eight dogs with atopic dermatitis completed an elimination diet. Combining the results of the diet trials and the challenges, 14 dogs were classified as food allergic (FA), 22 as nonfood-allergic and two as ambiguous cases. Amongst all dogs and amongst dogs with a clinical diagnosis of FA, 3% and 7% (respectively) were positive to Royal Canin Anallergenic ® , Vet-Concept Kanguru ® or Vet-Concept Dog Sana ® ; 8% and 7% to Hill's d/d Duck and Rice ® ; 8% and 21% to Hill's z/d Ultra Allergen Free ® ; 53% and 64% to Eukanuba Dermatosis FP ® ; and 32% and 43% to a home-cooked diet of horse meat, potatoes and zucchini. The specificity and sensitivity of Cyno-DIAL ® for diagnosing food allergy were 73% and 71%, respectively. Although Cyno-DIAL ® was considered potentially useful for identifying appropriate foods for elimination diet trials, it cannot be recommended for the diagnosis of food allergy. The Cyno-DIAL ® test performed better than some previously evaluated ELISA-based tests. © 2017 ESVD and ACVD.

  16. Indeterminate human immunodeficiency virus western blot results in Iranian patients with discordant screening assay results

    International Nuclear Information System (INIS)

    Ravanshad, M.; Sabahi, F.; Mahboudi, F.; Sabahi, F.

    2006-01-01

    The Western blot (WB) assay is the most widely accepted confirmatory assay for the detection and confirmation of antibodies to human immunodeficiency virus type 1 (HIV-1) and 2 (HIV-2). However, indeterminate WB reactivity to HIV-1 and HIV-2 proteins may occur in individuals who do not appear to be infected with HIV. In this study, we describe the results of indeterminate WB reactivity in Iranian patients with discordant screening assays. The samples were obtained from Iranian Blood Transfusion Center, Tehran, Iran and evaluated in the Biotechnology Process Development Center, Pasteur Institute of Iran, Tehran, Iran between 2003 and 2004. A total of 4707 were tested for the presence of HIV-1 antibodies. Six hundred and four (12.8%) patients tested for HIV were positive for HIV-1 antibody. Nine (1.49%) have discordant results among screening assays and indeterminate WB results as interpreted by Centers for Disease Control and Prevention (CDC) criteria. Most (66.7%) of these indeterminate WB results were due to p24 reactivity. However, 2(22.2%) display reactivity to both gp41 and gp120 proteins [Positive by World Health Organization (WHO) criteria]. Of 9 WB assays initially indeterminate by the CDC criteria and with follow-up samples 8(88.8%) became negative when retested subsequently while one (11.1%) remained indeterminate for more than a year and were thus considered negative. In addition all the indeterminate samples were negative when assessed by polymerase chain reaction assay. In general, there were was an 88.8% concordance between the CDC and WHO criteria for an indeterminate WB result. The CDC II criteria for an indeterminate WB result. The CDC II criteria best met the specified objectives for diagnosis in our setting. (author)

  17. 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.

  18. 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.

  19. Enrichment of PrPSc in Formalin Fixed Paraffin Embedded Tissues Prior to Analysis by Western Blot

    Science.gov (United States)

    Diagnosis of prion disease is primarily through immunodetection of the infectious agent. Typically, 2 distinct procedures are recommended for a definitive diagnosis with immunohistochemistry and Western blot providing the most information as to the specific isolate in question. In the past these app...

  20. 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

  1. 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.

  2. Solid-Color Stains on Western Redcedar and Redwood Siding

    Science.gov (United States)

    Mark Knaebe

    2013-01-01

    You have decided to put wood siding on your new house. Several questions are probably going through your mind: “What’s the best type of wood?” “Should I use paint or stain?” “Should I apply the finish before or after I install the siding?”

  3. Characterization of 65 Epitope-Specific Dystrophin Monoclonal Antibodies in Canine and Murine Models of Duchenne Muscular Dystrophy by Immunostaining and Western Blot

    Science.gov (United States)

    Shin, Jin-Hong; Yue, Yongping; Morris, Glenn E.; McIntosh, Mark A.; Duan, Dongsheng

    2014-01-01

    Epitope-specific monoclonal antibodies can provide unique insights for studying cellular proteins. Dystrophin is one of the largest cytoskeleton proteins encoded by 79 exons. The absence of dystrophin results in Duchenne muscular dystrophy (DMD). Over the last two decades, dozens of exon-specific human dystrophin monoclonal antibodies have been developed and successfully used for DMD diagnosis. Unfortunately, the majority of these antibodies have not been thoroughly characterized in dystrophin-deficient dogs, an outstanding large animal model for translational research. To fill the gap, we performed a comprehensive study on 65 dystrophin monoclonal antibodies in normal and dystrophic dogs (heart and skeletal muscle) by immunofluorescence staining and western blot. For comparison, we also included striated muscles from normal BL10 and dystrophin-null mdx mice. Our analysis revealed distinctive species, tissue and assay-dependent recognition patterns of different antibodies. Importantly, we identified 15 antibodies that can consistently detect full-length canine dystrophin in both immunostaining and western blot. Our results will serve as an important reference for studying DMD in the canine model. PMID:24516626

  4. Retrospective analysis of sheep scrapie by western blotting with formalin-fixed paraffin-embedded (FFPE) tissues.

    Science.gov (United States)

    Dorj, Gantsetseg; Okada, Hiroyuki; Miyazawa, Kohtaro; Masujin, Kentaro; Kimura, Kumiko; Mohri, Shirou; Yokoyama, Takashi

    2012-09-01

    An abnormal isoform of prion protein (PrP(Sc)) was extracted from formalin-fixed paraffin-embedded (FFPE) tissues from sheep and analyzed by western blotting. PrP(Sc) immunoreactivity against anti-PrP monoclonal antibody T2, which recognizes discontinuous PrP sequences, differed amongst individual scrapie sheep cases. This may reflect structural differences in PrP(Sc) that have been formalin-fixed prior to their extraction. This study indicates that western blotting by using FFPE tissues is useful for the retrospective analysis of transmissible spongiform encephalopathies in which only formalin-fixed samples are available and in conducting transmissible spongiform encephalopathies surveillance where freezing system is insufficient.

  5. Detection of early antibodies in human immunodeficiency virus infection by enzyme-linked immunosorbent assay, Western blot, and radioimmunoprecipitation.

    OpenAIRE

    Saah, A J; Farzadegan, H; Fox, R; Nishanian, P; Rinaldo, C R; Phair, J P; Fahey, J L; Lee, T H; Polk, B F

    1987-01-01

    A current concept of the serological response to human immunodeficiency virus (HIV) infection in humans is that antibodies to core antigens (p55, p24, and p15) are detectable earlier during initial stages of antibody production than antibodies against envelope antigens (gp160, gp120, and gp41). Comparative studies of Western blot (immunoblot), radioimmunoprecipitation assay (RIPA), and enzyme-linked immunosorbent assay (ELISA) during initial antibody production are limited to case reports and...

  6. 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.

  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. 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.

  9. 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.

  10. 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.

  11. Cross antigenicity of immunodominant polypeptides of somatic antigen of Oesophagostomum columbianum with other helminths by western blotting.

    Science.gov (United States)

    Dalal, Sunita; Prasad, Arvind; Nasir, Abdul; Saini, Vijesh Kumar

    2015-11-01

    Oesophagostomum columbianum in small ruminants in India is found as mixed infection commonly in sheep and goat. Haemonchus contortus, an abomasal nematode is found as concurrent infection with it. Eggs of Haemonchus and O. columbianum cannot be easily distinguished. Diagnosis of O. columbianum may only be possible if a non-cross antigenic polypeptide was available for immunodiagnosis. Somatic antigen (SoAg) of O. columbianum was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodominant polypeptides were identified by western blotting with homologous hyperimmune serum (HIS) and experimental sera of sheep or goat infected with other helminths. SoAg of O. columbianum was immunoaffinity purified. Sharp polypeptide bands of 130, 72 and 68 KDa were observed along with several faint bands of lower molecular weight. Western blot of purified SoAg of O. columbianum with homologous HIS showed reaction with all the protein bands of 17, 28, 30, 32, 35, 38, 50, 68, 100, 130, 150, and 170 kDa. For identification of non-cross antigenic polypeptide, immunoaffinity purified SoAg of O. columbianum was reacted to heterologous HIS against H. contortus, Paramphistomum epiclitum, and Fasciola gigantica in western blotting utilizing completely dry method (i-blot). Among high molecular weight polypeptides 100 and 150 kDa were non-cross antigenic and among low molecular weight except 50 kDa polypeptide, 17, 30, 32, 35, and 38 kDa of O. columbianum were not cross antigenic with other helminths. Hence, polypeptides of 17, 30, 32, 35 and 38 kDa as well as 100 and 150 kDa polypeptides of O. columbianum may be exploited for immunodiagnosis of the infection in sheep and goat with extensive studies on cross antigenicity.

  12. [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%.

  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. Western blot banding pattern in early Lyme borreliosis among patients from an endemic region of north-eastern Poland.

    Science.gov (United States)

    Flisiak, R; Wierzbicka, I; Prokopowicz, D

    1998-01-01

    Aim of this study was evaluation of Western blot banding patterns in different clinical forms of early Lyme borreliosis diagnosed in patients from north-eastern Poland, recognized as endemic for tick-borne diseases. Study was performed on serum samples of 48 patients with Lyme borreliosis and 26 healthy volunteers, as controls. Samples tested routinely for total antibody with enzyme immunoassay were subsequently analysed for specific antibodies with Western blot based on antigen extract of European strain of Borrelia burgdorferi. In patients, IgM antibodies were the most frequently directed against 41 kDa and 58 kDa antigens, whereas in control group only antibodies against 45 kDa and 58 kDa were present. Similar response was observed in respect to IgG antibodies. Evaluation of banding pattern in respect to clinical form of the disease revealed the highest prevalence of IgM and IgG anti-41 kDa antibodies in patients with erythema migrans and Lyme arthritis, and anti-58 kDa in neuroborreliosis patients, who had no anti-21 kDa antibodies. Relatively high frequency of IgG antibodies against 21, 30 and 93 kDa antigens was typical for neuroborreliosis. Bands count was significantly higher in different clinical forms of the disease than in controls, and it was the highest in neuroborreliosis. Combined analysis of Western blot results (IgM/IgG) enabled to achieve higher sensitivity (84%) and specificity (100%) than available with the most recommended EIA kits.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. [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.

  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. 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

  7. Quantitative Western ligand blotting reveals common patterns and differential features of IGFBP-fingerprints in domestic ruminant breeds and species.

    Science.gov (United States)

    Wirthgen, Elisa; Höflich, Christine; Spitschak, Marion; Helmer, Carina; Brand, Bodo; Langbein, Jan; Metzger, Friedrich; Hoeflich, Andreas

    2016-02-01

    The insulin-like growth factor binding proteins (IGFBPs) are determinants of local IGF-effects and thus have an impact on growth and metabolism in vertebrate species. In farm animals, IGFBPs are associated with traits such as growth rate, body composition, milk production, or fertility. It may be assumed, that selective breeding and characteristic phenotypes of breeds are related to differential expression of IGFBPs. Therefore, the aim of the present study was to investigate the effects of selective breeding on blood IGFBP concentrations of farm animals. Breeds of the sheep, goat, and cattle species were investigated. IGFBP-3, -2, and -4 were analyzed with quantitative Western ligand blotting (qWLB), enabling comprehensive monitoring of intact IGFBPs with IGF-binding capacity. We show that in sera of all species and breeds investigated, IGFBP-3, -2, and -4 were simultaneously detectable by qWLB analysis. IGFBP-3 and the total amount of IGFBPs were significantly increased (PWestern ligand blotting as an attractive tool for biomarker development and molecular phenotyping in farm animal breeds. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  8. Detection of anti-HIV-1 IgG antibodies in whole saliva by GACELISA and Western blot assays.

    Science.gov (United States)

    Matee, M I; Lyamuya, E F; Simon, E; Mbena, E C; Kagoma, C; Samaranayake, L P; Scheutz, F

    1996-05-01

    The present study, based on 158 HIV seropositives and 167 HIV seronegatives, demonstrates that saliva collected with the Omni-SAL device and tested with GACELISA (an IgG antibody capture ELISA) is an effective non-invasive alternative to serum for anti-HIV IgG antibody screening. The study also shows that a conventional serum Western blot kit can be used, with slight modifications, for confirmatory testing of saliva specimens. Collecting saliva with the Omni-SAL device had a very good acceptance rate among Tanzanian subjects, and although this diagnostic method is not yet known by the general public, 65% of the study participants preferred to give saliva instead of blood for HIV testing.

  9. Standardisation of Western blotting to detect HTLV-1 antibodies synthesised in the central nervous system of HAM/TSP patients

    Directory of Open Access Journals (Sweden)

    Luiz Claudio Pereira Ribeiro

    2013-09-01

    Full Text Available Intrathecal synthesis of human T-lymphotropic virus type 1 (HTLV-1 antibodies (Abs represents conclusive evidence of a specific immune response in the central nervous system of HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP patients. Western blotting (WB for HTLV Abs in serum is a confirmatory test for HTLV-1 infection. The aim of this study was to standardise the Western blot to demonstrate the intrathecal pattern of Abs against HTLV-1 proteins in HAM/TSP patients. Paired cerebrospinal fluid (CSF and serum samples were selected from 20 patients with definite HAM/TSP, 19 HTLV-1 seronegative patients and two HTLV-1 patients without definite HAM/TSP. The presence of reactive bands of greater intensity in the CSF compared to serum (or bands in only the CSF indicated the intrathecal synthesis of anti-HTLV-1 Abs. All definite HAM/TSP patients presented with an intrathecal synthesis of anti-HTLV-1 Abs; these Abs were not detected in the control patients. The most frequent intrathecal targets of anti-HTLV-1 Abs were GD21, rgp46-I and p24 and, to a lesser extent, p19, p26, p28, p32, p36, p53 gp21 and gp46. The intrathecal immune response against env (GD21 and rgp46-I and gag (p24 proteins represents the most important humoral pattern in HAM/TSP. This response may be used as a diagnostic marker, considering the frequent association of intrathecal anti-HTLV-1 Ab synthesis with HAM/TSP and the pathogenesis of this neurological disease.

  10. [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.

  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. 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.

  13. Prevalence of indeterminate human immunodeficiency virus western blot results in pregnant women attended at a public hospital in Presidente Prudente, Brazil

    Directory of Open Access Journals (Sweden)

    Denise Cremonezi

    Full Text Available The AIDS epidemic is spreading rapidly among women worldwide, offering increasing opportunities for vertical transmission of HIV. In Brazil, the prevalence of HIV infection among pregnant women is less than 1%. Therefore, the positive predictive value of an HIV EIA test tends to be lower than the more frequent indeterminate Western blot result. Pregnant women receiving antenatal care, from 2000 to 2004, at a public secondary hospital in the city of Presidente Prudente, São Paulo, Brazil, were systematically screened for HIV by means of two distinct EIA tests, in order to determine the prevalence of indeterminate Western blot results among pregnant women showing discordance in both HIV EIA tests and indirect immunofluorescence assay. Confirmatory indirect immunofluorescence was performed on material for all women with positive results in both EIA tests. Whenever there were positive results in EIA and IIA, the applicant was retested by the initial screening assay. Only those not showing concordance in results in EIA and IAA had a Western blot performed. The viral load was measured in pregnant women with positive or indeterminate Western blot results. Out of 9,786 sera, 105 (1.0% were positive in the two HIV EIA screening tests, confirmed by indirect immunofluorescence. Among these women, Western blot was interpreted as indeterminate in 11 (0.1% cases and their viral load was <50 copies/mL. We found a prevalence of 0.1% HIV indeterminate Western blots in pregnant women from Presidente Prudente and the surrounding region; none of these pregnant women had positive HIV viral loads.

  14. Exposure to Sarcocystis spp. in horses from Spain determined by Western blot analysis using Sarcocystis neurona merozoites as heterologous antigen.

    Science.gov (United States)

    Arias, M; Yeargan, M; Francisco, I; Dangoudoubiyam, S; Becerra, P; Francisco, R; Sánchez-Andrade, R; Paz-Silva, A; Howe, D K

    2012-04-30

    Horses serve as an intermediate host for several species of Sarcocystis, all of which utilize canids as the definitive host. Sarcocystis spp. infection and formation of latent sarcocysts in horses often appears to be subclinical, but morbidity can occur, especially when the parasite burden is large. A serological survey was conducted to determine the presence of antibodies against Sarcocystis spp. in seemingly healthy horses from the Galicia region of Spain. Western blot analyses using Sarcocystis neurona merozoites as heterologous antigen suggested greater than 80% seroprevalance of Sarcocystis spp. in a sample set of 138 horses. The serum samples were further tested with enzyme-linked immunosorbent assays (ELISAs) based on recombinant S. neurona-specific surface antigens (rSnSAGs). As expected for horses from the Eastern Hemisphere, less than 4% of the serum samples were positive when analyzed with either the rSnSAG2 or the rSnSAG4/3 ELISAs. An additional 246 horses were tested using the rSnSAG2 ELISA, which revealed that less than 3% of the 384 samples were seropositive. Collectively, the results of this serologic study suggested that a large proportion of horses from this region of Spain are exposed to Sarcocystis spp. Furthermore, the anti-Sarcocystis seroreactivity in these European horses could be clearly distinguished from anti-S. neurona antibodies using the rSnSAG2 and rSnSAG4/3 ELISAs. Copyright © 2011 Elsevier B.V. All rights reserved.

  15. Western blotting using Strongyloides ratti antigen for the detection of IgG antibodies as confirmatory test in human strongyloidiasis

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    Luciana Pereira Silva

    2003-07-01

    Full Text Available The present study was conducted to evaluate the frequency of antigenic components recognized by serum IgG antibodies in Western blotting (WB using a Strongyloides ratti larval extract for the diagnosis of human strongyloidiasis. In addition, the WB results were compared to the enzyme-linked immunosorbent assay (ELISA and the indirect immunofluorescence antibody test (IFAT results. Serum samples of 180 individuals were analyzed (80 with strongyloidiasis, 60 with other intestinal parasitoses, and 40 healthy individuals. S. ratti was obtained from fecal culture of experimentally infected Rattus rattus. For IFAT, S. ratti larvae were used as antigen and S. ratti larval antigenic extracts were employed in WB and ELISA. Eleven S. ratti antigenic components were predominantly recognized by IgG antibodies in sera of patients with strongyloidiasis. There was a positive concordance for the three tests in 87.5% of the cases of strongyloidiasis. The negative concordance in the three tests was 94% and 97.5%, in patients with other intestinal parasitoses and healthy individuals, respectively. In cases of positive ELISA and negative IFAT results, diagnosis could be confirmed by WB. ELISA, IFAT, and WB using S. ratti antigens showed a high rate of sensitivity and specificity. In conclusion, WB using S. ratti larval extract was able to recognize 11 immunodominant antigenic components, showing to be a useful tool to define the diagnosis in cases of equivocal serology.

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

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    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

  17. 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.

  18. 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.

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

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    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.

  20. 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.

  1. 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.

  2. 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.

  3. Identification of recombinant human EPO variants in greyhound plasma and urine by ELISA, LC-MS/MS and western blotting: a comparative study.

    Science.gov (United States)

    Timms, Mark; Steel, Rohan; Vine, John

    2016-02-01

    The recombinant human erythropoietins epoetin alfa (Eprex®), darbepoetin (Aranesp®) and methoxy polyethylene glycol-epoetin beta (Mircera®) were administered to greyhounds for 7, 10 and 14 days respectively. Blood and urine samples were collected and analysed for erythropoietin by ELISA, LC-MS/MS and western blotting. Limits of confirmation in plasma for western blotting and LC-MS/MS methods ranged from a low of 2.5mIU/mL, and closely matched the sensitivity of ELISA screening. Copyright © 2015 John Wiley & Sons, Ltd.

  4. 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)

  5. Detection of PrP(Sc) in formalin-fixed, paraffin-embedded tissue by Western blot differentiates classical scrapie, Nor98 scrapie, and bovine spongiform encephalopathy.

    Science.gov (United States)

    Loiacono, Christina M; Beckwith, Nadine; Kunkle, Robert A; Orcutt, Dennis; Hall, S Mark

    2010-09-01

    Transmissible, spongiform encephalopathies including bovine spongiform encephalopathy (BSE) and scrapie are fatal neurodegenerative disorders associated with the presence of an infectious abnormal isoform of normal mammalian proteins called prions. Identification of the prion protein associated with scrapie (PrP(Sc)) in the central nervous system is typically based upon immunoassays including immunohistochemistry (IHC) using formalin-fixed tissues or Western blot (WB) assays using fresh and/or frozen, non-formalin-fixed tissues. Each assay can discriminate between BSE, classical scrapie, and a previously reported strain of scrapie recently identified in the United States named Nor98 scrapie. Different tissue samples are required from the same animal to run these 2 different immunoassays. This may result in inconsistent test results for the same animal. Sampling problems such as collecting insufficient volumes of fresh tissue or less than optimal anatomic location of brainstem for IHC can affect the ability of the test procedures to offer definitive and discriminatory results. Recently, a WB method using formalin-fixed, paraffin-embedded (FFPE) tissue to identify PrP(Sc) was developed that successfully identified PrP(Sc) in sheep affected by classical scrapie. In the current study, the use of this technique to produce discriminatory results identifying classical BSE in bovine tissue and both classical and Nor98 scrapie in ovine tissue using paraffin-embedded brain samples is described. Protein-banding patterns from WB using FFPE tissue were similar to protein-banding patterns produced by WB assays utilizing fresh tissues from the same animals, and results correlated well with the IHC PrP(Sc)-positive staining present in the cerebellum and obex regions of brain samples from these animals.

  6. State-of-the-art housekeeping proteins for quantitative western blotting: Revisiting the first draft of the human proteome.

    Science.gov (United States)

    Lee, Hyun-Gwan; Jo, Jihoon; Hong, Hyun-Hee; Kim, Kee K; Park, Joong-Ki; Cho, Sung-Jin; Park, Chungoo

    2016-07-01

    Western blotting (WB) analysis is the most popular and widely used methodology for protein detection and characterization over recent decades. In accordance with the advancement of the technologies for the acquisition of WB signals, a quantitative value is used to present the abundance of target proteins in a complex sample, thereby requiring the use of specific proteins as internal references that represent total proteins. Heretofore, proteins encoded by housekeeping genes such as GAPDH, β-tubulin and β-actin have been commonly used as loading controls without any hesitation because their mRNA expression levels tend to be high and constant in many different cells and tissues. Experimentally, however, some of the housekeeping reference proteins are often displayed with inconsistent expression levels in both homogeneous and heterogeneous tissues, and, in terms of mRNA levels, they have a weak correlation to the abundance of proteins. To estimate accurate, reliable, and reproducible protein quantifications, it is crucial to define appropriate reference controls. For this paper, we explored the recently released large-scale, human proteomic database ProteomicsDB including 16 857 liquid chromatography tandem-mass-spectrometry data from 27 human tissues, and suggest 20 ubiquitously- and constitutively-expressed, putative internal-reference controls for the quantification of differential protein expressions. Intriguingly, the most commonly used, known housekeeping genes were entirely excluded in our newly defined candidates. Although the applications of the candidates under many different biological conditions and in other organisms are yet to be empirically verified, we propose reliable, potential loading controls for a WB analysis in this paper. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Detection of Potentially Diagnostic Leishmania Antigens with Western Blot Analysis of Sera from Patients with Cutaneous and Visceral Leishmaniases

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    Seyyed Javad SEYYEDTABAEI

    2017-06-01

    Full Text Available Background: Visceral leishmaniasis (VL and cutaneous leishmaniasis (CL are important public health problems in Iran. We aimed to evaluate the diagnostic potential of Western blot (WB compared with indirect immunofluorescence test (IFAT to serodiagnosis of leishmaniasis.Methods: This study was performed from 2010-2014 and participants were different parts of Iran. Serum samples were obtained from 43 patients with proven CL, 33 patients with proven VL, 39 patients with other parasitic diseases and 23 healthy individuals. Results: WB sensitivity for CL and VL was 100% and 91%, compared to IFA 4.6% and 87.8%, respectively. Sera from patients with CL and VL recognized numerous antigens with molecular weights ranging from 14 to 68 kDa and 12 to 94 kDa, respectively. The most sensitive antigens were 14 and 16 kDa for CL recognized by 100% of the sera from patients with proven CL and 12, 14 and 16 kDa for VL, recognized by 63.6%, 100% and 63.6% of the sera from patients with proven VL respectively. WB analysis is more sensitive than IFAT for the diagnosis of leishmaniasis particularly in cases of cutaneous leishmaniasis. The 12, 14 and 16 kDa can be valuable diagnostic molecules for serodiagnosis of leishmaniasis because at least two immunogenic molecules were simultaneously detected by all patient sera, as well as produced antibodies against these antigens have no cross-reactivity with other control groups.Conclusion: WB could be useful for screening and serodiagnosis of CL and VL in epidemiologic studies in endemic areas.

  8. Estrogen receptors alpha and beta in rat placenta: detection by RT-PCR, real time PCR and Western blotting

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    Al-Bader Maie D

    2006-03-01

    Full Text Available Abstract Background High levels of estrogens during pregnancy not only retard placental and fetal growth but can lead to reproductive tract abnormalities in male progeny. Estrogens act through estrogen receptors (ER to modulate the transcription of target genes. These ER exist in two isoforms, ER alpha and ER beta and recently several variants of these isoforms have been identified. Methods The expressions of ER isoforms and variants have been studied in rat placenta at 16, 19 and 21 days gestation (dg. Gene expression was assessed using RT-PCR and real time PCR while protein expression was studied using Western blotting followed by immunodetection. Placental homogenates were probed with: a monoclonal antibody raised against the steroid binding domain of the ER alpha (ER alpha -S, a monoclonal antibody raised against the hinge region of ER alpha (ER alpha -H and a polyclonal antibody raised against the amino terminus of ER beta. Results ER alpha and ER beta mRNA and protein were detected from as early as 16 dg. Two PCR products were detected for ER alpha, one for the wild type ER alpha, and a smaller variant. Real time PCR results suggested the presence of a single product for ER beta. The antibodies used for detection of ER alpha protein both identified a single 67 kDa isoform; however a second 54 kDa band, which may be an ER alpha variant, was identified when using the ER alpha -H antibody. The abundance of both ER alpha bands decreased significantly between 16 and 19 dg. As for ER beta, four bands (76, 59, 54 and 41 kDa were detected. The abundance of the 59 and 54 kDa bands decreased significantly between 16 and 19 dg. Conclusion This study shows that both ER protein isoforms and their variants are present in rat placenta. The decrease in their expression near parturition suggests that the placenta may be relatively unresponsive to estrogens at this stage.

  9. Application of Western blot analysis for the diagnosis of Encephalitozoon cuniculi infection in rabbits: example of a quantitative approach.

    Science.gov (United States)

    Desoubeaux, Guillaume; Pantin, Ana; Peschke, Roman; Joachim, Anja; Cray, Carolyn

    2017-02-01

    Diagnosis of Encephalitozoon cuniculi infection in rabbits remains a major veterinary issue. ELISA or immunofluorescence assays are the current reference standards of serological tests. However, these conventional techniques suffer from a lack of accuracy for distinguishing active from past infections, as a positive serostatus is common in clinically normal rabbits. In this study, we assessed the diagnostic performance of Western blot (WB) to detect both anti-E. cuniculi immunoglobulin G (IgG) and immunoglobulin M (IgM) in comparison with ELISA and to address the intensity of the immune response through a quantitative approach. Positive WB results were highly correlated with the E. cuniculi-related diseased status (P < 0.0001). Although it was more labor intensive and less standardized, quantitative WB provided detailed comparable analysis regarding the humoral response and diagnostic performance similar to ELISA testing with statistically higher sensitivity (88.4 vs. 76.1% for IgG detection and 84.3 vs. 70.4% for IgM, P < 0.01). Several specific WB bands were shown to be significantly associated with concomitant clinical signs, like the one located at 50 kDa (OR = 8.2, [2.4-27.7], P = 0.0008) for IgG and (OR = 27.9, [4.2-187.9], P = 0.0006) for IgM. Therefore, the quantitative WB may have application in veterinary diagnostic laboratories to increase the accuracy of the clinical diagnosis of E. cuniculi infection. In addition, this tool may help to further understand the development and function of the humoral immune response to this infectious agent.

  10. 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.

  11. [Standardization of Dot-ELISA for detection of anti-Trypanosoma cruzi antibodies, compared to ELISA and Western blot].

    Science.gov (United States)

    Cervantes-Landín, Alejandra Yunuen; Martínez-Martínez, Ignacio; Reyes, Pedro A; Shabib, Muslim; Espinoza-Gutiérrez, Bertha

    2014-01-01

    Chagas disease is considered endemic of Latin America. Because of migration of people from this region to non-endemic areas, such as the United States, Canada and Europe, it has become a major health problem. There are parasitology and serology tests for its diagnosis, but only the latter are useful during the chronic phase. Most of these tests require expensive equipment, which make them also inaccessible for laboratories in endemic areas. In the present work we standardize Dot-ELISA as a diagnostic test for Trypanosoma cruzi infection, since it is an easy, inexpensive and an accessible test. A total of 360 samples were tested: 96 sera from Chagas patients and 153 from healthy people; 40 blood samples spots collected and eluted from filter paper were also tested, as well as 71 serum samples of patients with non-related infections. Sensitivity, specificity and kappa index of Dot-ELISA test were calculated, in order to determine a correlation value of this technique compared to ELISA and Western blot that are already being used for diagnosis. Dot-ELISA obtained 97% sensitivity and 89% specificity, since it showed cross-reaction mainly with Leishmania spp., and a kappa index of 0,79. Dot-ELISA results correlate well with other tests that are already being used for diagnosis of Chagas disease. As it is easy and inexpensive, it may be useful as an additional diagnostic test or for field studies. Copyright © 2013 Elsevier España, S.L. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  12. Resolution and identification of major peanut allergens using a combination of fluorescence two-dimensional differential gel electrophoresis, western blotting and Q-TOF mass spectrometry.

    Science.gov (United States)

    Peanut allergy is triggered by several proteins known as allergens. The matching resolution and identification of major peanut allergens in 2D protein maps, was accomplished by the use of fluorescence two-dimensional differential gel electrophoresis (2D DIGE), Western blotting and quadrupole time-of...

  13. Blood donors with indeterminate anti-p24gag reactivity in HIV-1 western blot: absence of infectivity to transfused patients and in virus culture

    NARCIS (Netherlands)

    van der Poel, C. L.; Lelie, P. N.; Reesink, H. W.; van Exel-Oehlers, P. J.; Tersmette, M.; van den Akker, R.; Gonzalves, M.; Huisman, J. G.

    1989-01-01

    During a follow-up period of 23-40 months, 7 regular blood donors had persistently, and 4 had intermittently indeterminate anti-p24gag reactivity in human immunodeficiency virus (HIV)-1 Western Blot. Serological testing and viral cultures revealed that these donors had no signs of infection for

  14. A mass screening survey of cystic echinococcosis by ultrasonography, Western blotting, and ELISA among university students in Manisa, Turkey.

    Science.gov (United States)

    Kilimcioğlu, Ali Ahmet; Girginkardeşler, Nogay; Korkmaz, Metin; Özkol, Mine; Düzgün, Fatih; Östan, Ipek; Pabuşcu, Yüksel; Dinç, Gönül; Ok, Ulgen Zeki

    2013-12-01

    Cystic echinococcosis (CE) is one of the most important zoonotic diseases in a wide geographic area, including Turkey. In the present project, a total of 4275 students from Celal Bayar University, Manisa, Turkey, were screened by ultrasonography (US) and specific antibodies for CE were examined by Western blotting (WB) and ELISA in finger prick blood samples of 2034 of 4275 volunteered students. We aimed to report the apparent prevalence of CE based on different diagnostic procedures and to compare WB and ELISA with US in diagnosis of CE in a mass screening setting. Six new cases were diagnosed as CE by US during the survey. In addition to these cases, three students were also detected to have been previously operated and pathologically confirmed for hepatic CE. US revealed parenchymal changes in these cases in concordance with their operation history; so, the prevalence of CE by US was calculated as 0.21% (9/4275) (95%CI, 0.11-0.39%) among university students in Manisa. Bands were detected at 8, 28, 32, 38, 42, 47, 70 and 90kDa by WB and the cases were considered to be positive for CE when at least three of the bands were seen together. Apparent prevalence of CE by ELISA and WB were found to be 2.11% (43/2034) (95%CI, 1.57-2.83%) and 0.25% (5/2034) (95%CI, 0.10-0.57%), respectively. Of the six US positive cases, WB was positive in only one case with two cysts in the liver. All of four cases with liver involvement were positive by ELISA. The high prevalence of CE among university students in Manisa indicated that CE is a major health problem in this area of Turkey. Our results supported that WB is rather difficult and not feasible as a mass screening test and may not be effective for confirmation especially in asymptomatic cases. As a result, we recommend US to be used initially in mass screening surveys for CE followed by confirmation by ELISA for suspected cases. Further examination primarily by chest X-ray followed by computed tomography and/or magnetic

  15. Characterization of Sm14 related components in different helminths by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting analysis

    Directory of Open Access Journals (Sweden)

    Nilton Thaumaturgo

    2002-10-01

    Full Text Available Sm14 was the first fatty acid-binding protein homologue identified in helminths. Thereafter, members of the same family were identified in several helminth species, with high aminoacid sequence homology between them. In addition, immune crossprotection was also reported against Fasciola hepatica infection, in animals previously immunized with the Schistosoma mansoni vaccine candidate, r-Sm14. In the present study, data on preliminary sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting analysis of nine different helminth extracts focusing the identification of Sm14 related proteins, is reported. Out of these, three extracts - Ascaris suum (males and females, Echinostoma paraensei, and Taenia saginata - presented components that comigrated with Sm14 in SDS-PAGE, and that were recognized by anti-rSm14 policlonal serum, in Western blotting tests.

  16. 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.

  17. 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.

  18. [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.

  19. Living (stained) deep-sea foraminifera off Hachinohe (NE Japan, Western Pacific): environmental interplay in oxygen-depleted ecosystems

    NARCIS (Netherlands)

    Fontanier, C.; Duros, P.; Toyofuku, T.; Oguri, K.; Koho, K.A.; Buscail, R.; Grémare, A.; Radakovitch, O.; Deflandre, B.; de Nooijer, L.J.; Bichon, S.; Goubet, S.; Ivanovsky, A.; Chabaud, G.; Menniti, C.; Reichart, G.-J.; Kitazato, H.

    2014-01-01

    Live (Rose-Bengal stained) deep-sea foraminiferal faunashave been studied at five stations between 500–2000-m depthalong the NE Japanese margin (western Pacific) tounderstand how complex environmental conditions (e.g.,oxygen depletion, organic matter) control their structure(i.e., diversity,

  20. Glycophospholipid Formulation with NADH and CoQ10 Significantly Reduces Intractable Fatigue in Western Blot-Positive ‘Chronic Lyme Disease’ Patients: Preliminary Report

    Directory of Open Access Journals (Sweden)

    Garth L. Nicolson

    2012-03-01

    Full Text Available Background: An open label 8-week preliminary study was conducted in a small number of patients to determine if a combination oral supplement containing a mixture of phosphoglycolipids, coenzyme Q10 and microencapsulated NADH and other nutrients could affect fatigue levels in long-term, Western blot-positive, multi-symptom ‘chronic Lyme disease’ patients (also called ‘post-treatment Lyme disease’ or ‘post Lyme syndrome’ with intractable fatigue. Methods: The subjects in this study were 6 males (mean age = 45.1 ± 12.4 years and 10 females (mean age = 54.6 ± 7.4 years with ‘chronic Lyme disease’ (determined by multiple symptoms and positive Western blot analysis that had been symptomatic with chronic fatigue for an average of 12.7 ± 6.6 years. They had been seen by multiple physicians (13.3 ± 7.6 and had used many other remedies, supplements and drugs (14.4 ± 7.4 without fatigue relief. Fatigue was monitored at 0, 7, 30 and 60 days using a validated instrument, the Piper Fatigue Scale.Results: Patients in this preliminary study responded to the combination test supplement, showing a 26% reduction in overall fatigue by the end of the 8-week trial (p< 0.0003. Analysis of subcategories of fatigue indicated that there were significant improvements in the ability to complete tasks and activities as well as significant improvements in mood and cognitive abilities. Regression analysis of the data indicated that reductions in fatigue were consistent and occurred with a high degree of confidence (R2= 0.998. Functional Foods in Health and Disease 2012, 2(3:35-47 Conclusions: The combination supplement was a safe and effective method to significantly reduce intractable fatigue in long-term patients with Western blot-positive ‘chronic Lyme disease.’

  1. Western blotting analysis for quantitative detection of CYP2C19 expression in liver tissues in the setting of living donor liver transplantation.

    Science.gov (United States)

    Chiu, King-Wah; Nakano, Toshiaki; Tseng, Hui-Peng; Cheng, Yu-Fan; Jawan, Bruno; Eng, Hock-Liew; Goto, Shigeru; Chen, Chao-Long

    2012-05-01

    The cytochrome P450 (CYP) drug-metabolizing enzymes play an important role in cellular metabolism. Therapeutic failure or drug toxicity in the period after liver transplantation (LT) is influenced by the drug metabolizing capacity of the graft. The expression levels of CYP2C19 enzyme are often used as an indicator of the functioning of the CYP system. The aim of the present study was to assess the CYP2C19 protein expression in the setting of living donor liver transplantation (LDLT) by using western blotting analysis. We performed CYP2C19 genotyping of liver biopsy samples obtained from 24 donors and 8 recipients each in the pre- and post-LT periods, after which we analyzed the CYP enzyme activity by using western blotting analysis. The CYP2C19/β-actin ratio, which was an indicator of CYP expression, was 61.75% (23-100%) in donors, 59.13% (15-100%) in pre-LT recipients and 46.71% (12-67%) in post-LT recipients (p>0.05). The CYP2C19 expression levels associated with different genotypes were as follows: homozygous extensive metabolizers (HomEMs; n=24), 56.63±24.74%; heterozygous extensive metabolizers (HetEMs; n=15), 63.0±25.14% and poor metabolizers (PMs; n=1), 82.0% (p>0.05). Western blotting analysis showed low CYP2C19 protein expression not only in samples from the pre- and post-LT recipients but also from the donors.

  2. Comparative assessment of enzyme-linked immunosorbent assay and Western blot for the diagnosis of toxocariasis in patients with skin disorders.

    Science.gov (United States)

    Bellanger, A-P; Humbert, P; Gavignet, B; Deschaseaux, A D; Barisien, C; Roussel, S; Millon, L; Aubin, F; Piarroux, R

    2010-01-01

    Background The link between various chronic skin disorders and toxocariasis was previously demonstrated by case reports and several case-control studies. However, these previous studies were based only on the Toxocara canis excretory-secretory-enzyme-linked immunosorbent assay (TES-ELISA) serological technique, which is not specific due to cross-reactivity with parasites of the genera Anisakis or Ascaris. Immunoblot analysis is highly specific and can detect very low levels of Toxocara antibodies. Therefore, this technique may be useful in the identification of Toxocara infection in patients with chronic skin disorders. Objectives Because urticaria and pruritus/prurigo are skin conditions previously associated with toxocariasis, we carried out a prospective study using both TES-ELISA and Toxocara Western blot on 113 patients with either chronic urticaria (n = 84) or chronic pruritus (n = 29). Methods Patients were matched with controls according to gender, age and residence location (rural or urban area). Data were analysed using a Mantel-Haenszel chi(2) test. Results The proportion of positive TES-ELISA results was not significantly different for patients with chronic skin disorders (urticaria or pruritus/prurigo) from that of control subjects. However, the proportion of positive immunoblot results was significantly higher for patients with chronic urticaria than for control subjects (P = 0.009). Conclusions Our study demonstrates the need to perform Western blotting immunodiagnosis, whatever the TES-ELISA result, to improve diagnosis of human toxocariasis in patients with chronic urticaria caused by Toxocara infection.

  3. 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.

  4. 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.

  5. Determining the cleavage site for the mature antimicrobial peptide of Nile tilapia β-defensin using 2D electrophoresis, western blot, and mass spectrometry analysis.

    Science.gov (United States)

    Chang, Chin-I; Chen, Li-Hao; Hu, Yeh-Fang; Wu, Chia-Che; Tsai, Jyh-Ming

    2017-03-01

    Several proteomic techniques were used to determine the cleavage site of the mature antimicrobial peptide of Nile tilapia β-defensin. The computer-predicted Nile tilapia β-defensin ( 25 ASFPWSCLSLSGVCRKVCLPTELFFGPLGCGKGSLCCVSHFL 66 ) composed of 42 amino acids was chemically synthesized and prepared to produce an antibody for Western blotting. Total proteins from the skin of the Nile tilapia were separated on two-dimensional electrophoresis, and the spot of Nile tilapia β-defensin was recognized using Western blot analysis. It was then excised and extracted from the gel. The precise molecular mass of this spot was determined by LC-MS/MS spectrometry. Four major peptides were discovered, with molecular weights of 4293.2 Da, 4306.5 Da, 4678.9 Da, and 4715.0 Da. The calculated mass of the 40-amino-acid sequence ( 27 FPWSCLSLSGVCRKVCLPTELFFGPLGCGKGSLCCVSHFL 66 ) of Nile tilapia β-defensin starting from Phe27 and ending with Leu66 was 4293.18 Da, which completely matched the 4293.2 Da peptide that was obtained from the mass spectrometry analysis. This result confirmed that the cleavage site for the mature C-terminal Nile tilapia β-defensin is at residue Ser26-Phe27, not at Ala24-25 as predicted by computer analysis. This study provides a simple but reliable model to determine the cleavage site for a mature antimicrobial peptide. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Assessment and modification of a Western blot assay for detection of central nervous system tissue in meat products in the United States.

    Science.gov (United States)

    Salman, M D; Jemmi, T; Triantis, J; Dewell, R D

    2005-08-01

    Health hazards associated with meat contaminated by the bovine spongiform encephalopathy agent have led to the development of tests for the presence of this agent. The objective of this study was to optimize a neuron-specific enolase Western blot assay for use in the United States. We compared the original test with a modified protocol to evaluate the detection limit for the presence of central nervous system (CNS) tissue in experimentally inoculated samples and compared and evaluated the utility of these tests for detecting CNS tissue in retail sausages. Sensitivity and specificity of the original and modified protocols were evaluated using the kappa statistic to assess agreement between the results of the two protocols. The original protocol resulted in 100% specificity and 92% sensitivity for raw samples and 92% specificity and 72% sensitivity for cooked samples. The modified protocol resulted in 92% specificity and 89% sensitivity for raw samples and 83% specificity and 75% sensitivity for cooked samples. The kappa statistic for protocol comparison was 0.94 for raw samples and 0.74 for cooked samples. Both protocols correctly identified CNS tissue in positive controls for each replicate. Although the Western blot technique should be considered for screening for the presence of bovine CNS tissue in meat samples, the techniques should be further optimized to address problems of low sensitivity. A test with higher sensitivity is needed to protect consumers from food safety threats associated with bovine CNS tissue.

  7. Western blot analysis of antigens specifically recognized by natural immune responses of patients with Japanese encephalitis infections.

    Science.gov (United States)

    Patarapotikul, J; Pothipunya, S; Wanotayan, R; Hongyantarachai, A; Tharavanij, S

    1993-06-01

    Specific recognition of antigenic proteins of Japanese encephalitis virus (JEV) by JE patients was investigated by using non-reducing and reducing Western immunoblot analysis. Under non-reducing conditions, the profile of JEV proteins recognized comprised E (52 kDa), NS1 (45 and 41 kDa), NS3 (66.2 kDa) and NS5 (103 and 97.4 kDa). When recognition patterns of sera from JE and dengue patients were compared, only slight differences between JE and dengue sera were found (under non-reducing conditions), involving only the 66.2 kDa protein: to this protein, JE sera exhibited greater reactivity, but not in greater frequency, than did dengue sera. In contrast, cerebrospinal fluid (CSF) from JE patients showed more differences from JE sera: CSF antibody lacked recognition of the 41 kDa protein and had lower frequencies, as well as less reactivities to several other proteins. These results suggested that restricted populations of lymphocytes were localized in the central nervous system of JE patients. The effect of reducing agent (2 beta-mercaptoethanol) on the recognition patterns of those groups of sera was also analysed: the reducing agent affected all the proteins mentioned above, however, the effects were not uniform. It is proposed that JE and dengue sera may recognize different epitopes on some or all of these proteins. Such differences cannot be detected by Western immunoblot analysis, but it would be feasible to test this hypothesis using epitope mapping with synthetic peptides in a multi-pin ELISA. Analysis in this fine detail is essential for designing improved JE vaccines.

  8. 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...

  9. 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.

  10. Serum detection of IgG antibodies against Demodex canis by western blot in healthy dogs and dogs with juvenile generalized demodicosis.

    Science.gov (United States)

    Ravera, Ivan; Ferreira, Diana; Gallego, Laia Solano; Bardagí, Mar; Ferrer, Lluís

    2015-08-01

    The aim of this study was to investigate the presence of canine immunoglobulins (Ig) G against Demodex proteins in the sera of healthy dogs and of dogs with juvenile generalized demodicosis (CanJGD) with or without secondary pyoderma. Demodex mites were collected from dogs with CanJGD. Protein concentration was measured and a western blot technique was performed. Pooled sera from healthy dogs reacted mainly with antigen bands ranging from 55 to 72 kDa. Pooled sera from dogs with CanJGD without secondary pyoderma reacted either with 10 kDa antigen band or 55 to 72 kDa bands. Pooled sera from dogs with CanJGD with secondary pyoderma reacted only with a 10 kDa antigen band. The results of this study suggest that both healthy dogs and dogs with CanJGD develop a humoral response against different proteins of Demodex canis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Identification of liver protein targets modified by tienilic acid metabolites using a two-dimensional Western blot-mass spectrometry approach

    Science.gov (United States)

    Methogo, Ruth Menque; Dansette, Patrick M.; Klarskov, Klaus

    2007-12-01

    A combined approach based on two-dimensional electrophoresis-immuno-blotting and nanoliquid chromatography coupled on-line with electrospray ionization mass spectrometry (nLC-MS/MS) was used to identify proteins modified by a reactive intermediate of tienilic acid (TA). Liver homogenates from rats exposed to TA were fractionated using ultra centrifugation; four fractions were obtained and subjected to 2D electrophoresis. Following transfer to PVDF membranes, modified proteins were visualized after India ink staining, using an anti-serum raised against TA and ECL detection. Immuno-reactive spots were localized on the PVDF membrane by superposition of the ECL image, protein spots of interest were excised, digested on the membrane with trypsin followed by nLC-MS/MS analysis and protein identification. A total of 15 proteins were identified as likely targets modified by a TA reactive metabolite. These include selenium binding protein 2, senescence marker protein SMP-30, adenosine kinase, Acy1 protein, adenosylhomocysteinase, capping protein (actin filament), protein disulfide isomerase, fumarylacetoacetase, arginase chain A, ketohexokinase, proteasome endopeptidase complex, triosephosphate isomerase, superoxide dismutase, dna-type molecular chaperone hsc73 and malate dehydrogenase.

  12. Rendimiento diagnóstico del Western Blot para detectar simultáneamente anticuerpos en pacientes con cisticercosis, hidatidosis y fascioliasis humana

    Directory of Open Access Journals (Sweden)

    Kelly Davelois

    Full Text Available Objetivo. Determinar el rendimiento diagnóstico de la técnica de Western Blot para detectar simultáneamente anticuerpos en pacientes con cisticercosis, hidatidosis y fascioliasis humana. Materiales y métodos. Estudio transversal de evaluación de prueba diagnóstica. Se obtuvieron los antígenos de excreción-secreción de las larvas de Taenia solium, quistes de Echinococcus granulosus; y la forma adulta de Fasciola hepática; que luego fueron separados electroforéticamente en geles de poliacrilamida individuales, transferidos y fijados a una membrana de nitrocelulosa para ser enfrentados con sueros de pacientes con las tres parasitosis. La sensibilidad de la técnica se evaluó empleando 300 sueros individuales, 60 pools de dos parasitosis y 20 pools de tres parasitosis y la especificidad con 75 sueros de pacientes con otras parasitosis, 10 de pacientes con otras enfermedades y 15 sueros de personas no parasitadas. Resultados. La técnica reconoció trece glicoproteínas (GP: GP 35, 31, 24, 23, 18, 17, 14 y 13 kDa para cisticercosis, GP 8,16 y 21 kDa para hidatidosis y GP: 17 y 23 kDa para fascioliasis. La prueba detectó la presencia de anticuerpos alcanzando una sensibilidad de 96% (IC95%: 94,62-98,54% en la detección de una o las trece bandas, una especificidad de 100% (IC95%: 99,50 - 100,00%; individualmente, se tuvo una sensibilidad para cisticercosis de 97% (IC95%: 93,16-100%, para hidatidosis de 94% (IC95%: 88,85-99,15% y para fascioliasis de 96% (IC95%: 91,66-100%. Conclusiones. La prueba de Western blot es eficaz en la detección, simultanea de anticuerpos en pacientes con cisticercosis, hidatidosis y fascioliasis humana, y puede ser utilizada como prueba de descarte o confirmatoria en zonas endémicas.

  13. Myostatin inhibitors in sports drug testing: Detection of myostatin-neutralizing antibodies in plasma/serum by affinity purification and Western blotting.

    Science.gov (United States)

    Walpurgis, Katja; Thomas, Andreas; Schänzer, Wilhelm; Thevis, Mario

    2016-02-01

    Myostatin is a key regulator of skeletal muscle growth and inhibition of its signaling pathway results in an increased muscle mass and function. The aim of this study was to develop a qualitative detection assay for myostatin-neutralizing antibodies for doping control purposes by using immunological approaches. To detect different types of myostatin-neutralizing antibodies irrespective of their amino acid sequence, an immunological assay specific for antibodies directed against myostatin and having a human Fc domain was established. Affinity purification and Western blotting strategies were combined to allow extracting and identifying relevant analytes from 200 μL of plasma/serum in a non-targeted approach. The assay was characterized regarding specificity, linearity, precision, robustness, and recovery. The assay was found to be highly specific, robust, and linear from 0.1 to 1 μg/mL. The precision was successfully specified at three different concentrations and the recovery of the affinity purification was 58%. Within this study, an immunological detection assay for myostatin-neutralizing antibodies present in plasma/serum specimens was developed and successfully characterized. The presented approach can easily be modified to include other therapeutic antibodies and serves as proof-of-concept for the detection of antibody-based myostatin inhibitors in doping control samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. A Simple Method to Avoid Nonspecific Signal When Using Monoclonal Anti-Tau Antibodies in Western Blotting of Mouse Brain Proteins.

    Science.gov (United States)

    Petry, Franck R; Nicholls, Samantha B; Hébert, Sébastien S; Planel, Emmanuel

    2017-01-01

    In Alzheimer's disease and other tauopathies, tau displays several abnormal post-translation modifications such as hyperphosphorylation, truncation, conformation, and oligomerization. Mouse monoclonal antibodies have been raised against such tau modifications for research, diagnostic, and therapeutic purposes. However, many of these primary antibodies are at risk of giving nonspecific signals in common Western blotting procedures. Not because they are unspecific, but because the secondary antibodies used to detect them will also detect the heavy chain of endogenous mouse immunoglobulins (Igs), and give a nonspecific signal at the same molecular weight than tau protein (around 50 kDa). Here, we propose the use of anti-light chain secondary antibodies as a simple and efficient technique to prevent nonspecific Igs signals at around 50 kDa. We demonstrate the efficacy of this method by removing artifactual signals when using monoclonal antibodies directed at tau phosphorylation (AT100, 12E8, AT270), tau truncation (TauC3), tau oligomerization (TOMA), or tau abnormal conformation (Alz50), in wild-type, 3×Tg-AD, and tau knockout mice.

  15. IgG western blot for confirmatory diagnosis of equivocal cases of toxoplasmosis by EIA-IgG and fluorescent antibody test.

    Science.gov (United States)

    Khammari, Imen; Saghrouni, Fatma; Yaacoub, Alia; Gaied Meksi, Sondoss; Ach, Hinda; Garma, Lamia; Fathallah, Akila; Ben Saïd, Moncef

    2013-08-01

    The performance values of available techniques used in serodiagnosis of toxoplasmosis are satisfactory but they raise problems of equivocal and discordant results for very low IgG titers. Recently marketed, LDBio-Toxo II IgG Western blot (IB) showed an excellent correlation with the dye test. We estimated the proportion of equivocal and discordant results between the enzyme immunoassay Platelia Toxo IgG (EIA-IgG) and fluorescent antibody test (FAT) and assessed the usefulness of the IB as a confirmatory test. Out of 2,136 sera collected from pregnant women, 1,644 (77.0%) tested unequivocally positive and 407 (19.0%) were negative in both EIA-IgG and FAT. The remaining 85 (4%) sera showed equivocal or discordant results. Among them, 73 (85.9%) were positive and 12 (14.1%) were negative in IB. Forty-one (89.1%) equivocal sera in EIA-IgG and 46 (86.8%) equivocal sera in FAT were positive in IB. Reducing the cut-off values of both screening techniques improved significantly their sensitivity in detecting very low IgG titers at the expense of their specificity. In conclusion, equivocal results in routine-used techniques and their discordance in determination of the immune status in pregnancy women were not uncommon. IB test appeard to be highly useful in these situations as a confirmatory technique.

  16. 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.

  17. 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

    Full Text Available Objetivos. Evaluar la eficacia de la técnica de electroinmunotransferencia (EITB o Western blot utilizando antígenos de excreción-secreción de las formas adultas de Fasciola hepatica (Fh E/S Ag para el diagnóstico de la fasciolosis humana. Materiales y métodos. Los antígenos fueron obtenidos a las 18 horas de incubación en medio Minimum Essential Eagle y preparados a la concentración proteica de 0,15 ug/uL; los cuales, al ser enfrentados con un pool de sueros de pacientes con fasciolosis confirmada por el hallazgo de huevos del parásito en las heces, se detectaron los antígenos de 10, 12, 17, 23, 27, 30, 36, 43, 66 y 136 KDa, con los cuales se desarrolló la técnica de Western blot. La sensibilidad se evaluó empleando sueros de 67 pacientes con fasciolosis, y la especificidad con sueros de 57 pacientes con otras parasitosis y diez sueros de personas no parasitadas. Resultados. De los 67 sueros, 64 reaccionaron con la banda de 23 KDa y 61 con la banda de 17KDa. Estas dos bandas no fueron detectadas por ninguno de los sueros de pacientes con otras parasitosis, ni de personas no parasitadas, siendo por ello consideradas como específicas y diagnósticas. Conclusiones. La sensibilidad de la prueba, utilizando las bandas de 17 y 23 KDa, fue de 95,5 % cuando se presenta reacción positiva en una o en las dos bandas, siendo la especificidad para estos dos antígenos de 100 % con un valor predictivo positivo de 100 % y un valor predictivo negativo de 95,71 %.Objectives. 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. Materials and methods. 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

  18. Western blot data using two distinct anti-O-GlcNAc monoclonal antibodies showing unique glycosylation status on cellular proteins under 2-deoxy-d-glucose treatment

    Directory of Open Access Journals (Sweden)

    Tetsuya Okuda

    2017-02-01

    Full Text Available Protein modification by O-linked N-acetylglucosamine (O-GlcNAcylation is one of the post transcriptional modifications occurring on cellular proteins. This paper provides a data set relating to the O-GlcNAcylation of cellular proteins detected by RL2 and CTD110.6 antibodies, which are commonly used for detection of protein O-GlcNAcylation, in 2-deoxy-d-glucose (2DG-treated human teratocarcinoma NCCIT cells in support of the research article entitled “A novel, promoter-based, target-specific assay identifies 2-deoxy-d-glucose as an inhibitor of globotriaosylceramide biosynthesis” (Okuda et al., 2009 [1]. The main article described a suppressive effect of 2DG on an Sp1 target gene in NCCIT cells and discussed the relationship between the effect of 2DG and O-GlcNAcylation status of Sp1. The data in this paper complements this relationship by Western blotting and clearly showed that the 2DG treatment increased O-GlcNAcylation of cellular proteins in NCCIT cells, whereas the RL2 and CTD110.6 epitopes were detected in a different manner. The RL2 epitope was detected on Sp1 during 2DG treatment, and the level was transiently increased at 24 h. In contrast, the CTD110.6 epitope became detectable on Sp1 over 72 h after 2DG treatment, and then the other proteins containing CTD110.6 epitopes also appeared in the cell lysates and the anti-Sp1 antibody precipitates.

  19. Dogs vaccinated with gentamicin-attenuated Leishmania infantum or infected with wild-type parasite can be distinguished by Western blotting.

    Science.gov (United States)

    Daneshvar, H; Mahmmodi, Z; Kamiabi, H; Phillips, R S; Burchmore, R

    2014-05-01

    An attenuated line of Leishmania infantum (the H-line), developed through exposure to gentamicin, has been shown to protect dogs against canine visceral leishmaniasis. A specific diagnostic test to differentiate dogs vaccinated with the attenuated line from dogs infected with L. infantum wild-type (L. infantum WT) could be a valuable tool in evaluating the effectiveness of canine vaccination. In this study, 28 healthy dogs were allocated into four groups. In Group I and Group II (eight dogs per group), dogs were immunized subcutaneously (s.c.) with L. infantum H-line, and the dogs of Group II challenged s.c. with L. infantum WT, at 2 months post-immunization. In Group III, eight animals were challenged s.c. with L. infantum WT, and four dogs of Group IV were injected s.c. with PBS. We found that sera from vaccinated dogs recognize a 21 kDa antigen of promastigotes of L. infantum H-line but not of L. infantum WT, whereas sera from unvaccinated dogs challenged with L. infantum WT, recognized a 21 kDa antigen of promastigotes of L. infantum WT but not of L. infantum H-line. Sera from dogs challenged with L. infantum WT with prior vaccination with L. infantum H-line, recognized a 21 kDa antigen of both L. infantum WT and L. infantum H-line. These results suggest that the Western blot analysis of antibodies against 21 kDa antigens of L. infantum H-line and WT may be a useful technique for distinguishing between dogs vaccinated with L. infantum H-line and dogs naturally infected with L. infantum WT. © 2014 John Wiley & Sons Ltd.

  20. HIV-1/2 indeterminate Western blot results: follow-up of asymptomatic blood donors in Belo Horizonte, Minas Gerais, Brazil

    Directory of Open Access Journals (Sweden)

    CARNEIRO-PROIETTI A.B.F.

    1999-01-01

    Full Text Available The clinical and public health importance of indeterminate results in HIV-1/2 testing is still difficult to evaluate in volunteer blood donors. At Fundação Hemominas, HIV-1/2 ELISA is used as the screening test and, if reactive, is followed by Western blot (WB. We have evaluated 84 blood donors who had repeatedly reactive ELISA tests for HIV-1/2, but indeterminate WB results. Sixteen of the 84 donors (19.0% had history of sexually transmitted diseases; 18/84 (21.4% informed receiving or paying for sex; 3/84 (3.6% had homosexual contact; 2/26 women (7.6% had past history of multiple illegal abortions and 3/84 (3.6% had been previously transfused. Four out of 62 donors (6.5% had positive anti-nuclear factor (Hep2, with titles up to 1:640. Parasitological examination of the stool revealed eggs of S. mansoni in 4/62 (6.4% donors and other parasites in 8/62 (12.9%. Five (5.9% of the subjects presented overt seroconversion for HIV-1/2, 43/84 (51.2% had negative results on the last visit, while 36/84 (42.9% remained WB indeterminate. Although some conditions could be found associated with the HIV-1/2 indeterminate WB results and many donors had past of risky behavior, the significance of the majority of the results remains to be determined.

  1. Testing for the erythropoiesis-stimulating agent Sotatercept/ACE-011 (ActRIIA-Fc) in serum by means of Western blotting and LC-HRMS.

    Science.gov (United States)

    Walpurgis, Katja; Thomas, Andreas; Vogel, Matthias; Reichel, Christian; Geyer, Hans; Schänzer, Wilhelm; Thevis, Mario

    2016-11-01

    Sotatercept (formerly ACE-011) is a glycosylated, dimeric fusion protein composed of the extracellular domain of the human activin receptor type IIA (ActRIIA) and the Fc region of human IgG1. The protein-based drug candidate acts as a ligand trap which competitively binds to activin A and other members of the transforming growth factor beta superfamily, thus blocking signalling through ActRIIA. Since the inhibition of activin A was found to significantly increase bone formation and quality, Sotatercept was originally developed for the treatment of diseases involving bone loss. But as the protein therapeutic also stimulates erythropoiesis by a mechanism independent of the EPO receptor, it has been evaluated for the treatment of anaemia in rare blood diseases such as beta thalassemia. Due to its positive effects on erythropoiesis and bone formation, Sotatercept may also be misused as performance-enhancing agent in sports. Within this study, two complementary detection assays for Sotatercept and related ActRIIA-Fc fusion proteins in serum samples were developed. While the first assay combines affinity purification and Western blotting to generically detect ActRIIA-Fc fusion proteins irrespective of their amino acid sequence, the liquid chromatography-high resolution mass spectrometry (LC-HRMS) method is highly specific for proteolytic peptides originating from the receptor and Fc domain of Sotatercept. Both approaches can readily be modified to include other pharmaceutical proteins such as therapeutic antibodies, and serve as proof-of-concept for the capability of the approach to detect TGF-β inhibitors and Fc fusion proteins in doping control serum samples. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  2. 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?

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    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.

  3. Quantitative analysis of the IgG and IgG subclass immune responses to chromosomal Pseudomonas aeruginosa beta-lactamase in serum from patients with cystic fibrosis by western blotting and laser scanning densitometry

    DEFF Research Database (Denmark)

    Petersen, T D; Ciofu, O; Pressler, T

    1996-01-01

    lung infection with P aeruginosa was further investigated by correlating the a beta ab IgG subclasses with pulmonary function in patients with cystic fibrosis. METHODS: Immunoglobulin G (IgG) and IgG subclass a beta ab were investigated by western blotting and quantified by laser scanning densitometry...

  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. A comparison of extracted proteins of isolates of Dermatophilus congolensis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting.

    Science.gov (United States)

    Makinde, A A; Gyles, C L

    1999-07-01

    Antigenic diversity within a collection of 18 isolates of Dermatophilus congolensis from different Continents was examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blotting with sera from cattle with clinical dermatophilosis using whole cell extracts obtained by three methods and one extract of extracellular products of D. congolensis. One of the methods involving the release of a lysostaphin-solubilized protein (LSP) of whole cells of D. congolensis revealed a number of discrete and easily-identifiable bands in SDS-PAGE which were found suitable for characterizing protein patterns and was, therefore, subsequently used for a comparative analysis of the proteins of all the D. congolensis isolates. Six electropherotypes (ET) of D. congolensis were identified among the 18 isolates using the protein profiles based on the presence of four protein bands at Molecular weights (MW) 62, 28, 17.4 and 16.4 kDa. The ETs were found among isolates from different animal species and from different sources with ET1 consisting of three bovine and two equine isolates; ET2, two bovine and three ovine isolates; ET3, two bovine isolates; ET4, two bovine isolates; ET5, one bovine and one ovine isolates and ET6, two bovine isolates. Immunoblotting of the extracts of D. congolensis isolates with sera from cattle with clinical dermatophilosis infection demonstrated protein bands of MW ranging from 9 kDa to 188 kDa. Sera from chronic dermatophilosis infection demonstrated a 28 kDa protein which was immunodominant in the LSP extracts of all the 18 isolates of D. congolensis tested while sera from mild infections demonstrated mainly the 62 kDa protein in the same extracts. However, many protein bands were demonstrated in surface membrane (TSMP) and extracellular protein extracts with sera from only mildly infected animals. The protein patterns observed in all isolates of D. congolensis revealed global antigenic similarities and distinct differences

  6. Comparison of the efficiency of two commercial kits - ELFA and Western blot in estimating the phase of Toxoplasma gondii infection in pregnant women.

    Science.gov (United States)

    Sroka, Jacek; Wójcik-Fatla, Angelina; Zając, Violetta; Sawczyn, Anna; Cisak, Ewa; Karamon, Jacek; Dutkiewicz, Jacek; Bojar, Iwona

    2016-12-23

    Sera of 89 pregnant women were selected according to the results of ELFA IgM, IgG and avidity IgG, and tested with commercial tests IgM, IgG and avidity IgG Western Blot (WB) to compare the efficacy of both techniques in determining the phase of T. gondii infection. In total, 81 of 89 tested sera (91.0%) were classified as positive, both in the ELFA and WB tests for the presence of anti-Toxoplasma antibodies of class IgG, indicating a past infection, while the prevalence of anti-Toxoplasma positive reactions associated with the antibodies of class IgM indicating a recent infection was much lower - 31.5% and 20.2%, respectively. Sera of 81 women were also tested in the ELFA and WB tests for avidity, e.g. ability of forming high-molecular IgG antibody complexes. Low or medium results in these tests (in this study all classified as low), indicating a recent infection, were detected by ELFA and WB in 22.2% and 45.7% of the total examined samples, respectively. The Spearman's rank test for correlation, performed for recognition of quantitative data of the ELFA and WB tests (index, units or points), revealed a highly significant correlation between the ELFA and WB tests for homologous classes of antibodies, both for IgM and IgG (p0.05), except for the WB test for IgM antibodies, which showed a significant correlation with the ELFA test for IgG antibodies (p<0.01). A highly significant negative correlation between the ELFA and WB test for IgM antibodies and ELFA and WB tests for IgG avidity was demonstrated (p<0.01), except for a relationship between the WB test for IgM and WB for avidity, which was not significant. Such negative correlations are theoretically expected, as strong complexes with the participation of IgG antibodies are absent in the early phase of toxoplasmosis when early antibodies of IgM class are present. Summarizing, this study indicates the high usefulness of the commercial ELFA and WB tests in serodiagnostics of toxoplasmosis in pregnant women

  7. Comparison of the efficiency of two commercial kits – ELFA and Western blot in estimating the phase of Toxoplasma gondii infection in pregnant women

    Directory of Open Access Journals (Sweden)

    Jacek Sroka

    2016-09-01

    Full Text Available Sera of 89 pregnant women were selected according to the results of ELFA IgM, IgG and avidity IgG, and tested with commercial tests IgM, IgG and avidity IgG Western Blot (WB to compare the efficacy of both techniques in determining the phase of T. gondii infection. In total, 81 of 89 tested sera (91.0% were classified as positive, both in the ELFA and WB tests for the presence of anti- Toxoplasma antibodies of class IgG, indicating a past infection, while the prevalence of anti- Toxoplasma positive reactions associated with the antibodies of class IgM indicating a recent infection was much lower – 31.5% and 20.2%, respectively. Sera of 81 women were also tested in the ELFA and WB tests for avidity, e.g. ability of forming high-molecular IgG antibody complexes. Low or medium results in these tests (in this study all classified as low, indicating a recent infection, were detected by ELFA and WB in 22.2% and 45.7% of the total examined samples, respectively. The Spearman’s rank test for correlation, performed for recognition of quantitative data of the ELFA and WB tests (index, units or points, revealed a highly significant correlation between the ELFA and WB tests for homologous classes of antibodies, both for IgM and IgG (p0.05, except for the WB test for IgM antibodies, which showed a significant correlation with the ELFA test for IgG antibodies (p<0.01. A highly significant negative correlation between the ELFA and WB test for IgM antibodies and ELFA and WB tests for IgG avidity was demonstrated (p<0.01, except for a relationship between the WB test for IgM and WB for avidity, which was not significant. Such negative correlations are theoretically expected, as strong complexes with the participation of IgG antibodies are absent in the early phase of toxoplasmosis when early antibodies of IgM class are present. Summarizing, this study indicates the high usefulness of the commercial ELFA and WB tests in serodiagnostics of toxoplasmosis in

  8. Diagnostic performance of ELISA, IFAT and Western blot for the detection of anti-Leishmania infantum antibodies in cats using a Bayesian analysis without a gold standard.

    Science.gov (United States)

    Persichetti, Maria Flaminia; Solano-Gallego, Laia; Vullo, Angela; Masucci, Marisa; Marty, Pierre; Delaunay, Pascal; Vitale, Fabrizio; Pennisi, Maria Grazia

    2017-03-13

    Anti-Leishmania antibodies are increasingly investigated in cats for epidemiological studies or for the diagnosis of clinical feline leishmaniosis. The immunofluorescent antibody test (IFAT), the enzyme-linked immunosorbent assay (ELISA) and western blot (WB) are the serological tests more frequently used. The aim of the present study was to assess diagnostic performance of IFAT, ELISA and WB to detect anti-L. infantum antibodies in feline serum samples obtained from endemic (n = 76) and non-endemic (n = 64) areas and from cats affected by feline leishmaniosis (n = 21) by a Bayesian approach without a gold standard. Cut-offs were set at 80 titre for IFAT and 40 ELISA units for ELISA. WB was considered positive in presence of at least a 18 KDa band. Statistical analysis was performed through a written routine with MATLAB software in the Bayesian framework. The latent data and observations from the joint posterior were simulated in the Bayesian approach by an iterative Markov Chain Monte Carlo technique using the Gibbs sampler for estimating sensitivity and specificity of the three tests. The median seroprevalence in the sample used for evaluating the performance of tests was estimated at 0.27 [credible interval (CI) = 0.20-0.34]. The median sensitivity of the three different methods was 0.97 (CI: 0.86-1.00), 0.75 (CI: 0.61-0.87) and 0.70 (CI: 0.56-0.83) for WB, IFAT and ELISA, respectively. Median specificity reached 0.99 (CI: 0.96-1.00) with WB, 0.97 (CI: 0.93-0.99) with IFAT and 0.98 (CI: 0.94-1.00) with ELISA. IFAT was more sensitive than ELISA (75 vs 70%) for the detection of subclinical infection while ELISA was better for diagnosing clinical leishmaniosis when compared with IFAT (98 vs 97%). The overall performance of all serological techniques was good and the most accurate test for anti-Leishmania antibody detection in feline serum samples was WB.

  9. Testing UK blood donors for exposure to human parvovirus 4 using a time-resolved fluorescence immunoassay to screen sera and Western blot to confirm reactive samples.

    Science.gov (United States)

    Maple, Peter A C; Beard, Stuart; Parry, Ruth P; Brown, Kevin E

    2013-10-01

    Human parvovirus 4 (ParV4), a newly described member of the family Parvoviridae, like B19V, has been found in pooled plasma preparations. The extent, and significance, of ParV4 exposure in UK blood donors remain to be determined and reliable detection of ParV4 immunoglobulin (Ig)G, using validated methods, is needed. With ParV4 virus-like particles a ParV4 IgG time-resolved fluorescence immunoassay (TRFIA) was developed. There is no gold standard or reference assay for measuring ParV4 IgG and the utility of the TRFIA was first examined using a panel of sera from people who inject drugs (PWIDS)--a high-prevalence population for ParV4 infection. Western blotting was used to confirm the specificity of TRFIA-reactive sera. Two cohorts of UK blood donor sera comprising 452 sera collected in 1999 and 156 sera collected in 2009 were tested for ParV4 IgG. Additional testing for B19V IgG, hepatitis C virus antibodies (anti-HCV), and ParV4 DNA was also undertaken. The rate of ParV4 IgG seroprevalence in PWIDS was 20.7% and ParV4 IgG was positively associated with the presence of anti-HCV with 68.4% ParV4 IgG-positive sera testing anti-HCV-positive versus 17.1% ParV4 IgG-negative sera. Overall seropositivity for ParV4 IgG, in 608 UK blood donors was 4.76%. The ParV4 IgG seropositivity for sera collected in 1999 was 5.08%, compared to 3.84% for sera collected in 2009. No ParV4 IgG-positive blood donor sera had detectable ParV4 DNA. ParV4 IgG has been found in UK blood donors and this finding needs further investigation. © 2013 American Association of Blood Banks.

  10. Bicentric evaluation of six anti-toxoplasma immunoglobulin G (IgG) automated immunoassays and comparison to the Toxo II IgG Western blot.

    Science.gov (United States)

    Maudry, Arnaud; Chene, Gautier; Chatelain, Rémi; Patural, Hugues; Bellete, Bahrie; Tisseur, Bernard; Hafid, Jamal; Raberin, Hélène; Beretta, Sophie; Sung, Roger Tran Manh; Belot, Georges; Flori, Pierre

    2009-09-01

    A comparative study of the Toxoplasma IgG(I) and IgG(II) Access (Access I and II, respectively; Beckman Coulter Inc.), AxSYM Toxo IgG (AxSYM; Abbott Diagnostics), Vidas Toxo IgG (Vidas; bioMerieux, Marcy l'Etoile, France), Immulite Toxo IgG (Immulite; Siemens Healthcare Diagnostics Inc.), and Modular Toxo IgG (Modular; Roche Diagnostics, Basel, Switzerland) tests was done with 406 consecutive serum samples. The Toxo II IgG Western blot (LDBio, Lyon, France) was used as a reference technique in the case of intertechnique discordance. Of the 406 serum samples tested, the results for 35 were discordant by the different techniques. Using the 175 serum samples with positive results, we evaluated the standardization of the titrations obtained (in IU/ml); the medians (second quartiles) obtained were 9.1 IU/ml for the AxSYM test, 21 IU/ml for the Access I test, 25.7 IU/ml for the Access II test, 32 IU/ml for the Vidas test, 34.6 IU/ml for the Immulite test, and 248 IU/ml for the Modular test. For all the immunoassays tested, the following relative sensitivity and specificity values were found: 89.7 to 100% for the Access II test, 89.7 to 99.6% for the Immulite test, 90.2 to 99.6% for the AxSYM test, 91.4 to 99.6% for the Vidas test, 94.8 to 99.6% for the Access I test, and 98.3 to 98.7% for the Modular test. Among the 406 serum samples, we did not find any false-positive values by two different tests for the same serum sample. Except for the Modular test, which prioritized sensitivity, it appears that the positive cutoff values suggested by the pharmaceutical companies are very high (either for economical or for safety reasons). This led to imperfect sensitivity, a large number of unnecessary serological follow-ups of pregnant women, and difficulty in determining the serological status of immunosuppressed individuals.

  11. 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....

  12. 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...

  13. Validation of Anti-CSPα, SNAP25, Tyrosine Hydroxylase, Ubiquitin, Cleaved Caspase 3, and pSer PKC Motif Antibodies for Utilization in Western Blotting.

    Science.gov (United States)

    Shirafuji, Toshihiko; Ueyama, Takehiko; Tanaka, Shigeru; Hide, Izumi; Saito, Naoaki; Sakai, Norio

    2017-12-26

    There are many commercial antibodies with little information provided by their suppliers as to their reliability. Accordingly, commercial antibodies require proper validation before being used in scientific research. In this study, we validated several commercial antibodies, including anti-CSPα, SNAP25, tyrosine hydroxylase, ubiquitin, cleaved caspase 3, and pSer PKC motif. Anti-CSPα, SNAP25, and tyrosine hydroxylase antibodies could detect their endogenous target proteins with some degree of cross-reactivity. Furthermore, clear SNAP25 staining was observed with SNAP25 antibody. Antibodies directed against ubiquitin, cleaved caspase 3, and pSer PKC motif could detect poly-ubiquitination, apoptosis, and phosphorylation, respectively.

  14. 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.

  15. Expression of O6-Methylguanine-DNA Methyltransferase Examined by Alkyl-Transfer Assays, Methylation-Specific PCR and Western Blots in Tumors and Matched Normal Tissue

    Science.gov (United States)

    Ishiguro, Kimiko; Shyam, Krishnamurthy; Penketh, Philip G.; Baumann, Raymond P.; Sartorelli, Alan C.; Rutherford, Thomas J.; Ratner, Elena S.

    2013-01-01

    The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions, depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity being lower in tumor than in host tissue. Despite the established role of MGMT as a tumor resistance factor, consensus on how to assess MGMT expression in clinical samples is unsettled. The aim of this study is to examine the relationship between the values derived from distinctive MGMT measurements in 13, 12, 6 and 2 pairs of human tumors and matched normal adjacent tissue from the colon, kidney, lung and liver, respectively, and in human cell lines. The MGMT measurements included (a) alkyl-transfer assays using [benzene-3H]O6-benzylguanine as a substrate to assess functional MGMT activity, (b) methylation-specific PCR (MSP) to probe MGMT gene promoter CpG methylations as a measure of gene silencing, and (c) western immunoblots to analyze the MGMT protein. In human cell lines, a strict negative correlation existed between MGMT activity and the extent of promoter methylation. In tissue specimens, by contrast, the correlation between these two variables was low. Moreover, alkyl-transfer assays identified 3 pairs of tumors and normal tissue with tumor-selective reduction in MGMT activity in the absence of promoter methylation. Cell line MGMT migrated as a single band in western analyses, whereas tissue MGMT was heterogeneous around its molecular size and at much higher molecular masses, indicative of multi-layered post-translational modifications. Malignancy is occasionally associated with a mobility shift in MGMT. Contrary to the prevalent expectation that MGMT expression is governed at the level of gene silencing, these data suggest that other mechanisms that can lead to tumor-selective reduction in MGMT activity exist in human tissue. PMID:23946891

  16. 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...

  17. Quantitative analysis of the IgG and IgG subclass immune responses to chromosomal Pseudomonas aeruginosa beta-lactamase in serum from patients with cystic fibrosis by western blotting and laser scanning densitometry

    DEFF Research Database (Denmark)

    Petersen, T D; Ciofu, O; Pressler, T

    1996-01-01

    BACKGROUND: Antibodies against chromosomal beta-lactamase of Pseudomonas aeruginosa (a beta ab) are markers of the development of resistance of P aeruginosa to beta-lactam antibiotics in patients with cystic fibrosis and chronic lung infection. The role of these antibodies in patients with chronic...... lung infection with P aeruginosa was further investigated by correlating the a beta ab IgG subclasses with pulmonary function in patients with cystic fibrosis. METHODS: Immunoglobulin G (IgG) and IgG subclass a beta ab were investigated by western blotting and quantified by laser scanning densitometry....... A longitudinal study on 43 consecutive patients with cystic fibrosis who developed chronic lung infection with P aeruginosa was performed. RESULTS: IgG subclass a beta ab appeared in all patients with chronic infection with P aeruginosa. Eleven years after the onset of infection all the patients had IgG1, 79...

  18. Investigation of the effects of experimental autolysis on the detection of abnormal prion protein in lymphoid and central nervous system tissues from elk and sheep using the Western blotting method.

    Science.gov (United States)

    Huang, Hongsheng; Soutyrine, Andrei; Rendulich, Jasmine; O'Rourke, Katherine; Balachandran, Aru

    2011-01-01

    Tissues unsuitable for standard immunohistochemical and histopathological examinations for chronic wasting disease (CWD) in cervids and for scrapie in sheep are frequently submitted for testing. This study investigated the effects of experimental autolysis on the detection of abnormal prion protein (PrPsc) in lymphoid and central nervous system (CNS) tissues from elk and sheep. The PrPsc was detected using a Western blotting (WB) test following PrPsc enrichment using sodium phosphotungstic acid (PTA) precipitation (PTA-WB). A commercial enzyme-linked immunosorbent assay (ELISA) was used as a reference test for quantitative measurement. This study showed that the amount of PrPsc in lymphoid and CNS tssues from elk and sheep decreased gradually as a result of autolysis, but PrPsc was still detectable after 5 and 15 d incubation at 37°C by PTA-WB for all lymphoid and CNS samples. The results of the ELISA supported those of PTA-WB, particularly for CNS tissues. In conclusion, autolysis at 37°C for 15 d would not significantly affect the detection of PrPsc in lymphoid and CNS tissues by WB and ELISA and, particularly, PTA-WB is a valuable and alternative confirmatory test to detect PrPsc in autolyzed lymphoid and CNS samples.

  19. 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.

  20. Comparison of the level, distribution and form of disease-associated prion protein in variant and sporadic Creutzfeldt-Jakob diseased brain using conformation-dependent immunoassay and Western blot.

    Science.gov (United States)

    Choi, Young Pyo; Gröner, Albrecht; Ironside, James W; Head, Mark W

    2011-03-01

    Disease-associated prion protein (PrP(Sc)) can be distinguished from the cellular isoform (PrP(C)) by conformation-dependent immunoassay (CDI). This technique exploits the presence of an epitope, accessible in PrP(C), but only unmasked by denaturation in PrP(Sc). In this study, we investigated PrP(Sc) in different brain regions in variant and sporadic Creutzfeldt-Jakob disease (CJD) by using CDI, and directly compared the results with those obtained using the more commonly employed protease digestion and Western blotting. In general, there was good agreement between the results, although there were certain discrepancies in relative abundance when the regional distribution in variant CJD cases was considered. The results largely confirmed the previously described targeting of different brain regions by variant and sporadic CJD. Additionally, the combination of protease digestion and CDI detection demonstrated, for the first time, the presence of PrP(Sc) in variant CJD brains that is susceptible to proteolysis under standard conditions.

  1. 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.

  2. 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.

  3. Gram staining.

    Science.gov (United States)

    Coico, Richard

    2005-10-01

    Named after Hans Christian Gram who developed the method in 1884, the Gram stain allows one to distinguish between Gram-positive and Gram-negative bacteria on the basis of differential staining with a crystal violet-iodine complex and a safranin counterstain. The cell walls of Gram-positive organisms retain this complex after treatment with alcohol and appear purple, whereas gram-negative organisms decolorize following such treatment and appear pink. The method described here is useful for assessing bacterial contamination of tissue culture samples or for examining the Gram stain status and morphological features of bacteria isolated from mixed or isolated bacterial cultures.

  4. Comparison of T24H-his, GST-T24H and GST-Ts8B2 recombinant antigens in western blot, ELISA and multiplex bead-based assay for diagnosis of neurocysticercosis.

    Science.gov (United States)

    Hernández-González, Ana; Noh, John; Perteguer, María Jesús; Gárate, Teresa; Handali, Sukwan

    2017-05-15

    Currently, the reference standard assay for the serodiagnosis of neurocysticercosis (NCC) is the lentil lectin-bound glycoproteins/enzyme-linked immunoelectrotransfer blot (LLGP-EITB). The main disadvantage of this technique is the complexity of obtaining and purifying the LLGP extract. This could be solved by replacement with highly specific recombinant antigens from Taenia solium. Based on previous studies, we selected and produced the recombinant Ts8B2 and T24H proteins and applied them to three diagnostic techniques: western blot (WB), enzyme-linked immunosorbent assay (ELISA) and the multiplex bead-based assay (MBA). The Ts8B2 and T24H cDNA sequences were expressed in a prokaryotic system and the corresponding expression products purified; three recombinant proteins were further characterized: T24H-his, GST-T24H and GST-Ts8B2. The proteins on WB, ELISA and MBA were tested against 149 sera from patients with NCC confirmed by brain imaging, 40 sera from patients with other parasitic diseases, and 131 sera from US. individuals without evidence of neurocysticercosis (clinical/serological/brain imaging). The sensitivity and specificity of each antigen by WB were calculated by counting the number of true positive, false positive, true negative and false negative results. Using the receiver operating characteristic (ROC) curves, the cut-off values for the ELISA and MBA were established as well as the sensitivity and specificity of each assay. All three antigens showed a high sensitivity on WB in active NCC cases with two or more viable cysts and low sensitivity for cases with single viable cyst or calcified lesions and inactive NCC. WB showed the highest specificity and sensitivity out of the three diagnostic techniques. The recombinant T24H-his was the best diagnostic reagent in WB (100% sensitivity, 99.4% specificity), exhibiting similar results to the LLGP-EITB, against the same panel of NCC sera. The GST-T24H antigen worked better than the others in ELISA and MBA

  5. 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).

  6. Screening for simian foamy virus infection by using a combined antigen Western blot assay: evidence for a wide distribution among Old World primates and identification of four new divergent viruses

    International Nuclear Information System (INIS)

    Hussain, Althaf I.; Shanmugam, Vedapuri; Bhullar, Vinod B.; Beer, Brigitte E.; Vallet, Dominique; Gautier-Hion, Annie; Wolfe, Nathan D.; Karesh, William B.; Kilbourn, Annelisa M.; Tooze, Zeena; Heneine, Walid; Switzer, William M.

    2003-01-01

    Simian foamy viruses (SFVs) belong to a genetically and antigenically diverse class of retroviruses that naturally infect a wide range of nonhuman primates (NHPs) and can also be transmitted to humans occupationally exposed to NHPs. Current serologic detection of SFV infection requires separate Western blot (WB) testing by using two different SFV antigens [SFV AGM (African green monkey) and SFV CPZ (chimpanzee)]. However, this method is labor intensive and validation is limited to only small numbers of NHPs. To facilitate serologic SFV testing, we developed a WB assay that combines antigens from both SFV AGM and SFV CPZ . The combined-antigen WB (CA-WB) assay was validated with 145 serum samples from 129 NHPs (32 African and Asian species) and 16 humans, all with known SFV infection status determined by PCR. Concordant CA-WB results were obtained for all 145 PCR-positive or -negative primate and human specimens, giving the assay a 100% sensitivity and specificity. In addition, no reactivity was observed in sera from persons positive for human immunodeficiency virus or human T cell lymphotropic virus (HIV/HTLV) (n = 25) or HIV/HTLV-negative U.S. blood donors (n = 100). Using the CA-WB assay, we screened 360 sera from 43 Old World primate species and found an SFV prevalence of about 68% in both African and Asian primates. We also isolated SFV from the blood of four seropositive primates (Allenopithecus nigroviridis, Trachypithecus francoisi, Hylobates pileatus, and H. leucogenys) not previously known to be infected with SFV. Phylogenetic analysis of integrase sequences from these isolates confirmed that all four SFVs represent new, distinct, and highly divergent lineages. These results demonstrate the ability of the CA-WB assay to detect infection in a large number of NHP species, including previously uncharacterized infections with divergent SFVs

  7. Immunocytochemical electron microscopic study and western blot analysis of myosin, paramyosin and miniparamyosin in the striated muscle of the fruit fly Drosophila melanogaster and in obliquely striated and smooth muscles of the earthworm Eisenia foetida.

    Science.gov (United States)

    Royuela, M; Fraile, B; Cervera, M; Paniagua, R

    1997-04-01

    Miniparamyosin is a paramyosin isoform (55-60 kDa) that has been isolated in insects (Drosophila) and immunolocalized in several species of arthropods, molluscs, annelids and nematodes. In this study, the presence and distribution of this protein, in comparison with that of paramyosin and myosin, has been examined in the striated muscle (tergal depressor of trochanter) of Drosophila melanogaster, and the obliquely striated muscle (body wall) and the smooth muscle (outer layer of the pseudoheart) of the earthworm Eisenia foetida by means of immunocytochemical electron microscopic study and Western blot analysis miniparamyosin paramyosin and myosin antibodies from Drosophila. In the striated muscle of D. melanogaster, the three proteins were immunolocalized along the length of the thick filaments (A-bands). The distribution of immunogold particles along these filaments was uniform. The relative proportions miniparamyosin/paramyosin/myosin (calculated by counting the number of immunogold particles) were: 1/10/68. In the obliquely striated muscle of E. foetida, immunoreactions to the three proteins were also found in the thick filaments, and the relative proportions miniparamyosin/paramyosin/myosin were 1/2.4/6.9. However, whereas the distribution of both myosin and miniparamyosin along the thick filament length was uniform, paramyosin immunolabelling was more abundant in the extremes of thick filaments (the outer zones of A-bands in the obliquely striated muscle), where the thick filaments become thinner than in the centre (the central zone of A-bands), where these filaments are thicker. The relative proportions of paramyosin in the outer and of paramyosin in the central zones of A-bands were 4/1. This irregular distribution of paramyosin along the thick filament length might be actual but it may also be explained by the fusiform shape of thick filaments in the earthworm: assuming that paramyosin is covered by myosin, paramyosin antigens would be more exposed in the

  8. Dimethylformamide interferes with Coomassie dye staining of proteins on blue native gel electrophoresis.

    Science.gov (United States)

    Raghupathy, V; Oommen, Anna; Ramachandran, Anup

    2014-06-15

    Blue native gel electrophoresis (BN-PAGE) is used extensively for characterization of mitochondrial respiratory complexes and uses the binding of Coomassie brilliant blue G-250 to visualize proteins. Oxidative modification of sulfhydryl groups of such proteins can be evaluated by labeling with iodoacetamide conjugated to biotin (BIAM) and detected with streptavidin peroxidase on Western blots following BN-PAGE. However, dissolving BIAM in dimethylformamide, a recommended solvent, reduces Coomassie blue G staining to proteins during BN-PAGE. This interference is prevented by dissolving BIAM in dimethyl sulfoxide. Precautions in the use of the dye for protein staining subsequent to BIAM labeling are discussed. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Pleural fluid Gram stain

    Science.gov (United States)

    Gram stain of pleural fluid ... mixing it with a violet stain (called a Gram stain). A laboratory specialist uses a microscope to ... reveals an abnormal collection of pleural fluid. The Gram stain can help identify the bacteria that might ...

  10. 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...

  11. 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…

  12. Differential staining of bacteria: gram stain.

    Science.gov (United States)

    Moyes, Rita B; Reynolds, Jackie; Breakwell, Donald P

    2009-11-01

    In 1884, Hans Christian Gram, a Danish doctor, developed a differential staining technique that is still the cornerstone of bacterial identification and taxonomic division. This multistep, sequential staining protocol separates bacteria into four groups based on cell morphology and cell wall structure: Gram-positive cocci, Gram-negative cocci, Gram-positive rods, and Gram-negative rods. The Gram stain is useful for assessing bacterial contamination of tissue culture samples or for examining the Gram stain status and morphological features of bacteria isolated from mixed or isolated bacterial cultures. (c) 2009 by John Wiley & Sons, Inc.

  13. Port-Wine Stains

    Science.gov (United States)

    ... Safe Videos for Educators Search English Español Port-Wine Stains KidsHealth / For Parents / Port-Wine Stains What's ... Manchas de vino de oporto What Are Port-Wine Stains? A port-wine stain is a type ...

  14. Acid-fast stain

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003766.htm Acid-fast stain To use the sharing features on this page, please enable JavaScript. The acid-fast stain is a laboratory test that determines ...

  15. Phenylglyoxal-Based Visualization of Citrullinated Proteins on Western Blots

    Directory of Open Access Journals (Sweden)

    Sanne M. M. Hensen

    2015-04-01

    Full Text Available Citrullination is the conversion of peptidylarginine to peptidylcitrulline, which is catalyzed by peptidylarginine deiminases. This conversion is involved in different physiological processes and is associated with several diseases, including cancer and rheumatoid arthritis. A common method to detect citrullinated proteins relies on anti-modified citrulline antibodies directed to a specific chemical modification of the citrulline side chain. Here, we describe a versatile, antibody-independent method for the detection of citrullinated proteins on a membrane, based on the selective reaction of phenylglyoxal with the ureido group of citrulline under highly acidic conditions. The method makes use of 4-azidophenylglyoxal, which, after reaction with citrullinated proteins, can be visualized with alkyne-conjugated probes. The sensitivity of this procedure, using an alkyne-biotin probe, appeared to be comparable to the antibody-based detection method and independent of the sequence surrounding the citrulline.

  16. Stabilized Romanowsky blood stain.

    Science.gov (United States)

    Gilliland, J W; Dean, W W; Stastny, M; Lubrano, G J

    1979-05-01

    It has been shown that the degradation of thiazine dyes which normally occurs in methanolic solution, as in the case of Romanowsky blood stains, can be prevented by making the solution acidic. In a certain range of acidity, the stain precipitates in the form of monothiazine eosinate, but by making the solution sufficiently acidic, eosin is protonated and the precipitate cannot form. These observations have been used to develop a blood stain which is stable, even at elevated temperatures, for several months. For use the stain is neutralized by a specially formulated fixative solution.

  17. Design and synthesis of new fluorescent probe for rapid and highly sensitive detection of proteins via electrophoretic gel stain.

    Science.gov (United States)

    Suzuki, Yoshio; Takagi, Nobuyuki; Chimuro, Tomoyuki; Shinohara, Atsushi; Sakaguchi, Nao; Hiratsuka, Atsunori; Yokoyama, Kenji

    2011-06-01

    A new fluorescent molecular probe, 2,2'-(1E,1'E)-2,2'-(4-(dicyanomethylene)-4H-pyrane-2,6-diyl)bis(ethene-2,1-diyl)bis(sodium benzenesulfonate) salt (1), possessing the cyanopyranyl moieties and two benzene sulfonic acid groups was designed and synthesized to detect proteins in solution and for high-throughput SDS-PAGE. Compound 1 exhibited no fluorescence in the absence of proteins; however, it exhibited strong fluorescence on the addition of bovine serum albumin as a result of intramolecular charge transfer. Compared with the conventional protocols for in-gel protein staining, such as SYPRO Ruby and silver staining, 1 achieves higher sensitivity, even though it offers a simplified, higher throughput protocol. In fact, the total time required for protein staining was 60-90 min under optimum conditions much shorter than that required by the less-sensitive silver staining or SYPRO Ruby staining protocols. Moreover, 1 was successfully applied to protein identification by mass spectrometry via in-gel tryptic digestion, Western blotting, and native PAGE together with protein staining by 1, which is a modified protocol of blue native PAGE (BN-PAGE). Thus, 1 may facilitate high-sensitivity protein detection, and it may be widely applicable as a convenient tool in various scientific and medical fields. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. [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.

  19. Iron Stain on Wood

    Science.gov (United States)

    Mark Knaebe

    2013-01-01

    Iron stain, an unsightly blue–black or gray discoloration, can occur on nearly all woods. Oak, redwood, cypress, and cedar are particularly prone to iron stain because these woods contain large amounts of tannin-like extractives. The discoloration is caused by a chemical reaction between extractives in the wood and iron in steel products, such as nails, screws, and...

  20. Endocervical gram stain

    Science.gov (United States)

    ... no symptoms. Alternative Names Gram stain of cervix; Gram stain of cervical secretions References Marrazzo JM, Apicella MA. Neisseria gonorrhoeae (gonorrhea). In: Bennett JE, Dolin R, Blaser MJ, eds. Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases, Updated Edition . 8th ...

  1. Dramatic Stained Glass.

    Science.gov (United States)

    Prater, Michael

    2002-01-01

    Describes an art project that is appropriate for students in fifth through twelfth grade in which they create Gothic-style stained-glass windows. Discusses how college students majoring in elementary education created stained-glass windows. Addresses how to adapt this lesson for younger students. (CMK)

  2. Stool Gram stain

    Science.gov (United States)

    ... of stool; Feces Gram stain References Allos BM. Campylobacter infections. In: Goldman L, Schafer AI, eds. Goldman- ... Bacterial Infections Read more Foodborne Illness Read more Gastroenteritis Read more A.D.A.M., Inc. is ...

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. "Stained Glass" Landscape Windows

    Science.gov (United States)

    Vannata, Janine

    2008-01-01

    Both adults and children alike marvel at the grand vivid stained-glass windows created by American artist Louis Comfort Tiffany. Today he is commonly recognized as one of America's most influential designers and artists throughout the last nineteenth and early twentieth century. In the lesson described in this article, students created their own…

  9. Stained Glass and Flu

    Centers for Disease Control (CDC) Podcasts

    2017-02-01

    Dr. Robert Webster, an Emeritus member of the Department of Infectious Diseases at St. Jude Children's Research Hospital, discusses his cover art story on stained glass and influenza.  Created: 2/1/2017 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 2/1/2017.

  10. Port-wine stain

    Science.gov (United States)

    ... About MedlinePlus Show Search Search MedlinePlus GO GO About MedlinePlus Site Map FAQs Customer Support Health Topics Drugs & Supplements Videos & Tools Español You Are Here: Home → Medical Encyclopedia → Port-wine stain URL of this page: //medlineplus.gov/ency/ ...

  11. Ghost mycobacteria on Gram stain.

    Science.gov (United States)

    Trifiro, S; Bourgault, A M; Lebel, F; René, P

    1990-01-01

    The Gram stain is a key tool in diagnostic microbiology. Its usefulness with respect to mycobacteria is undefined. The neutrality of mycobacteria other than Mycobacterium tuberculosis on Gram staining of various clinical specimens is described. Images PMID:1688872

  12. Ghost mycobacteria on Gram stain.

    OpenAIRE

    Trifiro, S; Bourgault, A M; Lebel, F; René, P

    1990-01-01

    The Gram stain is a key tool in diagnostic microbiology. Its usefulness with respect to mycobacteria is undefined. The neutrality of mycobacteria other than Mycobacterium tuberculosis on Gram staining of various clinical specimens is described.

  13. Gram stain of urethral discharge

    Science.gov (United States)

    Urethral discharge Gram stain; Urethritis - Gram stain ... Augenbraun MH, McCormack WM. Urethritis. In: Bennett JE, Dolin R, Blaser MJ, eds. Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases, Updated Edition . ...

  14. Expression of TGFβ superfamily components and other markers of oocyte quality in oocytes selected by brilliant cresyl blue staining: relevance to early embryonic development.

    Science.gov (United States)

    Ashry, Mohamed; Lee, KyungBon; Mondal, Mohan; Datta, Tirtha K; Folger, Joseph K; Rajput, Sandeep K; Zhang, Kun; Hemeida, Nabil A; Smith, George W

    2015-03-01

    Brilliant cresyl blue (BCB) is a super-vital stain that has been used to select competent oocytes in different species. One objective of the present study was to assess the relationship between BCB staining, which correlates with an oocyte's developmental potential, and the transcript abundance for select TGFβ-superfamily components, SMAD2/3 and SMAD1/5 phosphorylation levels, and oocyte (JY1) and cumulus-cell (CTSB, CTSK, CTSS, and CTSZ) transcript markers in bovine oocytes and/or adjacent cumulus cells. The capacity of exogenous follistatin or JY1 supplementation or cathepsin inhibitor treatment to enhance development of embryos derived from low-quality oocytes, based on BCB staining, was also determined. Cumulus-oocyte complexes (COCs) from abattoir-derived ovaries were subjected to BCB staining, and germinal-vesicle-stage oocytes and cumulus cells were harvested from control, BCB+, and BCB- (low-quality oocyte) groups for real-time PCR or Western-blot analysis. Remaining COCs underwent in vitro maturation, in vitro fertilization, and embryo culture in the presence or absence of the above exogenous supplements. Levels of FST, JY1, BMP15, and SMAD1, 2, 3, and 5 transcripts were higher in BCB+ oocytes whereas CTSB, CTSK, CTSS, and CTSZ mRNA abundance was higher in cumulus cells surrounding BCB- oocytes. Western-blot analysis revealed higher SMAD1/5 and SMAD2/3 phosphorylation in BCB+ than BCB- oocytes. Embryo-culture studies demonstrated that follistatin and cathepsin inhibitor treatment, but not JY-1 treatment, improve the developmental competence of BCB- oocytes. These results contribute to a better understanding of molecular indices of oocyte competence. © 2015 Wiley Periodicals, Inc.

  15. Staining properties and stability of a standardised Romanowsky stain.

    Science.gov (United States)

    Marshall, P N; Bentley, S A; Lewis, S M

    1978-03-01

    An evaluation of the standardised Romanowsky stain of Marshall et al. has been made in a routine haematology laboratory. It was noted that this stain had several advantages over the May-Grünwald Giemsa stain used in most British laboratories. These advantages include ease and speed of preparation, a shorter staining time, and reproducibility of results. These results are described in detail. The stability of the stock stain solution and of the 'working' stain (stock + buffer) has been studied by, respectively, thin-layer chromatography and visible spectroscopy. No change was detected in the composition of the stock solution at ambient temperature over a period of six months. Stability was unaffected by the composition of the container (polyethylene, PyrexTM, or soda-glass) or by daylight. The 'working' solution was stable for 3 hours. Thereafter a precipitate is formed, consisting of thiazine dyes and eosin in a molar ratio of approximately 2:1.

  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. A new bacterial staining method involving Gram stain with theoretical considerations of the staining mechanism.

    Science.gov (United States)

    Noda, Y; Tôei, K

    1992-01-01

    In order to investigate the mechanism of Gram staining of bacteria, tests with anionic dyes followed by treatment with cationic octyltrimethylammonium (OTMA) were carried out. The study revealed that tetrabromophenolphthalein ethylester (TBPE) gave the most reliable staining of Gram-negative bacteria with negative staining of Gram-positive bacteria. Tests on many species of bacteria showed that TBPE positive bacteria were Gram-negative and vice versa, without exception.

  18. An evaluation of some commerical Romanowsky stains.

    Science.gov (United States)

    Marshall, P N; Bentley, S A; Lewis, S M

    1975-08-01

    The staining properties of 43 commerical Romanowsky-type stains have been studied. Considerable differences in the appearance of stained blood films were observed with different batches of these stains, the staining of red cells being particularly variable. Attempts have been made to correlate staining patterns with stain composition as revealed by thin-layer chromatography and sulphated ash analyses. In this way it has been possible to define some essential requirements for satisfactory staining.

  19. Image-based stained glass.

    Science.gov (United States)

    Brooks, Stephen

    2006-01-01

    We present a method of restyling an image so that it approximates the visual appearance of a work of stained glass. To this end, we develop a novel approach which involves image warping, segmentation, querying, and colorization along with texture synthesis. In our method, a given input image is first segmented. Each segment is subsequently transformed to match real segments of stained glass queried from a database of image exemplars. By using real sources of stained glass, our method produces high quality results in this nascent area of nonphotorealistic rendering. The generation of the stained glass requires only modest amounts of user interaction. This interaction is facilitated with a unique region-merging tool.

  20. 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.

  1. Sensitivity of prestaining RNA with ethidium bromide before electrophoresis and performance of subsequent northern blots using heterologous DNA probes.

    Science.gov (United States)

    Zhao, Yun; Du, Linfang; Zhang, Nianhui

    2013-06-01

    Adding ethidium bromide (EtBr) at low concentrations to RNA samples before running formaldehyde-agarose gels affords the advantages of checking RNA integrity and evaluating the quality of size-separation at any time during electrophoresis or immediately after either electrophoresis or blotted the separated RNA onto the membrane without significantly compromising mobility, transfer, or hybridization. In this study, we systematically examined the factors that affect the sensitivity of RNA prestaining by heating RNA samples that include EtBr before electrophoresis under different denaturation conditions. We also examined the efficiency of the hybridization of EtBr-prestained RNA with heterologous DNA probes. The results showed that the fluorescent intensity of EtBr-prestained RNA was affected not only by the EtBr concentration as previously reported but also by the RNA amount, denaturation time, and denaturation temperature. Prior staining of RNA with 40 μg/mL EtBr significantly decreased the efficiency of Northern blot hybridization with heterologous DNA probes. We propose that to best combine staining sensitivity and the efficiency of Northern blot hybridization with heterologous DNA probes, the concentration of EtBr used to prestain RNA should not exceed 30 μg/mL. The efficiency of the hybridization of EtBr-prestained RNA was affected not only by factors that affect staining sensitivity but also by the type of probe used.

  2. 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.

  3. 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.

  4. Salt stains from evaporating droplets

    NARCIS (Netherlands)

    Shahidzadeh, N.; Schut, M.F.L.; Desarnaud, J.; Prat, M.; Bonn, D.

    2015-01-01

    The study of the behavior of sessile droplets on solid substrates is not only associated with common everyday phenomena, such as the coffee stain effect, limescale deposits on our bathroom walls, but also very important in many applications such as purification of pharmaceuticals, deicing of

  5. 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

  6. Etika Berbusana Mahasiswa Stain Samarinda

    Directory of Open Access Journals (Sweden)

    Ida Suryani Wijaya

    2012-06-01

    Full Text Available Ethics is about behavior of human being, such as which one is right or wrong. The ethics is always affecting the human life. The ethics gives people orientation how he/she do manything every time every day. Islamic ethics consists of the way how someone interact each other; how someone should do or not to do, how to sit, how to walk, how to eat or drink, how to sleep, or how to get dressed. Al-Qur’an uses three terms to define about dressing, they are: libas, tsiyah, and sarahi. Dressing has a function as covering the body, as assessoris, as the way to do Islamic taqwa, and as an identiy. Dressing ethics of the female students of STAIN Samarinda has been regulated by the rector regulation No 19 of the year 2002 about relation and dressing ethics for the students of STAIN Samarinda.

  7. Microwave-stimulated staining of plastic embedded bone marrow sections with the Romanowsky-Giemsa stain: improved staining patterns.

    Science.gov (United States)

    Boon, M E; Kok, L P; Moorlag, H E; Gerrits, P O; Suurmeijer, A J

    1987-07-01

    Staining plastic sections with the Romanowsky-Giemsa method is both time-consuming and difficult. This paper reports how the staining time can be reduced to 25 min using microwave irradiation of the staining solution. It is shown that staining results depend on the fixative used, staining temperature, dye concentration and pH of the staining solution as well as on several parameters of the microwave irradiation technique. The staining patterns are improved when compared with those obtained by conventional staining of plastic sections. The colors are more brilliant and greater contrasts are observed. Basophilia, polychromasia, and orthochromasia accompanying red cell maturation are more pronounced. For white cell maturation the initial appearance of specific granules (neutrophil, basophil, and eosinophil) is more evident. Thus, cell classification is easily accomplished using the described technique. It is suggested that microwave-stimulated staining be considered for routine use.

  8. Standardization of the Romanowsky-Giemsa stain: the influence of staining time on the RG-staining pattern.

    Science.gov (United States)

    Schulte, E

    1987-01-01

    In this paper the influence of staining time on the staining pattern of Romanowsky-Giemsa (RG) type stains is investigated. Smears of rabbit bone marrow and of human venous blood were stained with azure B-eosin Y, azure A-eosin Y and with the cationic dyes alone under varying conditions of staining time and dye concentration. The stained smears were investigated by integrating microdensitometry. DNA-polyacrylamide (PAA) model films were stained with azure A-eosin Y, the extinction of the stained model films was determined by spectrophotometry. With increasing staining time the color of the cell nuclei changed from blue to an intense purple, the texture of the nuclear chromatin became more prominent. Prolonged staining resulted in over-staining of the cell nuclei with loss of a distinct chromatin texture. Besides such factors as dye concentration and pH of the staining solution standardization of staining time may be considered necessary for the reproducibility of the RG staining pattern.

  9. Accelerated staining technique using kitchen microwave oven

    Directory of Open Access Journals (Sweden)

    Archana Mukunda

    2015-01-01

    Full Text Available Introduction: Histopathological diagnosis of specimens is greatly dependent on good sample preparation and staining. Both of these processes is governed by diffusion of fluids and dyes in and out of the tissue, which is the key to staining. Diffusion of fluids can be accelerated by the application of heat that reduces the time of staining from hours to the minute. We modified an inexpensive model of kitchen microwave oven for staining. This study is an attempt to compare the reliability of this modified technique against the tested technique of routine staining so as to establish the kitchen microwave oven as a valuable diagnostic tool. Materials and Methods: Sixty different tissue blocks were used to prepare 20 pairs of slides for 4 different stains namely hematoxylin and eosin, Van Gieson′s, 0.1% toluidine blue and periodic acid-Schiff. From each tissue block, two bits of tissues were mounted on two different slides. One slide was stained routinely, and the other stained inside a microwave. A pathologist evaluated the stained slides and the results so obtained were analyzed statistically. Results: Microwave staining considerably cut down the staining time from hours to seconds. Microwave staining showed no loss of cellular and nuclear details, uniform-staining characteristics and was of excellent quality. Interpretation and Conclusion: The cellular details, nuclear details and staining characteristics of microwave stained tissues were better than or equal to the routine stained tissue. The overall quality of microwave-stained sections was found to be better than the routine stained tissue in majority of cases.

  10. Indeterminate HIV-1 Western Blots: Etiology, Natural History, and Psychological Reactions

    Science.gov (United States)

    1992-09-16

    ME.1O Skin diseases 0-no: l-seborrhea; 2-recurrent staph: 3- psoriasis : 4-vitil igo 5-other. . .. . .. ... 6-multiple ME. iP Mononucleosis 0-no: 1...Amsterdam, The Netherlands : VIII Internatonal C~oaerenc~c on AIDS, uly 1992. ~.Haff eid RM, Wande!! M. Goldstein L.. Shrlw & SCoopersaft Study Group...AIDS/Ill STD World Congress Amsterdam, the Netherlands 19-24 July 1992 Secretariat Space RA _____________Date Received ____________Abstrzct

  11. Utility of Western Blot Analysis for the Diagnosis of Cutaneous Leishmaniasis

    Directory of Open Access Journals (Sweden)

    Marzieh ASHRAFMANSOURI

    2015-12-01

    Full Text Available Background: Cutaneous leishmaniasis (CL is a parasitic disease with a relatively wide distribution in different areas of the world, including Iran. The parasite is mainly diagnosed microscopically, but serological approaches might be useful for diagnosis as well.  This study aimed to assess the efficacy of an immunoblotting system for serodiagnosis of cutaneous leishmaniasis in Iran.Methods: Sixty-one sera samples from parasitologically confirmed CL patients and 50 sera samples from healthy controls along with 50 sera sample from non-CL patients were collected. Native strain of Leishmania major was cultured in Schnei­der medium and soluble Leishmania antigens were prepared from amastigotes-like parasites. All of sera samples were evaluated by an immunoblot­ting system.Results: Components of 14 to 135 kDa were detectable by the sera of CL pa­tients. From 61 sera of CL patients, 59 cases (96.7% detected a 63 kDa subunit and 51 cases (83.6% recognized a 32-35 kDa component. Among all subunits, the 63 kDa band showed the highest sensitivity (96.7% and a 75 kDa band had the highest (98% specificity.Conclusion: Immunoblotting has a satisfactory performance in diagnosis of CL and this test can be used, as an aid, for proper diagnosis of CL.

  12. 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.

  13. Optimization of Western blotting analysis for the isolation and detection of membrane xenobiotic transporter ABCG2.

    Science.gov (United States)

    Szczygieł, Małgorzata; Markiewicz, Marcin; Szafraniec, Milena; Zuziak, Roxana; Urbańska, Krystyna; Fiedor, Leszek

    2017-01-01

    All organisms are exposed to numerous stress factors, which include harmful xenobiotics. The diversity of these compounds is enormous, thus in the course of evolution diverse biological defense mechanisms at various levels of organization have developed. One of them engages an evolutionarily conserved family of transporters from the ABC superfamily, found in most species - from bacteria to humans. An important example of such a transporter is the breast cancer resistance protein (BCRP/ABCG2), a typical integral membrane protein. It plays a key role in the absorption, distribution and elimination of a wide variety of xenobiotics, including drugs used in chemotherapy, and is involved in multidrug resistance. It also protects against phototoxic chlorophyll derivatives of dietary origin. BCRP is a hemitransporter which consists of one transmembrane domain, made of six alpha-helices forming a characteristic pore structure, and one ATP-binding domain, which provides the energy from ATP hydrolysis, required for active transport of the substrates. The isolation of BCRP is still not an easy task, because its insolubility in water and the presence of membrane rafts pose serious methodological and technical challenges during the purification. The aim of this study was to optimize the methods for detection and isolation of BCRP-enriched fractions obtained from animal tissue samples. In this report we describe an optimization of isolation of a BCRP-enriched membrane fraction, which is suitable for further protein quantitative and qualitative analysis using the molecular biology tools.

  14. TSE strain differentiation in mice by immunohistochemical PrPSc profiles and triplex Western blot

    NARCIS (Netherlands)

    Keulen, van L.J.M.; Langeveld, J.P.M.; Dolstra, C.H.; Jacobs, J.G.; Bossers, A.; Zijderveld, van F.G.

    2015-01-01

    TSE strains are routinely identified by their incubation period and vacuolation profile in the brain after intracerebral inoculation and serial passaging in inbred mouse lines. There are some major drawbacks to this method that are related to the variation in vacuolation that exists in the brains of

  15. 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.

  16. 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...

  17. Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins.

    Science.gov (United States)

    Ramsey, Kathryn A; Rushton, Zachary L; Ehre, Camille

    2016-06-14

    Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

  18. Microdissection of stained archival tissue.

    Science.gov (United States)

    Gupta, S K; Douglas-Jones, A G; Morgan, J M

    1997-08-01

    In many tissues the preinvasive stage of neoplastic progression can be identified histologically as dysplasia or in situ disease. There is much interest in defining the molecular events associated with the early stages of neoplasia. Retrieval of histologically recognisable preinvasive neoplastic tissue uncontaminated by inflammatory or stromal cells is important for genetic studies using polymerase chain reaction (PCR) assay. A novel method for microdissection is described in which 10 microns sections are dewaxed, stained with haematoxylin and eosin, dried, covered with Sellotape, and the tissue cut out using a scalpel blade under direct visual control. The method is quick, eliminates problems of operator tremor, preserves the architecture of the micro-dissected tissue (for photographic documentation) and requires no special equipment. The presence of Sellotape and adhesive in the reaction mixture has no detrimental effect on the ability to extract DNA or to perform PCR.

  19. Comparison of immunohistochemical and modified Giemsa stains ...

    African Journals Online (AJOL)

    In 2 cases immunostain could not demonstrate the bacteria but they were identified with modified Giemsa stain while in 5 cases the bacteria were identified by immunostain but not with modified Giemsa stain. The sensitivity of modified Giemsa stain was 85% (CI 66.5-98.8) while the specificity was 89% (CI 60.4 – 97.8).

  20. [Diagnostic stain of helminth eggs (author's transl)].

    Science.gov (United States)

    Cerva, L

    1976-12-01

    A description is given of a diagnostic method for the staining of eggs and larvae of intestinal helminth in smears of both fresh and fixed stool samples. The contents of the eggs and larvae stain red, the background various shades of blue. The most contrasting staining was obtained with thin-walled eggs.

  1. Histological stain evaluation for machine learning applications

    Directory of Open Access Journals (Sweden)

    Jimmy C Azar

    2013-01-01

    Full Text Available Aims: A methodology for quantitative comparison of histological stains based on their classification and clustering performance, which may facilitate the choice of histological stains for automatic pattern and image analysis. Background: Machine learning and image analysis are becoming increasingly important in pathology applications for automatic analysis of histological tissue samples. Pathologists rely on multiple, contrasting stains to analyze tissue samples, but histological stains are developed for visual analysis and are not always ideal for automatic analysis. Materials and Methods: Thirteen different histological stains were used to stain adjacent prostate tissue sections from radical prostatectomies. We evaluate the stains for both supervised and unsupervised classification of stain/tissue combinations. For supervised classification we measure the error rate of nonlinear support vector machines, and for unsupervised classification we use the Rand index and the F-measure to assess the clustering results of a Gaussian mixture model based on expectation-maximization. Finally, we investigate class separability measures based on scatter criteria. Results: A methodology for quantitative evaluation of histological stains in terms of their classification and clustering efficacy that aims at improving segmentation and color decomposition. We demonstrate that for a specific tissue type, certain stains perform consistently better than others according to objective error criteria. Conclusions: The choice of histological stain for automatic analysis must be based on its classification and clustering performance, which are indicators of the performance of automatic segmentation of tissue into morphological components, which in turn may be the basis for diagnosis.

  2. 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.

  3. Standardization of Romanowsky stains. The relationship between stain composition and performance.

    Science.gov (United States)

    Marshall, P N; Bentley, S A; Lewis, S M

    1978-03-01

    A panel of 17 eminent haematologists has assessed the performance of 5 Romanowsky stains prepared from pure component dyes, comparing the suitability and acceptability of these stains for the preparation of routine blood and bone-marrow films. It was found that the results obtained using the stain described by Marshall et al (1975) were comparable to those obtained using a modification of the stain described by Wittekind et al (1976). The performance of the 3 other stains was less acceptable. Variations in stain formulation have been correlated with stain performance.

  4. A procedure for Alcian blue staining of mucins on polyvinylidene difluoride membranes.

    Science.gov (United States)

    Dong, Weijie; Matsuno, Yu-ki; Kameyama, Akihiko

    2012-10-16

    The isolation and characterization of mucins are critically important for obtaining insight into the molecular pathology of various diseases, including cancers and cystic fibrosis. Recently, we developed a novel membrane electrophoretic method, supported molecular matrix electrophoresis (SMME), which separates mucins on a polyvinylidene difluoride (PVDF) membrane impregnated with a hydrophilic polymer. Alcian blue staining is widely used to visualize mucopolysaccharides and acidic mucins on both blotted membranes and SMME membranes; however, this method cannot be used to stain mucins with a low acidic glycan content. Meanwhile, periodic acid-Schiff staining can selectively visualize glycoproteins, including mucins, but is incompatible with glycan analysis, which is indispensable for mucin characterizations. Here we describe a novel staining method, designated succinylation-Alcian blue staining, for visualizing mucins on a PVDF membrane. This method can visualize mucins regardless of the acidic residue content and shows a sensitivity 2-fold higher than that of Pro-Q Emerald 488, a fluorescent periodate Schiff-base stain. Furthermore, we demonstrate the compatibility of this novel staining procedure with glycan analysis using porcine gastric mucin as a model mucin.

  5. Characterization of ELISA Antibody-Antigen Interaction using Footprinting-Mass Spectrometry and Negative Staining Transmission Electron Microscopy

    Science.gov (United States)

    Lin, Margaret; Krawitz, Denise; Callahan, Matthew D.; Deperalta, Galahad; Wecksler, Aaron T.

    2018-03-01

    We describe epitope mapping data using multiple covalent labeling footprinting-mass spectrometry (MS) techniques coupled with negative stain transmission electron microscopy (TEM) data to analyze the antibody-antigen interactions in a sandwich enzyme-linked immunosorbant assay (ELISA). Our hydroxyl radical footprinting-MS data using fast photochemical oxidation of proteins (FPOP) indicates suppression of labeling across the antigen upon binding either of the monoclonal antibodies (mAbs) utilized in the ELISA. Combining these data with Western blot analysis enabled the identification of the putative epitopes that appeared to span regions containing N-linked glycans. An additional structural mapping technique, carboxyl group footprinting-mass spectrometry using glycine ethyl ester (GEE) labeling, was used to confirm the epitopes. Deglycosylation of the antigen resulted in loss of potency in the ELISA, supporting the FPOP and GEE labeling data by indicating N-linked glycans are necessary for antigen binding. Finally, mapping of the epitopes onto the antigen crystal structure revealed an approximate 90° relative spatial orientation, optimal for a noncompetitive binding ELISA. TEM data shows both linear and diamond antibody-antigen complexes with a similar binding orientation as predicted from the two footprinting-MS techniques. This study is the first of its kind to utilize multiple bottom-up footprinting-MS techniques and TEM visualization to characterize the monoclonal antibody-antigen binding interactions of critical reagents used in a quality control (QC) lot-release ELISA. [Figure not available: see fulltext.

  6. Indirect immunofluorescence staining of cultured neural cells.

    Science.gov (United States)

    Barbierato, Massimo; Argentini, Carla; Skaper, Stephen D

    2012-01-01

    Immunofluorescence is a technique allowing the visualization of a specific protein or antigen in cells or tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein isothiocyanate. There are two major types of immunofluorescence staining methods: (1) direct immunofluorescence staining in which the primary antibody is labeled with fluorescence dye and (2) indirect immunofluorescence staining in which a secondary antibody labeled with fluorochrome is used to recognize a primary antibody. This chapter describes procedures for the application of indirect immunofluorescence staining to neural cells in culture.

  7. Standard specimens for stain calibration: application to Romanowsky-Giemsa staining.

    Science.gov (United States)

    Turner, J N; Weir, B; Collins, D N

    1990-01-01

    Standardized specimens with reproducible staining properties were fabricated from extracts of biological objects (bovine liver, nucleoprotamine and defatted muscle). The standard specimens were stained with two formulations of the Romanowsky-Giemsa stain (RG), using the same azure B and eosin Y. One formulation used methanol and Sorensen's buffer and the other DMSO and Hepes buffer as solvents. The standard specimens were stained either in the composite stain or in the individual dyes dissolved in the same solvents and at the same concentration as the composite stain. Solution spectroscopy demonstrated different spectra for the two formulations with some wavelength regions varying by more than an order of magnitude. The RG spectra were also very different from those of the individual dyes dissolved at the RG concentration in the respective solvents. The stained standard specimens were analyzed by microspectrophotometry and were found to have spectra similar to those of cell smears. Furthermore, the standard specimens were shown to be a repeatable substrate for stain uptake. The transmitted light intensity from random fields of the same standardized specimen varied +/- 5%. When specimens were stained at the same time, the specimen-to-specimen variation depended on preparation conditions and the measurement wavelength, but was as good as +/- 5% for some conditions. The quantitative stain performance of both formulations was studied and compared. The standardized specimens provide a tool for the quantitative study of staining processes and specimen preparation procedures and for stain calibration.

  8. Multicenter Assessment of Gram Stain Error Rates.

    Science.gov (United States)

    Samuel, Linoj P; Balada-Llasat, Joan-Miquel; Harrington, Amanda; Cavagnolo, Robert

    2016-06-01

    Gram stains remain the cornerstone of diagnostic testing in the microbiology laboratory for the guidance of empirical treatment prior to availability of culture results. Incorrectly interpreted Gram stains may adversely impact patient care, and yet there are no comprehensive studies that have evaluated the reliability of the technique and there are no established standards for performance. In this study, clinical microbiology laboratories at four major tertiary medical care centers evaluated Gram stain error rates across all nonblood specimen types by using standardized criteria. The study focused on several factors that primarily contribute to errors in the process, including poor specimen quality, smear preparation, and interpretation of the smears. The number of specimens during the evaluation period ranged from 976 to 1,864 specimens per site, and there were a total of 6,115 specimens. Gram stain results were discrepant from culture for 5% of all specimens. Fifty-eight percent of discrepant results were specimens with no organisms reported on Gram stain but significant growth on culture, while 42% of discrepant results had reported organisms on Gram stain that were not recovered in culture. Upon review of available slides, 24% (63/263) of discrepant results were due to reader error, which varied significantly based on site (9% to 45%). The Gram stain error rate also varied between sites, ranging from 0.4% to 2.7%. The data demonstrate a significant variability between laboratories in Gram stain performance and affirm the need for ongoing quality assessment by laboratories. Standardized monitoring of Gram stains is an essential quality control tool for laboratories and is necessary for the establishment of a quality benchmark across laboratories. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  9. Purified azure B as a reticulocyte stain.

    Science.gov (United States)

    Marshall, P N; Bentley, S A; Lewis, S M

    1976-01-01

    A comparison has been made between reticulocyte preparations stained with purified azure B and with several commerically available batches of brilliant cresyl blue and new methylene blue. Marked variations were observed in the composition and staining performances of the various batches of the two commerically available dyes. Although there were no significant differences in reticulocyte counts obtained with these two dyes, varying amounts of an extraneous, particulate dye deposit were present in these preparations, making accuracte counting both tedious and timeconsuming. Purified azure B, on the other hand, gave reproducibly stained, deposit-free preparations. Reticulocyte counts obtained from azure B preparations correlated almost exactly with those determined using new methylene blue. Purified azure B is therefore recommended as a convenient reticulocyte stain for routine use. Images PMID:64475

  10. Research on pre-staining gel electrophoresis

    International Nuclear Information System (INIS)

    Zhong Ruibo; Liu Yushuang; Zhang Ping; Liu Jingran; Zhao Guofen; Zhang Feng

    2014-01-01

    Background: Gel electrophoresis is a powerful biochemical separation technique. Most biological molecules are completely transparent in the visible region of light, so it is necessary to use staining to show the results after gel electrophoresis, and the general steps of conventional staining methods are time-consuming. Purpose: We try to develop a novel approach to simplify the gel electrophoresis: Pre-Staining Gel Electrophoresis (PSGE), which can make the gel electrophoresis results monitored in real time. Methods: Pre-stain the protein samples with Coomassie Brilliant Blue (CBB) for 30 min before loading the sample into the gel well. Results and Conclusion: PSGE can be successfully used to analyze the binding efficiency of Bovine Serum Albumin (BSA) and amphiphilic polymer via chemical coupling and physical absorption, and the double PSGE also shows a great potential in bio-analytical chemistry. (authors)

  11. New Grocott Stain without Using Chromic Acid

    International Nuclear Information System (INIS)

    Shiogama, Kazuya; Kitazawa, Kayo; Mizutani, Yasuyoshi; Onouchi, Takanori; Inada, Ken-ichi; Tsutsumi, Yutaka

    2015-01-01

    We established a new “ecological” Grocott stain for demonstrating fungi, based upon a 4R principle of refusal, reduction, reuse, and recycle of waste management. Conventional Grocott stain employs environmentally harsh 5% chromic acid for oxidization. Initially, we succeeded in reducing the concentration of chromic acid from 5% to 1% by incubating the solution at 60°C and using five-fold diluted chromic acid solution at which point it was reusable. Eventually, we reached the refusal level where 1% periodic acid oxidization was efficient enough, when combined with preheating of sections in the electric jar, microwave oven, or pressure pan. For convenience sake, we recommend pressure pan heating in tap water for 10 min. Stainability of fungi in candidiasis and aspergillosis was comparable with conventional Grocott stain, while Mucor hyphae showed enhanced staining. The modified sequence was further applicable to detecting a variety of mycotic pathogens in paraffin sections. Our environmentally-friendly Grocott stain also has the advantage of avoiding risk of human exposure to hexavalent chromium solution in the histopathology laboratory. The simple stain sequence is can be easily applied worldwide

  12. Stain Removal Assessment of Two Manual Toothbrushes with an Interproximal Tooth Stain Index.

    Science.gov (United States)

    Farrell, Svetlana; Grender, Julie M; Terézhalmy, Geza; Archila, Luis R

    2015-01-01

    To assess a newly developed index to measure interproximal stain and evaluate the stain removal efficacy of two commercially available manual toothbrushes. This was a randomized, examiner-blind, parallel-group, two-treatment clinical trial of two weeks' duration. Subjects qualified for the study if they had an average Modified Lobene Stain Index of ≥ 1.5 from two anterior teeth. At baseline, subjects brushed in front of a mirror for one minute under supervision. All subjects were provided with a standard 0.243% sodium fluoride dentifrice and were randomly assigned either an Oral-B Pulsar manual brush (OBP) or a Colgate Whitening manual brush (CW) to use for two weeks. Stain was reassessed after two weeks of product use. Stain measurements were conducted using the Modified Lobene Stain Index and the new Interproximal Modified Lobene Stain Index, which allows for assessment of stain in hard-to-reach areas using the same area and intensity scales as the Modified Lobene Stain Index. Use of the two manual brushes resulted in statistically significant reductions in surface stain relative to baseline after two weeks of use. Median stain reductions were 78% and 60% for the OBP and CW, respectively, as measured by the Modified Lobene Stain Index. The mean changes in the composite scores from baseline to week two were 1.85 and 1.57 for the two treatment groups, respectively. Statistically significant reductions from baseline were also found for the intensity and extent of stain measures (p brush and 83% reduction with the CW brush. For the gingival sites, the median stain removal percentages were 83% and 50%, respectively For the body region, a median stain removal of 100% was found for both treatment groups. No statistically significant differences were found between the two groups for the mean composite scores for either index. Both manual brushes showed effective stain removal, including interproximal hard-to-reach sites. The Interproximal Modified Lobene Stain Index

  13. 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

  14. Ethidium bromide fluorescence of 28S ribosomal RNA can be used to normalize samples in northern or dot blots when analyzing small drug-induced changes in specific mRNA.

    Science.gov (United States)

    Duhl, D M; Gillespie, D D; Sulser, F

    1992-05-01

    Quantitative analysis of Northern blots is frequently accomplished with the aid of an internal standard. Most common is probing for an additional message the steady-state levels of which do not change in response to the experimental conditions and the signal of which is sufficiently removed from that of the target gene after gel electrophoresis. However, this strategy is not always feasible. When total RNA is immobilized on nylon, 28S ribosomal RNA on the blot can be used as an internal standard and quantitated by scanning the negative photograph of the blotted RNA stained with ethidium bromide. This procedure can also be used for RNA dot blots. The method is quick, reliable, will work with laser or white-light densitometers, and can serve as a universal internal standard, eliminating the need for additional probes.

  15. Potential Value of YAP Staining in Rhabdomyosarcoma.

    Science.gov (United States)

    Ahmed, Atif A; Habeebu, Sultan S; Sherman, Ashley K; Ye, Shui Q; Wood, Nicole; Chastain, Katherine M; Tsokos, Maria G

    2018-03-01

    Rhabdomyosarcoma (RMS) is a common malignancy of soft tissue, subclassified as alveolar (ARMS), pleomorphic (PRMS), spindle cell/sclerosing (SRMS), and embryonal (ERMS) types. The Yes-associated protein (YAP) is a member of the Hippo pathway and a transcriptional regulator that controls cell proliferation. We have studied the immunohistochemical expression of YAP in different RMSs, arranged in tissue microarray (TMA) and whole slide formats. Pertinent clinical data including patient age, gender, tumor location, and clinical stage were collected. Out of 96 TMA cases, 30 cases (31%) were pleomorphic, 27 (28%) were embryonal, 24 (25%) alveolar, and 15 (16%) spindle cell. Positive nuclear YAP staining was seen in the PRMS (17/30, 56.7%), SRMS (7/15, 46.7%), ERMS (19/27 or 70%), and less in ARMS (37.5%). YAP nuclear staining was significantly more prevalent in ERMS than ARMS ( p=0.02). Of the 41 whole slide cases, nuclear staining was detected in all ARMS but was restricted in distribution to 30% staining. These results highlight the role of YAP in RMS tumorigenesis, a fact that can be useful in engineering targeted therapy. Restricted nuclear YAP staining (<30% of cells) may be of value in the diagnosis of ARMS.

  16. Adversarial Stain Transfer for Histopathology Image Analysis.

    Science.gov (United States)

    Bentaieb, Aicha; Hamarneh, Ghassan

    2018-03-01

    It is generally recognized that color information is central to the automatic and visual analysis of histopathology tissue slides. In practice, pathologists rely on color, which reflects the presence of specific tissue components, to establish a diagnosis. Similarly, automatic histopathology image analysis algorithms rely on color or intensity measures to extract tissue features. With the increasing access to digitized histopathology images, color variation and its implications have become a critical issue. These variations are the result of not only a variety of factors involved in the preparation of tissue slides but also in the digitization process itself. Consequently, different strategies have been proposed to alleviate stain-related tissue inconsistencies in automatic image analysis systems. Such techniques generally rely on collecting color statistics to perform color matching across images. In this work, we propose a different approach for stain normalization that we refer to as stain transfer. We design a discriminative image analysis model equipped with a stain normalization component that transfers stains across datasets. Our model comprises a generative network that learns data set-specific staining properties and image-specific color transformations as well as a task-specific network (e.g., classifier or segmentation network). The model is trained end-to-end using a multi-objective cost function. We evaluate the proposed approach in the context of automatic histopathology image analysis on three data sets and two different analysis tasks: tissue segmentation and classification. The proposed method achieves superior results in terms of accuracy and quality of normalized images compared to various baselines.

  17. Detection Of Concrete Deterioration By Staining

    Science.gov (United States)

    Guthrie, Jr., George D.; Carey, J. William

    1999-09-21

    A method using concentrated aqueous solutions of sodium cobaltinitrite and a rhodamine dye is described which can be used to identify concrete that contains gels formed by the alkali-silica reaction (ASR), and to identify degraded concrete which results in a porous or semi-permeable paste due to carbonation or leaching. These solutions present little health or environmental risk, are readily applied, and rapidly discriminate between two chemically distinct gels; K-rich, Na--K--Ca--Si gels are identified by yellow staining, and alkali-poor, Ca--Si gels are identified by pink staining.

  18. News from the biological stain commission

    DEFF Research Database (Denmark)

    Kiernan, J.A.; Lyon, Hans Oluf

    2008-01-01

    In the three earlier editions of News from the Biological Stain Commission (BSC), under the heading of "Regulatory affairs," the BSC's International Affairs Committee reported on the work of Technical Committee 212, Clinical Laboratory Testing and in Vitro Diagnostic Test Systems of the Internati......In the three earlier editions of News from the Biological Stain Commission (BSC), under the heading of "Regulatory affairs," the BSC's International Affairs Committee reported on the work of Technical Committee 212, Clinical Laboratory Testing and in Vitro Diagnostic Test Systems...

  19. Identification of Cyclospora cayetanensis in stool using different stains.

    Science.gov (United States)

    Negm, A Y

    1998-08-01

    Cyclospora cayetanensis, a newly emerging coccidian protozoa is world-wide in distribution. In the present study, different concentrations and staining techniques were used for identification of Cyclospora. Formol-ether sedimentation and Sheather's sugar flotation were used as concentration techniques and the different stains used were: the modified Ziehl-Neelsen, Giemsa, safranin-methylene blue, modifications of trichrome stain, calcoflour white and finally phenol-auramine. The safranin stain was the best, as it stained all the oocysts of Cyclospora uniformly, besides being rapid and easily applicable in the laboratories. Phenol-auramine stained the oocysts well, where both the wall and internal contents fluoresced brightly. With the calcoflour white stain, only the wall of oocysts took that fluorescent stain. The modified Ziehl-Neelsen stained some of the oocysts well, yet great variability in the staining pattern was noticed. Cyclospora oocysts were not efficiently stained with either trichrome modifications or Giemsa stains.

  20. Photoacoustic imaging of port-wine stains

    NARCIS (Netherlands)

    Kolkman, Roy G. M.; Mulder, Miranda J.; Glade, Conrad P.; Steenbergen, Wiendelt; van Leeuwen, Ton G.

    2008-01-01

    BACKGROUND AND OBJECTIVE: To optimize laser therapy of port-wine stains (PWSs), information about the vasculature as well as lesion depth is valuable. In this study we investigated the use of photoacoustic imaging (PAI) to obtain this information. STUDY DESIGN/MATERIALS AND METHODS: PAI uses pulsed

  1. Photoacoustic Imaging of Port-Wine Stains

    NARCIS (Netherlands)

    Kolkman, R.G.M.; Mulder, M.J.; Mulder, Miranda J.; Glade, Conrad P.; Steenbergen, Wiendelt; van Leeuwen, Ton

    2008-01-01

    Background and Objective: To optimize laser therapy of port-wine stains (PWSs), information about the vasculature as well as lesion depth is valuable. In this study we investigated the use of photoacoustic imaging (PAI) to obtain this information. - Study Design/Materials and Methods: PAI uses

  2. A comparative assessment of commonly employed staining ...

    African Journals Online (AJOL)

    Following an increase in the number of reports of Cryptosporidium infections and the problems encountered in detecting these organisms in faecal smears, a comparative assessment of a modification of the Sheather's flotation technique and other commonly employed staining procedures proved the modified Sheather's ...

  3. A comparative assessment of commonly employed staining ...

    African Journals Online (AJOL)

    1991-03-16

    Mar 16, 1991 ... methylene blue;8 fluorescence (auramine-phenoI9 and direct immunofluorescence1o. • ll. ); negative staining (periodic acid-. Schiff12. ); and flotation (Sheather's ..... using a direct immunofluorescence assay. Pediatr Infect Dis 1986; 5: 139-142. 12. Horen WP. Detection of Cryptosporidium in human faecal ...

  4. The Language of Stained-Glass Windows

    Science.gov (United States)

    Brew, Charl Anne

    2010-01-01

    The splendor and beauty of stained glass punctuates any room. In this article, the author describes a cross-curriculum project which incorporated the French classes' research and written study of France in the Middle Ages. For the project the author suggested Sainte-Chapelle which is considered a reliquary and was built by Louis IX to house the…

  5. Corneal staining after treatment with topical tetracycline

    NARCIS (Netherlands)

    Lapid-Gortzak, Ruth; Nieuwendaal, Carla P.; Slomovic, Allan R.; Spanjaard, Lodewijk

    2006-01-01

    PURPOSE: The purpose of this paper is to report a case of corneal staining after treatment with topical tetracycline. METHODS: A patient with crystalline keratopathy caused by Streptococcus viridans after corneal transplantation was treated topically with tetracycline eye drops, based on results of

  6. 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...

  7. 21 CFR 864.1850 - Dye and chemical solution stains.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Dye and chemical solution stains. 864.1850 Section... (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Biological Stains § 864.1850 Dye and chemical solution stains. (a) Identification. Dye and chemical solution stains for medical purposes are mixtures of...

  8. Laser Treatment of Port Wine Stains

    Science.gov (United States)

    Majaron, Boris; Nelson, J. Stuart

    Port wine stain (PWS), also called nevus flammeus, is a congenital, cutaneous vascular malformation involving post-capillary venules which produce a light pink to red to dark-red-violet discoloration of human skin [1]. PWS occurs in an estimated 3 children per 1000 live births, affecting males and females and all racial groups equally [2]. There appears to be no hereditary predilection for PWS within families. There are no known risk factors or ways to prevent PWS.

  9. Diagnosis of Blastocystis hominis by different staining techniques.

    Science.gov (United States)

    Khalifa, A M

    1999-01-01

    One hundred and fifty stool samples were collected from diarrheic patients of different ages, and examined for Blastocystis hominis by direct smears and concentrated by Sheather's sugar flotation. Staining was done by: Giemsa, two modifications of trichrome stain, modified Ziehl-Neelsen, safranin-methylene blue and two-auramine stains. Out of the 150 cases nine were positive for blastocystosis. The best stains were safranin-methylene blue and modified Ziehl-Neelsen stains. They had the advantage of staining cysts and amoeboid forms besides being rapid and easy to perform. The modified trichrome stains identified 8 ie, less specific and were time consuming. The auramine dyes stained the cyst, both the wall and internal body fluoresced brightly. Giemsa stain was not an efficient stain. Scanning and transmission electron microscopy (SEM, TEM) were performed to study the fine ultrastructure.

  10. Microspectrophotometric studies of Romanowsky stained blood cells. II. Comparison of the performance of two standardized stains.

    Science.gov (United States)

    Marshall, P N; Galbraith, W; Navarro, E F; Bacus, J W

    1981-11-01

    This paper describes a comparison of the performance of two standardized Romanowsky blood stains, namely those of Marshall et al. and Wittekind et al., both containing azure B and eosin alone. Stain performance is assessed objectively by the use of three complementary techniques, all based on the visible absorbance spectra of stained cellular substrates. The first of these techniques is a simple comparison of the shapes and heights of the absorbance spectra. The second technique uses the CIE Colorimetric System, and thus permits the quantitation of colour in a manner that agrees with human observation. CIE co-ordinates (chromaticity points, luminance) are calculated directly from absorbance spectra. The third technique is that of spectral subtraction, which yields a set of factors which describe the quantities of component dyes which are bound by the object. This technique, unlike the other two, requires a priori knowledge of the dyes used in the stains, and their spectra when bound to cellular substrates. Although the differences between the two methods are subtle, and hard for the subjective observer to define, the objective methods described here do show statistically significant differences. Wittekind's stain produces less intense staining, except for lymphocyte and monocyte cytoplasms. To the human eye, the differential coloration of these two substrates is more pronounced, but the difference between all nuclei and cytoplasm is less marked. The major difference in the uptake of dye components is in the small quantities of eosin dimer that are bound in this technique.

  11. Comparison of methylene blue/gentian violet stain to Gram's stain for the rapid diagnosis of gonococcal urethritis in men.

    Science.gov (United States)

    Taylor, Stephanie N; DiCarlo, Richard P; Martin, David H

    2011-11-01

    We compared a simple, one-step staining procedure using a mixture of methylene blue and gentian violet to Gram stain for the detection of gonococcal urethritis. The sensitivity and specificity of both Gram stain and methylene blue/gentian violet stain were 97.3% and 99.6%, respectively. There was a 100% correlation between the 2 methods.

  12. 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.

  13. [Detection and documentation of masked blood stains with infrared technique].

    Science.gov (United States)

    Du Chesne, A; Bajanowski, T; Brinkmann, B

    1993-01-01

    On dark textiles the visualization of blood stains with the naked eye is either difficult or impossible. In experimental stains and in case work stains we have applied an infrared (IR) video camera in combination with a video printer. As an alternative, an IR goggle was used which could also be connected with a video printer. The results obtained on a variety of different stains and stain carriers are encouraging. Stains showing poor contrast usually become more contrasted. Stains which are partly masked can become complete. Masked stains can become visible. The system is not effective in all combinations of stains and carriers. But it solves a great proportion of formerly problematic cases. Documentation of results is quite easy if a videoprinter is used.

  14. Fetal Outcome in Meconium Stained Deliveries

    Science.gov (United States)

    Mundhra, Rajlaxmi; Agarwal, Manika

    2013-01-01

    Objective: To evaluate the foetal outcome in Meconium Stained Amniotic Fluid (MSAF). Material and Methods: This prospective observational study was carried out in the Department of Obstetrics and Gynaecology, North Eastern Indira Gandhi Regional Institute of Health And Medical Sciences, Shillong, India, over a period of eighteen months, from January 2010 to June 2011. A total of 355 pregnant women who had completed more than 37 weeks of gestation, with singleton pregnancies and cephalic presentations were included in this study. One hundred and sixty five cases with MSAF, were thus selected and they were compared with 190 randomly selected controls. Results: Among 165 cases, 27.88 % of the cases had regular visits to the Institute at least 3 times previously, 72.12% cases had no previous visit at all. Primigravidas accounted for a majority of cases and approximately 50% cases had gestational ages of more than 40 weeks Pregnancies complicated with pregnancy induced hypertension had statistically significant higher rates of meconium staining among cases (16.97%), as compared to those among controls (7.89%). 21.81% cases had foetal heart rate abnormalities, as were detected by electronic foetal monitoring and presence of foetal bradycardia was statistically higher in cases compared to that in controls. Casearean section rates were nearly double in cases (49.09%). Neonatal outcome was poor in terms of low Apgar score at birth, birth asphyxia, Meconium Aspiration Syndrome (MAS) and increased neonatal admission among cases as compared to that among controls. Conclusion: Meconium stained amniotic fluid is really worrisome from both, obstetrician’s and paediatrician’s points of view, as it increases the caesarean rates, causes birth asphyxia, MAS and increases neonatal intensive care unit admissions. PMID:24551662

  15. 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.

  16. Robust fadeout profile of an evaporation stain

    Science.gov (United States)

    Witten, T. A.

    2009-06-01

    We propose an explanation for the commonly seen fading in the density of a stain remaining after a droplet has dried on a surface. The density decreases as a power p of the distance from the edge. For thin, dilute drops of general shape this power is determined by a flow stagnation point in the distant interior of the drop. The power p depends on the local evaporation rate J(0) at the stagnation point and the liquid depth h(0) there: p = 1 - 2~ (h(0)/\\bar h)(\\bar J/J(0)) , where \\bar h and \\bar J are averages over the drop surface.

  17. Romanowsky staining in cytopathology: history, advantages and limitations.

    Science.gov (United States)

    Krafts, K P; Pambuccian, S E

    2011-04-01

    If the entire discipline of diagnostic cytopathology could be distilled into a single theme, it would be the Papanicolaou stain. Yet it was the Romanowsky stain upon which the discipline of cytopathology was founded. Both stains are used today in the cytopathology laboratory, each for a different and complementary purpose. We trace the history of cytopathological stains and discuss the advantages and limitations of Romanowsky-type stains for cytological evaluation. We also provide suggestions for the advantageous use of Romanowsky-type stains in cytopathology.

  18. Western Sufism

    DEFF Research Database (Denmark)

    Sedgwick, Mark

    , of thought both familiar and less familiar: Neoplatonic emanationism, perennialism, pantheism, universalism, and esotericism. Western Sufism, then, is the product not of the new age but of Islam, the ancient world, and centuries of Western religious and intellectual history. Drawing on sources from antiquity...

  19. Novel methods of cytokine detection: Real-time PCR, ELISPOT, and intracellular cytokine staining

    Directory of Open Access Journals (Sweden)

    Eliza Turlej

    2009-05-01

    Full Text Available Cytokines are small hormone-like proteins that play important roles in immune system control. Cytokines regulate the proliferation and differentiation of cells and hematopoiesis and act as mediators in the inflammatory reaction. Changes in cytokine levels are found in many diseases, such as sepsis, bowel inflammatory disease, autoimmune diseases, as well as graft-versus-host disease. Cytokines levels can be detected using in vivo, in vitro, and ex vivo techniques. The level of cytokine produced can be measured by immunoenzymatic test (ELISA in supernatant after cell culture with the addition of stimulant and in plasma by techniques that measure the level of cytokine secretion in cells (e.g. immunohistochemical staining, ELISPOT, and intracellular cytokine staining, and by molecular biological methods (RPA, real-time PCR, in situ hybridization, and Northern blot. Detection of cytokine mRNA in tissues is useful in the direct determination of heterogenic populations of cytokine-producing cells. Nowadays the most frequently used methods for measuring cytokine level are ELISPOT, intracellular cytokine staining with flow cytometry detection, and real-time PCR. These methods have an important clinical role in vaccine efficacy, in viral, bacterial, and verminous diagnostics, and in determining the efficacy of cancer treatment.

  20. Laser therapy in plastic surgery: decolorization in port wine stains

    Science.gov (United States)

    Peszynski-Drews, Cezary; Wolf, Leszek

    1996-03-01

    For the first time laserotherapy is described as a method of port wine stain decolorization in plastic surgery. The authors present their 20-year experience in the treatment of port wine stains with the argon laser and dye laser.

  1. Port wine stain on a child's face (image)

    Science.gov (United States)

    Port wine stains are always present at birth. In an infant, they are flat, pink, vascular lesions. Common locations ... may be present anywhere on the body. Port wine stains may appear in association with other syndromes.

  2. The degradation of Romanowsky-type blood stains in methanol.

    Science.gov (United States)

    Dean, W W; Stastny, M; Lubrano, G J

    1977-01-01

    The oxidative demethylation of Romanowsky-type stains in methanol has been examined quantitatively with respect to its effect upon the staining of blood smears. Spectral changes in bound dye, observed through two color filters, have been measured for the nuclei and cytoplasm of segmented neutrophils and monocytes utilizing the LARC automated differential analyzer. Stain decomposition in methanol results in a large loss in staining intensity with little change in color. The loss in intensity has been correlated with the observed spectral changes in the degraded stain. High-performance liquid chromatographic analysis of degraded stain samples has shown the products of methanolic degradation to be different from those obtained in aqueous polychroming reactions. To maintain a stain of defined thiazine dye composition and thus defined staining properties, refrigeration is recommended.

  3. Several staining techniques to enhance the visibility of Acanthamoeba cysts.

    Science.gov (United States)

    El-Sayed, Nagwa Mostafa; Hikal, Wafaa Mohamed

    2015-03-01

    Acanthamoeba is one of the most common free-living amoebae. It is widespread in the environment and can infect humans causing keratitis. Delayed diagnosis or misdiagnosis leads to extensive corneal inflammation and profound visual loss. Therefore, accurate and rapid diagnosis of Acanthamoeba keratitis is essential for successful treatment and good prognosis. This study was designed to use different staining techniques to facilitate the identification of Acanthamoeba cysts. Acanthamoeba cysts were isolated by cultivation of either corneal scraping specimens or tap water samples onto non-nutrient agar plates seeded with Escherichia coli. Subcultures were done from positive cultures until unique cysts were isolated. Acanthamoeba cysts were stained temporarily using iodine, eosin, methylene blue, and calcofluor white (CFW) stains and as permanent slides after processing for mounting using modified trichrome, Gimenez and Giemsa staining. These stains were compared on the basis of staining quality including clarity of morphological details, differentiation between cytoplasm and nuclei, color and contrast, and also other characteristics of the staining techniques, including ease of handling, time taken for the procedure, and cost effectiveness. The cysts of Acanthamoeba were recognized in the form of double-walled cysts: the outer wall (ectocyst) that was being differentiated from the variably stained surrounding background and the inner wall (endocyst) that was sometimes stellated, polygonal, round, or oval and visualized as separate from the spherical, sometimes irregular, outline of the ectocyst. Regarding the temporary stains, it was found that they were efficient for visualizing the morphological details of Acanthamoeba cysts. In CFW staining, Acanthamoeba cysts appeared as bluish-white or turquoise oval halos although the internal detail was not evident. On the other hand, the results of permanent-stained slides showed the most consistent stain for identification of

  4. Dye purity and dye standardization for biological staining

    DEFF Research Database (Denmark)

    Lyon, H O

    2002-01-01

    for separating, identifying and assaying dye components. In the second part of the review, descriptions are given of the standardized staining method approach using standard staining methods for assessing stains, and practical responses to stain impurity including commercial quality control, third-party quality...... control and standardization of reagents, protocols and documentation. Finally, reference is made to the current state of affairs in the dye field....

  5. Comparison between Giemsa and Van Geison stains in ...

    African Journals Online (AJOL)

    Trichrome stain (such as Van Geison) is usually used in histopathology laboratory for demonstration of collagenic fibers. Lack of selectivity and tendency of stain to fade makes van Gieson not ideal for collagen demonstration. This study was aimed to compare between Giemsa's and van Gieson's stains in collagen fibers ...

  6. Modified ultrafast Papanicolaou staining technique: A comparative study

    Science.gov (United States)

    Thakur, Moni; Guttikonda, Venkateswara Rao

    2017-01-01

    Introduction: Ultrafast Papanicolaou stain (UFP) was introduced as a hybrid of Romanowsky and Papanicolaou (PAP) stain. It enhances the quality and reduces the time. In the present study, a modified staining technique was adapted where Gill's Hematoxylin was replaced by Harris Hematoxylin. Aims: The aim of the study was to assess the use of the modified ultrafast Papanicolaou (MUFP) stain for fine needle aspiration cytology (FNAC) of head and neck swellings in comparison with the routine PAP stain, hematoxylin and eosin (H and E), and Giemsa. Materials and Methods: Forty FNACs of head and neck swellings were collected. FNAC procedure was performed by standard method; two smears were fixed in 95% propanol and stained with PAP and H and E. Two smears were air dried, 1 was stained with Giemsa, and 1 was rehydrated with normal saline, fixed in alcoholic formalin, and stained with MUFP. Four parameters were considered and scored background, cell morphology, nuclear staining, and overall staining pattern). Results: The quality of MUFP smears were better when compared to routine PAP, H and E, and Giemsa, and was statistically significant by Wilcoxon matched pair test. Conclusions: MUFP stain in comparison to routine PAP, H and E, and Giemsa provides an excellent and suitable alterative in cytological staining for the study of various organs. PMID:28701828

  7. Modified ultrafast Papanicolaou staining technique: A comparative study

    Directory of Open Access Journals (Sweden)

    Moni Thakur

    2017-01-01

    Full Text Available Introduction: Ultrafast Papanicolaou stain (UFP was introduced as a hybrid of Romanowsky and Papanicolaou (PAP stain. It enhances the quality and reduces the time. In the present study, a modified staining technique was adapted where Gill's Hematoxylin was replaced by Harris Hematoxylin. Aims: The aim of the study was to assess the use of the modified ultrafast Papanicolaou (MUFP stain for fine needle aspiration cytology (FNAC of head and neck swellings in comparison with the routine PAP stain, hematoxylin and eosin (H and E, and Giemsa. Materials and Methods: Forty FNACs of head and neck swellings were collected. FNAC procedure was performed by standard method; two smears were fixed in 95% propanol and stained with PAP and H and E. Two smears were air dried, 1 was stained with Giemsa, and 1 was rehydrated with normal saline, fixed in alcoholic formalin, and stained with MUFP. Four parameters were considered and scored background, cell morphology, nuclear staining, and overall staining pattern. Results: The quality of MUFP smears were better when compared to routine PAP, H and E, and Giemsa, and was statistically significant by Wilcoxon matched pair test. Conclusions: MUFP stain in comparison to routine PAP, H and E, and Giemsa provides an excellent and suitable alterative in cytological staining for the study of various organs.

  8. Comparison between Giemsa and Van Geison stains in ...

    African Journals Online (AJOL)

    Rukevwe S. Abraka

    2016-09-14

    Sep 14, 2016 ... Trichrome stain (such as Van Geison) is usually used in histopathology laboratory for demonstration of collagenic fibers. Lack of selectivity and tendency of stain to fade makes van Gieson not ideal for collagen demonstration. This study was aimed to compare between Giemsa's and van Gieson's stains.

  9. 7 CFR 28.442 - Middling Yellow Stained Color.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Middling Yellow Stained Color. 28.442 Section 28.442... Stained Color. Middling Yellow Stained Color is American Upland cotton which in color is deeper than Middling Tinged Color. [57 FR 34498, Aug. 5, 1992] below color grade cotton ...

  10. 7 CFR 28.441 - Strict Middling Yellow Stained Color.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Strict Middling Yellow Stained Color. 28.441 Section... Strict Middling Yellow Stained Color. Strict Middling Yellow Stained Color is color which is deeper than that of Strict Middling Tinged Color. [57 FR 34498, Aug. 5, 1992] ...

  11. Myeloperoxidase staining in the diagnosis of aggressive periodontitis.

    Science.gov (United States)

    Singh, Sukhdeep; Acharya, Anirudh B; Kumar, S C Veerendra

    2011-04-01

    To evaluate neutrophil myeloperoxidase (MPO) staining procedure as a reliable, affordable and easily available diagnostic assay for aggressive periodontitis. Fifteen subjects were recruited in the study wherein five each were diagnosed as aggressive periodontitis and chronic periodontitis respectively, and five were periodontally healthy. Three millilitres (ml) of venous blood was collected using Vacutainers containing ethylene diamine tetra acetate (EDTA) and was subjected to MPO staining procedure. Histological picture was evaluated using a visual analogue scale (VAS). MPO stained specimen of all the patients showed positive MPO staining of the neutrophils. The intensity of the stain of MPO granules was more in aggressive periodontitis specimen as compared to the chronic periodontitis patient specimen and healthy subject specimen. The staining characteristics were comparable for chronic periodontitis patients and healthy subject. This study shows that there is a potential and probable place for MPO staining as an economical, relatively convenient and easily available assay in the diagnosis of aggressive periodontitis.

  12. Refractile mycobacteria in Romanowsky-stained bone marrow smears. A comparison of acid-fast-stained tissue sections and Romanowsky-stained smears.

    Science.gov (United States)

    Torlakovic, E; Clayton, F; Ames, E D

    1992-03-01

    The appearance of mycobacteria was studied in Wright-stained bone marrow preparations of human immunodeficiency virus-infected patients and compared with acid-fast-stained trephine biopsy sections and culture results. Mycobacterium avium complex in Romanowsky-stained preparations may be seen as extracellular and intracellular clear or red refractile beaded rods and nonrefractile "negative images." Refractile mycobacteria were seen in 17 of 20 culture-positive cases. Acid-fast stain of the trephine biopsy demonstrated organisms in only 11 of the 20 cases. Thus, six cases were culture positive and contained refractile rods but had no acid-fast organisms on the trephine biopsy. No false-positive results were seen with Romanowsky stain; the three false-negative results for refractility also were negative with acid-fast stain. Examination of Romanowsky-stained smears or imprints for refractile mycobacteria provides a reliable and sensitive method to identify mycobacteria in this population. Romanowsky-stained bone marrow aspirate and imprint smears should be examined for refractile bacilli when mycobacterial infection is suspected.

  13. Western Samoa.

    Science.gov (United States)

    1985-12-01

    This discussion of Western Samoa, which lies 2575 km northeast of Auckland, New Zealand, focuses on the following: geography; the people; history; government; political conditions; the economy; foreign relations; and relations the US. The population of Western Samoa, as of 1985, totals 163,000 with an annual growth rate of 0.9%. The infant mortality rate is 13/1000; life expectancy is 65 years. The main islands are formed ranges of extinct volcanoes. Volcanic activity last occurred in 1911. More than 2000 years age, waves of Polynesians migrated from Southeast Asia to the Samoan Islands. Samoans are the 2nd largest Polynesian group, after the Maoris of New Zealand, and speak a Polynesian dialect. Samoans have tended to retain their traditional ways despite exposure to European influence for more than 150 years. Most Samoans live within the traditional social system based on an extended family group, headed by a chief. Western Samoans are Christian. Education is free but not compulsory. In 1967, 95% of the children of primary school age attended school. From 1947 to 1961, a series of constitutional advances, assisted by visits from UN missions, brought Western Samoa from dependent status to self-government and finally to independence. The 1960 constitution is based on the British pattern of parliamentary democracy, modified to take Samoan customs into account. The present head of state holds his position for life. Future heads of state will be elected by the Legislative Assembly for 5-year terms. The Parliament consists of the Legislative Assembly and the head of state. The Supreme Court is the superior court of record and has full jurisdiction in civil, criminal, and constitutional matters. The "matai" of chief system still dominates the politics of Western Samoa, although several political parties have been formed and seem to be taking root. The "matai" system is a predominantly conservative force but does provide for change. Western Samoa is predominantly

  14. Erbium doped stain etched porous silicon

    International Nuclear Information System (INIS)

    Gonzalez-Diaz, B.; Diaz-Herrera, B.; Guerrero-Lemus, R.; Mendez-Ramos, J.; Rodriguez, V.D.; Hernandez-Rodriguez, C.; Martinez-Duart, J.M.

    2008-01-01

    In this work a simple erbium doping process applied to stain etched porous silicon layers (PSLs) is proposed. This doping process has been developed for application in porous silicon solar cells, where conventional erbium doping processes are not affordable because of the high processing cost and technical difficulties. The PSLs were formed by immersion in a HF/HNO 3 solution to properly adjust the porosity and pore thickness to an optimal doping of the porous structure. After the formation of the porous structure, the PSLs were analyzed by means of nitrogen BET (Brunauer, Emmett and Teller) area measurements and scanning electron microscopy. Subsequently, the PSLs were immersed in a saturated erbium nitrate solution in order to cover the porous surface. Then, the samples were subjected to a thermal process to activate the Er 3+ ions. Different temperatures and annealing times were used in this process. The photoluminescence of the PSLs was evaluated before and after the doping processes and the composition was analyzed by Fourier transform IR spectroscopy

  15. 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...

  16. Survival of Pseudomonas aeruginosa in modified Romanowsky staining solutions.

    Science.gov (United States)

    Duffield, Richard; Wong, Hui-San; Trott, Darren J; Hill, Peter B

    2015-08-01

    Anecdotal reports suggest that rapid staining solutions can become contaminated with micro-organisms, especially Pseudomonas aeruginosa. To determine whether inoculation of rapid Romanowsky-type stains with P. aeruginosa results in viable bacterial contamination, which could lead to cross-contamination of slides during cytological staining. Pseudomonas aeruginosa was inoculated into clean and organically contaminated staining solutions (fixative, eosin and methylene blue) and positive (broth) and negative (bleach) control solutions. Subsequent viability and survival were detected by measuring colony-forming units per millilitre at various time points up to 2 weeks. Each sample was stained and microscopically examined to determine whether bacteria were visible. No bacteria could be cultured at any time point from the bleach or fixative solution. In clean eosin and methylene blue staining solutions, viable bacteria were recovered for up to 1 h, but by 24 h all bacteria were dead. In staining solutions contaminated with hair and dead skin cells, bacteria survived in methylene blue for up to 1 h, and viable bacteria persisted in the eosin stain for 2 weeks. In solutions containing viable organisms, the bacteria could be observed by microscopic examination; no bacteria were visible when the solutions contained no viable organisms. Pseudomonas aeruginosa can survive in commonly used staining solutions for variable periods of time, but is unable to proliferate. Although theoretically this might complicate cytological interpretation and subsequent diagnosis, the likelihood of this in clinical practice appears remote when the correct staining technique is used. © 2015 ESVD and ACVD.

  17. Harmonization of the intracellular cytokine staining assay.

    Science.gov (United States)

    Welters, Marij J P; Gouttefangeas, Cécile; Ramwadhdoebe, Tamara H; Letsch, Anne; Ottensmeier, Christian H; Britten, Cedrik M; van der Burg, Sjoerd H

    2012-07-01

    Active immunotherapy for cancer is an accepted treatment modality aiming to reinforce the T-cell response to cancer. T-cell reactivity is measured by various assays and used to guide the clinical development of immunotherapeutics. However, data obtained across different institutions may vary substantially making comparative conclusions difficult. The Cancer Immunotherapy Immunoguiding Program organizes proficiency panels to identify key parameters influencing the outcome of commonly used T-cell assays followed by harmonization. Our successes with IFNγ-ELISPOT and peptide HLA multimer analysis have led to the current study on intracellular cytokine staining (ICS). We report the results of three successive panels evaluating this assay. At the beginning, 3 out of 9 participants (33 %) were able to detect >6 out of 8 known virus-specific T-cell responses in peripheral blood of healthy individuals. This increased to 50 % of the laboratories in the second phase. The reported percentages of cytokine-producing T cells by the different laboratories were highly variable with coefficients of variation well over 60 %. Variability could partially be explained by protocol-related differences in background cytokine production leading to sub-optimal signal-to-noise ratios. The large number of protocol variables prohibited identification of prime guidelines to harmonize the assays. In addition, the gating strategy used to identify reactive T cells had a major impact on assay outcome. Subsequent harmonization of the gating strategy considerably reduced the variability within the group of participants. In conclusion, we propose that first basic guidelines should be applied for gating in ICS experiments before harmonizing assay protocol variables.

  18. A useful single-solution polychrome stain for plant material...Brook Cyte-Chrome I.

    Science.gov (United States)

    Stanley L Krugman; Julia F. Littlefield

    1968-01-01

    Fresh and chemically fixed sectioned plant material can be quickly stained by applying a Brook Cyte Chrome I polychrome stain. Staining time averaged only about 10 minutes. And exact timing of staining and de-staining is not as critical as with most of the commonly used stains. The overall quality is comparable to that of the traditional stains.

  19. Hoffman's violet and dahlia as specific stains for animal chromosomes.

    Science.gov (United States)

    Dutt, M K

    1979-03-01

    The paper deals with staining of the chromosomes of animal testicular materials with two basic dyes, Hoffman's violet and dahlia of the triphenylmethane group, following iodine-dye procedure. The important finding, as presented herein, is that iodinated alcohol after staining can be substituted with various acids, both organic as well as inorganic, all of which act as trapping agent preventing leaching of the dye that binds with the chromosomal DNA. It is clear from this study that RNA is not involved by this process of staining, since treatment of stained sections with cold phosphoric acid at 5 degrees C for 20--25 min and then stained also reveals perfect colouration of the chromosomes without any cytoplasmic staining. The in vitro absorption properties of Hoffman's violet have also been presented herein. The probable mechanism of action of these dyes has been suggested.

  20. Efficacy test of a toothpaste in reducing extrinsic dental stain

    Science.gov (United States)

    Agustanti, A.; Ramadhani, S. A.; Adiatman, M.; Rahardjo, A.; Callea, M.; Yavuz, I.; Maharani, D. A.

    2017-08-01

    This clinical trial compared the external dental stain reduction achieved by tested toothpaste versus placebo in adult patients. In this double-blind, parallel, randomised clinical trial, 45 female volunteers with a mean age of 20 years old were included. All study subjects front teeth were topically applicated with Silver Diamine Fluoride (SDF) to create external dental stains. Subjects were randomized into test (n=22) and control (n=23) groups. Toothpastes were used for two days to analyse the effects of removing external stains on the labial surfaces of all anterior teeth. VITA Easyshade Advance 4.0 was used to measure dental extrinsic stains changes. The analysis showed statistically significant efficacy of the tested toothpaste in reducing external dental stain caused by SDF, comparing to the placebo toothpaste, after one and two days of usage. The tested toothpaste was effective in reducing dental stain.

  1. Techniques for controlling variability in gram staining of obligate anaerobes.

    Science.gov (United States)

    Johnson, M J; Thatcher, E; Cox, M E

    1995-01-01

    Identification of anaerobes recovered from clinical samples is complicated by the fact that certain gram-positive anaerobes routinely stain gram negative; Peptostreptococcus asaccharolyticus, Eubacterium plautii, Clostridium ramosum, Clostridium symbiosum, and Clostridium clostridiiforme are among the nonconformists with regard to conventional Gram-staining procedures. Accurate Gram staining of American Type Culture Collection strains of these anaerobic bacteria is possible by implementing fixing and staining techniques within a gloveless anaerobic chamber. Under anaerobic conditions, gram-positive staining occurred in all test organisms with "quick" fixing techniques with both absolute methanol and formalin. The results support the hypothesis that, when anaerobic bacteria are exposed to oxygen, a breakdown of the physical integrity of the cell wall occurs, introducing Gram stain variability in gram-positive anaerobes. PMID:7538512

  2. Factors influencing extract of Hibiscus sabdariffa staining of rat testes.

    Science.gov (United States)

    Bassey, R B; Bakare, A A; Peter, A I; Oremosu, A A; Osinubi, A A

    2012-08-01

    Some plant extracts can be used in biology and medicine to reveal or identify cellular components and tissues. We investigated the effects of time and concentration on staining of histological sections of rat testes by an acidified extract of Hibiscus sabdariffa. An ethanolic extract of H. sabdariffa was diluted using 1% acetic acid in 70% ethanol to stain histological sections of testes at concentrations of 0.2, 0.1 and 0.05 g/ml for 5, 10, 15, 30, 45 and 60 min. The sections of testes were stained deep red. The staining efficiency of H. sabdariffa was greater at a high concentration and required less time to achieve optimal staining. H. sabdariffa is a strongly basic dye that can be used for various diagnostic purposes. Staining time and concentration must be considered to achieve optimal results.

  3. 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...

  4. 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...

  5. 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.

  6. Diagnosing periprosthetic infection: false-positive intraoperative Gram stains.

    Science.gov (United States)

    Oethinger, Margret; Warner, Debra K; Schindler, Susan A; Kobayashi, Hideo; Bauer, Thomas W

    2011-04-01

    Intraoperative Gram stains have a reported low sensitivity but high specificity when used to help diagnose periprosthetic infections. In early 2008, we recognized an unexpectedly high frequency of apparent false-positive Gram stains from revision arthroplasties. The purpose of this report is to describe the cause of these false-positive test results. We calculated the sensitivity and specificity of all intraoperative Gram stains submitted from revision arthroplasty cases during a 3-month interval using microbiologic cultures of the same samples as the gold standard. Methods of specimen harvesting, handling, transport, distribution, specimen processing including tissue grinding/macerating, Gram staining, and interpretation were studied. After a test modification, results of specimens were prospectively collected for a second 3-month interval, and the sensitivity and specificity of intraoperative Gram stains were calculated. The retrospective review of 269 Gram stains submitted from revision arthroplasties indicated historic sensitivity and specificity values of 23% and 92%, respectively. Systematic analysis of all steps of the procedure identified Gram-stained but nonviable bacteria in commercial broth reagents used as diluents for maceration of periprosthetic membranes before Gram staining and culture. Polymerase chain reaction and sequencing showed mixed bacterial DNA. Evaluation of 390 specimens after initiating standardized Millipore filtering of diluent fluid revealed a reduced number of positive Gram stains, yielding 9% sensitivity and 99% specificity. Clusters of false-positive Gram stains have been reported in other clinical conditions. They are apparently rare related to diagnosing periprosthetic infections but have severe consequences if used to guide treatment. Even occasional false-positive Gram stains should prompt review of laboratory methods. Our observations implicate dead bacteria in microbiologic reagents as potential sources of false-positive Gram

  7. Near-UV laser treatment of extrinsic dental enamel stains.

    Science.gov (United States)

    Schoenly, J E; Seka, W; Featherstone, J D B; Rechmann, P

    2012-04-01

    The selective ablation of extrinsic dental enamel stains using a 400-nm laser is evaluated at several fluences for completely removing stains with minimal damage to the underlying enamel. A frequency-doubled Ti:sapphire laser (400-nm wavelength, 60-nanosecond pulse duration, 10-Hz repetition rate) was used to treat 10 extracted human teeth with extrinsic enamel staining. Each tooth was irradiated perpendicular to the surface in a back-and-forth motion over a 1-mm length using an ∼300-µm-diam 10th-order super-Gaussian beam with fluences ranging from 0.8 to 6.4 J/cm(2) . Laser triangulation determined stain depth and volume removed by measuring 3D surface images before and after irradiation. Scanning electron microscopy evaluated the surface roughness of enamel following stain removal. Fluorescence spectroscopy measured spectra of unbleached and photobleached stains in the spectral range of 600-800 nm. Extrinsic enamel stains are removed with laser fluences between 0.8 and 6.4 J/cm(2) . Stains removed on sound enamel leave behind a smooth enamel surface. Stain removal in areas with signs of earlier cariogenic acid attacks resulted in isolated and randomly located laser-induced, 50-µm-diam enamel pits. These pits contain 0.5-µm diam, smooth craters indicative of heat transfer from the stain to the enamel and subsequent melting and water droplet ejection. Ablation stalling of enamel stains is typically observed at low fluences (<3 J/cm(2) ) and is accompanied by a drastic reduction in porphyrin fluorescence from the Soret band. Laser ablation of extrinsic enamel stains at 400 nm is observed to be most efficient above 3 J/cm(2) with minimal damage to the underlying enamel. Unsound underlying enamel is also observed to be selectively removed after irradiation. Copyright © 2012 Wiley Periodicals, Inc.

  8. Studies on the blue-staining fungi of pine wood

    Directory of Open Access Journals (Sweden)

    A. Strzelczyk

    2014-11-01

    Full Text Available The aim of this was were to examine associations of bule-staining fungi which occur on pine wood to determine the interactions between fungi and to check the suscebility of these fungi to commonly used fungieides. The stron antagonism of members of the Trichoderma genus against the blue-staining fungi was demonstrated, Members of genera Pullularia, Hormiscium and Hormodendrum were strongy inhibited by stains of Trichoderma Ophiostoma strains were less susceptible to inhibition by this antagonist.

  9. 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.

  10. [Histochemical stains for minerals by hematoxylin-lake method].

    Science.gov (United States)

    Miyagawa, Makoto

    2013-04-01

    The present study was undertaken to establish the experimental animal model by histological staining methods for minerals. After intraperitoneal injections of minerals, precipitates deposited on the surface of the liver. Liver tissues were fixed in paraformaldehyde, embedded in paraffin and cut into thin sections which were used as minerals containing standard section. Several reagents for histological stains and spectrophotometry for minerals were applied in both test-tube experiments and stainings of tissue sections to test for minerals. Hematoxylin-lake was found of capable of staining minerals in tissue. A simple technique used was described for light microscopic detection of minerals.

  11. Histopathological evaluation of ocular microsporidiosis by different stains

    Directory of Open Access Journals (Sweden)

    Sharma Savitri

    2006-06-01

    Full Text Available Abstract Background There is limited data on comparing stains in the detection of microsporidia in corneal biopsies. Hence we wanted to evaluate various stains for their ability to detect microsporidia in corneal tissue sections. Methods Four cases diagnosed with microsporidiosis on Hematoxylin and Eosin and Periodic Acid Schiff's stained sections of the corneal button between January 2002 and December 2004, were included. Further sections were prospectively stained with calcofluor white, Gram, Giemsa, Masson's trichrome, acridine orange, Gomori's methenamine silver, Gram's chromotrope and modified acid fast stain. The stained sections were analyzed for the spore characteristics in terms of size, shape, color contrast, cell wall morphology, waist band in cytoplasm and ease of detection. Results All sections showed microsporidial spores as 3 – 5 μm, oval bodies. 1% acid fast, Gram's chromotrope and GMS stains provided a reliable diagnosis of microsporidia as diagnostic waist band could be identified and good contrast helped distinguish the spores from inflammatory debris. Conclusion Considering the ease of performance, cost effectiveness and rapidity of the technique, 1% acid fast stain and Gram's chromotrope stain are ideal for the detection of microsporidia.

  12. Reliability of a rapid hematology stain for sputum cytology

    OpenAIRE

    Gonçalves, Jéssica; Pizzichini, Emilio; Pizzichini, Marcia Margaret Menezes; Steidle, Leila John Marques; Rocha, Cristiane Cinara; Ferreira, Samira Cardoso; Zimmermann, Célia Tânia

    2014-01-01

    Objective: To determine the reliability of a rapid hematology stain for the cytological analysis of induced sputum samples. Methods: This was a cross-sectional study comparing the standard technique (May-Grünwald-Giemsa stain) with a rapid hematology stain (Diff-Quik). Of the 50 subjects included in the study, 21 had asthma, 19 had COPD, and 10 were healthy (controls). From the induced sputum samples collected, we prepared four slides: two were stained with May-Grünwald-Giemsa, and two w...

  13. Black stain and dental caries in Filipino schoolchildren.

    Science.gov (United States)

    Heinrich-Weltzien, Roswitha; Monse, Bella; van Palenstein Helderman, Wim

    2009-04-01

    Black stain is defined as dark pigmented exogenous substance in lines or dots parallel to the gingival margin and firmly adherent to the enamel at the cervical third of the tooth crowns in the primary and permanent dentition. This study was conducted to assess the prevalence of black stain on teeth of Filipino children and to determine a possible association between black stain and caries levels. The study was designed to test the following hypotheses: (i) the prevalence of black stain does not differ between children from schools with oral health intervention programs and those from schools without an intervention program, (ii) the prevalence of black stain does not differ in children attending easily accessible and remote schools, (iii) caries prevalence and caries experience do not differ in children with and without black stain and (iv) the caries distribution at the surface level does not differ in children with and without black stain. In total, 32 elementary schools were included. 19 schools with a comprehensive school-based preventive oral health program, seven schools with a basic preventive program and six control schools. All sixth graders of these schools (n=1748) aged 11.7+/-1.1 years were clinically examined for black stain. DMFT was assessed in 1121 children by seven calibrated dentists using WHO criteria. DMFS was scored in 627 children by two calibrated dentists. Black stain was found in 16% of this population. The prevalence of black stain did not differ significantly between children attending schools with different oral health intervention programs. Thus, hypothesis 1 was accepted. The prevalence of black stain was significantly higher (Pcaries prevalence and caries experience than children without black stain. Thus, hypothesis 3 was rejected. No difference was found in the DMFS pattern of occlusal, smooth and proximal surfaces between children with and without black stain. Thus hypothesis 4 was accepted. The presence of black stain is

  14. Confusion over live/dead stainings for the detection of vital microorganisms in oral biofilms--which stain is suitable?

    Science.gov (United States)

    Netuschil, Lutz; Auschill, Thorsten M; Sculean, Anton; Arweiler, Nicole B

    2014-01-11

    There is confusion over the definition of the term "viability state(s)" of microorganisms. "Viability staining" or "vital staining techniques" are used to distinguish live from dead bacteria. These stainings, first established on planctonic bacteria, may have serious shortcomings when applied to multispecies biofilms. Results of staining techniques should be compared with appropriate microbiological data. Many terms describe "vitality states" of microorganisms, however, several of them are misleading. Authors define "viable" as "capable to grow". Accordingly, staining methods are substitutes, since no staining can prove viability.The reliability of a commercial "viability" staining assay (Molecular Probes) is discussed based on the corresponding product information sheet: (I) Staining principle; (II) Concentrations of bacteria; (III) Calculation of live/dead proportions in vitro. Results of the "viability" kit are dependent on the stains' concentration and on their relation to the number of bacteria in the test. Generally this staining system is not suitable for multispecies biofilms, thus incorrect statements have been published by users of this technique.To compare the results of the staining with bacterial parameters appropriate techniques should be selected. The assessment of Colony Forming Units is insufficient, rather the calculation of Plating Efficiency is necessary. Vital fluorescence staining with Fluorescein Diacetate and Ethidium Bromide seems to be the best proven and suitable method in biofilm research.Regarding the mutagenicity of staining components users should be aware that not only Ethidium Bromide might be harmful, but also a variety of other substances of which the toxicity and mutagenicity is not reported. - The nomenclature regarding "viability" and "vitality" should be used carefully.- The manual of the commercial "viability" kit itself points out that the kit is not suitable for natural multispecies biofilm research, as supported by an

  15. A bead-based western for high-throughput cellular signal transduction analyses

    Science.gov (United States)

    Treindl, Fridolin; Ruprecht, Benjamin; Beiter, Yvonne; Schultz, Silke; Döttinger, Anette; Staebler, Annette; Joos, Thomas O.; Kling, Simon; Poetz, Oliver; Fehm, Tanja; Neubauer, Hans; Kuster, Bernhard; Templin, Markus F.

    2016-01-01

    Dissecting cellular signalling requires the analysis of large number of proteins. The DigiWest approach we describe here transfers the western blot to a bead-based microarray platform. By combining gel-based protein separation with immobilization on microspheres, hundreds of replicas of the initial blot are created, thus enabling the comprehensive analysis of limited material, such as cells collected by laser capture microdissection, and extending traditional western blotting to reach proteomic scales. The combination of molecular weight resolution, sensitivity and signal linearity on an automated platform enables the rapid quantification of hundreds of specific proteins and protein modifications in complex samples. This high-throughput western blot approach allowed us to identify and characterize alterations in cellular signal transduction that occur during the development of resistance to the kinase inhibitor Lapatinib, revealing major changes in the activation state of Ephrin-mediated signalling and a central role for p53-controlled processes. PMID:27659302

  16. Anolyte as an alternative bleach for stained cotton fabrics ...

    African Journals Online (AJOL)

    ... as the two- and three-factor interactions. The results from the study indicated that Anolyte was less effective than sodium hypochlorite as a stain remover for blood, tea, soot/mineral oil and blackcurrant juice. It was noted that the temperature of bleach liquids had an influence on the removal of stains by both bleach liquids.

  17. News from the Biological Stain Commission No. 10

    DEFF Research Database (Denmark)

    Lyon, H O

    2011-01-01

    In the 10th issue of News from the Biological Stain Commission (BSC), under the heading of Regulatory affairs, the Biological Stain Commission's International Affairs Committee presents information from the meeting of ISO/TC 212/WG 1 held in London, UK, on 16-17 November 2009. Furthermore...

  18. News from the Biological Stain Commission no. 12

    DEFF Research Database (Denmark)

    Lyon, H O

    2012-01-01

    In this 12(th) issue of News from the Biological Stain Commission (BSC) under the heading of Regulatory affairs, the Biological Stain Commission's International Affairs Committee presents information from the meetings of ISO/TC 212/WG 1 Quality and competence in the medical laboratory and ISO...

  19. News from the Biological Stain Commission no. 13

    DEFF Research Database (Denmark)

    Lyon, H O

    2013-01-01

    In the 13(th) issue of News from the Biological Stain Commission (BSC) under the heading of Regulatory affairs, the Biological Stain Commission's International Affairs Committee presents information from the first plenary meeting of the International Standards Organization ISO/TC 212 Clinical lab...... laboratory testing and in vitro diagnostic test systems held on 17-19 October 2011 in Las Vegas, Nevada....

  20. Alcian blue-stained particles in a eutrophic lake

    DEFF Research Database (Denmark)

    Worm, J.; Søndergaard, Morten

    1998-01-01

    We used a neutral solution of Alcian Blue to stain transparent particles in eutrophic Lake Frederiksborg Slotss0, Denmark. Alcian Blue-stained particles (ABSP) appeared to be similar to the so-called transparent exopolymer particles (TEP) identified with an acidic solution of Alcian Blue. Our...

  1. Lawsonia inermis And Hibiscus sabdariffa : Posible Histological Stains

    African Journals Online (AJOL)

    The ability of various concentrations of aqueous extracts of Lawsonia inermis and Hibiscus sabdariffa to stain histological tissues was demonstrated. The results with sections of tongue and kidney of the laboratory rat, cut at 6microns thickness showed that only the cellular cytoplasm was stained. However, combinations of ...

  2. The use of special stains in liver biopsy interpretation: Implications ...

    African Journals Online (AJOL)

    Materials and Methods: The formalin fixed paraffin embedded blocks of liver biopsies reported in two histopathology laboratories between 2008 and 2013 were retrieved. These were stained with H and E and the following standard special stains for liver tissue histology – Perl's Prussian blue, reticulin, Sirius red, Shikata ...

  3. anolyte as an alternative bleach for stained cotton fabrics

    African Journals Online (AJOL)

    user

    Serowe College of Education. Private Bag 009. Serowe. Botswana ... and cons that may affect the environment as well as the quality of the final ..... improved as the pH became higher. Effects of anolyte, sodium hypochlorite and distilled water on the removal of tea stain on cotton. Leverette (2013:1) describes a tea stain as a.

  4. Lasers or light sources for treating port-wine stains

    DEFF Research Database (Denmark)

    Faurschou, Annesofie; Olesen, Anne Braae; Leonardi-Bee, Jo

    2011-01-01

    Port-wine stains are birthmarks caused by malformations of blood vessels in the skin. Port-wine stains manifest themselves in infancy as a flat, red mark and do not regress spontaneously but may, if untreated, become darker and thicker in adult life. The profusion of various lasers and light...

  5. Decreased mortality associated with prompt Gram staining of blood cultures.

    Science.gov (United States)

    Barenfanger, Joan; Graham, Donald R; Kolluri, Lavanya; Sangwan, Gaurav; Lawhorn, Jerry; Drake, Cheryl A; Verhulst, Steven J; Peterson, Ryan; Moja, Lauren B; Ertmoed, Matthew M; Moja, Ashley B; Shevlin, Douglas W; Vautrain, Robert; Callahan, Charles D

    2008-12-01

    Gram stains of positive blood cultures are the most important factor influencing appropriate therapy. The sooner appropriate therapy is initiated, the better. Therefore, it is reasonable to expect that the sooner Gram stains are performed, the better. To determine the value of timely Gram stains and whether improvement in Gram stain turnaround time (TAT) is feasible, we compared data for matched pairs of patients with cultures processed promptly ( or =1 hour TAT) and then monitored TAT by control charting.In 99 matched pairs, average difference in time to detection of positive blood cultures within a pair of patients was less than 0.1 hour. For the less than 1 hour TAT group, the average TAT and crude mortality were 0.1 hour and 10.1%, respectively; for the 1 hour or longer TAT group, they were 3.3 hours and 19.2%, respectively (P Gram stains.

  6. 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.

  7. 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.

  8. 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...

  9. Mapping stain distribution in pathology slides using whole slide imaging

    Directory of Open Access Journals (Sweden)

    Fang-Cheng Yeh

    2014-01-01

    Full Text Available Background: Whole slide imaging (WSI offers a novel approach to digitize and review pathology slides, but the voluminous data generated by this technology demand new computational methods for image analysis. Materials and Methods: In this study, we report a method that recognizes stains in WSI data and uses kernel density estimator to calculate the stain density across the digitized pathology slides. The validation study was conducted using a rat model of acute cardiac allograft rejection and another rat model of heart ischemia/reperfusion injury. Immunohistochemistry (IHC was conducted to label ED1 + macrophages in the tissue sections and the stained slides were digitized by a whole slide scanner. The whole slide images were tessellated to enable parallel processing. Pixel-wise stain classification was conducted to classify the IHC stains from those of the background and the density distribution of the identified IHC stains was then calculated by the kernel density estimator. Results: The regression analysis showed a correlation coefficient of 0.8961 between the number of IHC stains counted by our stain recognition algorithm and that by the manual counting, suggesting that our stain recognition algorithm was in good agreement with the manual counting. The density distribution of the IHC stains showed a consistent pattern with those of the cellular magnetic resonance (MR images that detected macrophages labeled by ultrasmall superparamagnetic iron-oxide or micron-sized iron-oxide particles. Conclusions: Our method provides a new imaging modality to facilitate clinical diagnosis. It also provides a way to validate/correlate cellular MRI data used for tracking immune-cell infiltration in cardiac transplant rejection and cardiac ischemic injury.

  10. 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.

  11. 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.

  12. A sensitive cytochemical staining method for glucose-6-phosphate dehydrogenase activity in individual erythrocytes. I. Optimalization of the staining procedure

    NARCIS (Netherlands)

    van Noorden, C. J.; Vogels, I. M.; James, J.; Tas, J.

    1982-01-01

    A sensitive cytochemical staining method for glucose-6-phosphate dehydrogenase activity in individual human erythrocytes is described. This staining method can be used for the rapid routine discrimination of patients with a deficiency of the enzyme in its homozygote or heterozygote form, but also

  13. Novel Process for Laser Stain Removal from Archaeological Oil Paintings

    Science.gov (United States)

    El-Nadi, Lotfia; El-Feky, Osama; Abdellatif, Galila; Darwish, Sawsan

    2013-03-01

    Some samples of oil paintings (5 × 5 cm) were prepared on wooden panel with four types of fungi commonly encountered on oil paintings were selected for this study. Each of the fungi is associated with different colored stains. Fungus Alternaria tenuis is associated by a dense black stain, Chetomium globosum by a brownish gray stain, Aspergillus flavus by a yellowish stain, and Fusaruim oxysporum by a pinkish stain. Fungi growing on oil paintings affect the surface characteristics by forming a variety of colored patches typically composed of many complex chemical substances that are produced during metabolic processes. These colored stains may be encrusted in spores, present in mycelium or secreted to a substance such as oil paintings surfaces. While the fungal stains can sometimes be extracted with appropriate solvents, there are some stains that resist solvent extraction entirely. Developing new solvent system that might attack the paint structure, and is time consuming and requires a great deal of trial and error. Mechanical stain removal is also problematic in that it often produces abrasion of the surface, markedly deteriorating the artwork, and is extra ordinarily fine and tedious. For these reasons, we decided to examine an alternative physical technique as a new approach to deal with stain removal. Since the stains are due to the existence of fungi, we thought it a good idea to remove them by singlet oxygen. We applied the photo dynamic process through which the fungi stains were covered with organic dye derivatives in solution under controlled illumination in the lab. The samples were then irradiated by low power Laser light from a He-Ne laser, the dye will be photodecomposed and produce singlet oxygen. We report in this work the results obtained as a function of: - The concentration and types of the organic dye in solution, - The presence of certain amounts of liquids added to the solution, - The scanning speed of the laser beam on the sample surface

  14. Rapid staining techniques in cytopathology: a review and comparison of modified protocols for hematoxylin and eosin, Papanicolaou and Romanowsky stains.

    Science.gov (United States)

    Jörundsson, Einar; Lumsden, John H.; Jacobs, Robert M.

    1999-01-01

    The objective of this study was to review and compare rapid protocols for fixation and staining of cytologic smears. We used fresh surgical specimens from dogs and horses to evaluate and modify, if necessary, previously described rapid staining protocols. Slides were wet-fixed, rehydrated or air-dried. Rapid Papanicolaou, hematoxylin and eosin (H&E), and Romanowsky stains were applied, including modification of Diff-Quick stain. The modified rapid staining protocols were simple to use and gave results within 5 minutes that were comparable to those obtained with traditional methods. Advantages of rehydrated vs wet-fixed smears included consistent preparations, a clean background, and equally good or superior nuclear detail.

  15. Molecular evidence for the occurrence of beet western yellows virus on chickpea in Morocco.

    NARCIS (Netherlands)

    Fortass, M.; Wilk, van der F.; Heuvel, van de J.F.J.M.; Goldbach, R.W.

    1997-01-01

    A luteovirus isolate infecting chickpea in Morocco was experimentally transmitted by Myzus persicae to Physalis floridana, on which it produced mild symptoms. When tested in western blots against antisera to known legume luteoviruses, this isolate reacted strongly to beet western yellows virus

  16. Effect of Melamine Sponge on Tooth Stain Removal.

    Science.gov (United States)

    Otsuka, Takero; Kawata, Toshitsugu

    2015-01-01

    To investigate the stain removal ability of melamine sponge before aesthetic tooth whitening in extracted teeth. Melamine sponge of thickness 40 mm was compressed and the destruction of the partition wall structure during the compression process was examined under a stereoscopic microscope. An extracted human tooth was cleaned by normal polishing or with melamine sponge for 90 s. To evaluate the stain level, the tooth surfaces were photographed under a stereoscopic microscope at 0, 30, 60 and 90 s. The residual stained region was traced in a high-magnification photograph, and the stain intensity was presented as a change, relative to the intensity before the experiment (0 s). Mechanical cleaning by toothbrushing produced polishing scratches on the tooth surface, whereas use of the melamine sponge resulted in only minimal scratches. As the compression level increased, the stain-removing effect tended to become stronger. Melamine sponge can remove stains from the tooth surface more effectively and less invasively compared to a conventional toothbrush. As no new scratches are made on the tooth surface when using a melamine sponge brush, the risk of re-staining is reduced. Cleaning using a melamine sponge brush can be easily and effectively performed at home and in a dental office.

  17. Microscopic analysis of MTT stained boar sperm cells

    Directory of Open Access Journals (Sweden)

    B.M. van den Berg

    2015-06-01

    Full Text Available The ability of sperm cells to develop colored formazan by reduction of MTT was used earlier to develop a spectrophotometric assay to determine the viability of sperm cells for several mammalian species. It was the objective of the present study to visualize microscopically the location of the formazan in boar sperm cells. The MTT staining process of boar sperm cells can be divided into a series of morphological events. Incubation of the sperm cells in the presence of MTT resulted after a few min in a diffuse staining of the midpiece of the sperm cells. Upon further incubation the staining of the midpiece became more intense, and gradually the formation of packed formazan granules became more visible. At the same time, a small formazan stained granule appeared medially on the sperm head, which increased in size during further incubation. After incubation for about 1 h the midpiece granules were intensely stained and more clearly distinct as granules, while aggregation of sperm cells occurred. Around 90% of the sperm cells showed these staining events. At the end of the staining the formazan granules have disappeared from both the sperm cells and medium, whereas formazan crystals appeared as thin crystal threads, that became heavily aggregated in the incubation medium. It was concluded that formazan is taken up by lipid droplets in the cytoplasm. Further, the use of the MTT assay to test for sperm viability should be regarded as a qualitative assay, whereas its practical use at artificial insemination (AI Stations is limited.

  18. Are there metallic traces in black extrinsic dental stain?

    Science.gov (United States)

    Parnas, Limor; Chevion, Mordechai; Berenshtein, Eduard; Faibis, Sarit; Moskovitz, Moti

    2013-05-01

    The detection of ferric ions in samples of black extrinsic dental stain led to the idea that it is comprised of insoluble ferric compounds. The present study examined the chemical composition of black extrinsic dental stain. Plaque was collected from 17 children with black extrinsic dental stain (study group A) and from 15 children without black extrinsic stain (control group), using sterile graphite curettes; and from 4 children with black extrinsic stain (study group B), using a standard sterile metal curette. Samples were analyzed with a scanning electron microscope (SEM) and subjected to quantitative chemical analysis (energy dispersive spectrometry). Except for calcium and phosphorus levels, no significant differences were found between the chemical composition of black extrinsic dental stain and dental plaque. Metallic ions were not detected in samples collected with a graphite curette (study group A), but were detected in samples collected with a metal curette (study group B). Metallic ions do not seem to be the origin of black extrinsic dental stain. Previous reports of the presence of metallic ions are probably due to contamination of the samples by the collection method.

  19. DNA Comet Giemsa Staining for Conventional Bright-Field Microscopy

    Directory of Open Access Journals (Sweden)

    Andreyan Osipov

    2014-04-01

    Full Text Available This study was undertaken to evaluate the compatibility of Giemsa staining protocol with the comet assay. We showed, for the first time, that DNA comets can be visualized and analyzed using Giemsa staining. We generated DNA damage dose response curves for human peripheral blood lymphocytes exposed to X-ray radiation using the comet assay with either SybrGreen I or Giemsa stain. The dose response curves were fitted by linear regressions (R2 > 0.977. The SybrGreen I results showed only ~1.2-fold higher slope coefficient (method sensitivity compared to the Giemsa results. The unexpectedly high sensitivity of Giemsa staining for the comet assay is due to the Romanowsky-Giemsa effect, the stain photo-stability and the higher resolution of bright-field imaging compared to fluorescence imaging. Our results demonstrate that Giemsa staining can effectively be used for measuring DNA damage by the comet assay. The low cost and availability of Giemsa stain makes this method affordable for any low budget research and will facilitate new applications of the comet assay in biology and medicine.

  20. DNA Comet Giemsa Staining for Conventional Bright-Field Microscopy

    Science.gov (United States)

    Osipov, Andreyan; Arkhangelskaya, Ekaterina; Vinokurov, Alexei; Smetanina, Nadezhda; Zhavoronkov, Alex; Klokov, Dmitry

    2014-01-01

    This study was undertaken to evaluate the compatibility of Giemsa staining protocol with the comet assay. We showed, for the first time, that DNA comets can be visualized and analyzed using Giemsa staining. We generated DNA damage dose response curves for human peripheral blood lymphocytes exposed to X-ray radiation using the comet assay with either SybrGreen I or Giemsa stain. The dose response curves were fitted by linear regressions (R2 > 0.977). The SybrGreen I results showed only ~1.2-fold higher slope coefficient (method sensitivity) compared to the Giemsa results. The unexpectedly high sensitivity of Giemsa staining for the comet assay is due to the Romanowsky-Giemsa effect, the stain photo-stability and the higher resolution of bright-field imaging compared to fluorescence imaging. Our results demonstrate that Giemsa staining can effectively be used for measuring DNA damage by the comet assay. The low cost and availability of Giemsa stain makes this method affordable for any low budget research and will facilitate new applications of the comet assay in biology and medicine. PMID:24727376

  1. A novel washing algorithm for underarm stain removal

    Science.gov (United States)

    Acikgoz Tufan, H.; Gocek, I.; Sahin, U. K.; Erdem, I.

    2017-10-01

    After contacting with human sweat which comprise around 27% sebum, anti-perspirants comprising aluminium chloride or its compounds form a jel-like structure whose solubility in water is very poor. In daily use, this jel-like structure closes sweat pores and hinders wetting of skin by sweat. However, when in contact with garments, they form yellowish stains at the underarm of the garments. These stains are very hard to remove with regular machine washing. In this study, first of all, we focused on understanding and simulating such stain formation on the garments. Two alternative procedures are offered to form jel-like structures. On both procedures, commercially available spray or deo-stick type anti-perspirants, standard acidic and basic sweat solutions and artificial sebum are used to form jel-like structures, and they are applied on fabric in order to get hard stains. Secondly, after simulation of the stain on the fabric, we put our efforts on developing a washing algorithm specifically designed for removal of underarm stains. Eight alternative washing algorithms are offered with varying washing temperature, amounts of detergent, and pre-stain removal procedures. Better algorithm is selected by comparison of Tristimulus Y values after washing.

  2. LANTHANUM STAINING OF THE SURFACE COAT OF CELLS

    Science.gov (United States)

    Shea, Stephen M.

    1971-01-01

    Among the techniques which have been reported to stain the surface coat of cells, for electron microscopy, is lanthanum staining en bloc. Similarly, the presence of the cationic dye, Alcian blue 8GX, in a primary glutaraldehyde fixative has been reported to improve the preservation of the surface coat of cells of many types; however, the preserved coat is not very electron opaque unless thin sections are counterstained. The present paper shows that for several rat tissues lanthanum staining en bloc is an effective electron stain for the cell surface, giving excellent contrast, if combined sequentially with prefixation in an aldehyde fixative containing Alcian blue. The cationic substance cetylpyridinium chloride was found to have a similar effect to that of Alcian blue in enhancing the lanthanum staining of the surface coat material of the brush border of intestinal epithelial cells. The patterns of lanthanum staining obtained for the tissues studied strikingly resemble those reported in the literature where tissues are stained by several standard methods for demonstrating mucosubstances at the ultrastructural level. This fact and the reproduction of the effect of Alcian blue by cetylpyridinium chloride constitute a persuasive empirical argument that the material visualized is a mucopolysaccharide or mucopolysaccharide-protein complex. PMID:4108476

  3. Silver and Cyanine Staining of Oligonucleotides in Polyacrylamide Gel.

    Science.gov (United States)

    Tang, Weizhong; Zhou, Huafu; Li, Wei

    2015-01-01

    To explore why some oligonucleotides in denaturing polyacrylamide gel could not be silver-stained, 134 different oligonucleotides were analyzed using denaturing polyacrylamide gel electrophoresis stained with silver and asymmetric cyanine. As a result, we found that the sensitivity of oligos (dA), (dC), (dG) and (dT) to silver staining could be ranged as (dA) > (dG) > (dC) > (dT) from high to low. It was unexpected that oligo (dT) was hard to be silver-stained. Moreover, the silver staining of an oligonucleotide containing base T could be partially or completely inhibited by base T. The inhibition of silver staining by base T was a competitive inhibition which could be affected by the amounts of the argyrophil nucleobase and base T, the cis-distance between the argyrophil nucleobase and base T, and the gel concentration. The changes of the intensity of an oligonucleotide band caused by the changes of DNA base composition were diverse and interesting. The intensity of some oligonucleotide bands would significantly change when the changes of DNA base composition accumulated to a certain extent (usually ≥ 4 nt). The sensitivity of cyanine staining of ≤ 11-nt long oligonucleotides could be enhanced about 250-fold by fixing the gels with methanol fixing solution.

  4. News from the Biological Stain Commission No. 11

    DEFF Research Database (Denmark)

    Lyon, H O; Horobin, R W

    2012-01-01

    The 11th issue of News from the Biological Stain Commission (BSC) provides our first impressions of the REACH and ECHA programs. We intend to give a more thorough account of what these important programs actually mean in later editions of News from the Biological Stain Commission. Under the heading...... of Regulatory Affairs, the Biological Stain Commission's International Affairs Committee presents information from the opening session of the meeting of the International Standards Organization ISO/TC 212 Clinical laboratory testing and in vitro diagnostic test systems held on 2-4 June 2010 in Seoul, Republic...

  5. Simvastatin impairs the induction of pulmonary fibrosis caused by a western style diet: a preliminary study.

    Science.gov (United States)

    Kruzliak, Peter; Hare, David L; Zvonicek, Vaclav; Klimas, Jan; Zulli, Anthony

    2015-11-01

    The role of an atherogenic diet in causing pulmonary fibrosis has received little attention and simvastatin has been shown to reduce pulmonary fibrosis in animal models. To determine if an atherogenic diet can induce pulmonary fibrosis and whether simvastatin treatment is beneficial by up-regulating heat shock protein 70 and 90. New Zealand white rabbits (n = 15) were divided: Group 1 (control); Group 2 (MC) received a normal rabbit diet with 1% methionine plus 0.5% cholesterol (atherogenic diet). Group 3 received the same diet as the MC group plus 5 mg/kg/day simvastatin orally (MCS). After 4 weeks, the lungs were collected and analysed. Picrosirus red staining of lung interstitial collagen content showed that the atherogenic diet increased fibrosis 2.9-fold (P Western blot analysis showed that the atherogenic diet significantly reduced lung Hsp70 protein by 22% (P diet stimulates pulmonary fibrosis and reduces lung Hsp70/Hsp90 protein concentration. Simvastatin impairs this by mechanisms unrelated to Hsp70/Hsp90, but possibly a reduction in angiotensin II receptor or alpha adrenergic receptor pathways. These results could have implications in idiopathic pulmonary fibrosis. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  6. 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.

  7. The Gram stain after more than a century.

    Science.gov (United States)

    Popescu, A; Doyle, R J

    1996-05-01

    The Gram stain, the most important stain in microbiology, was described more than a century ago. Only within the past decade, however, has an understanding of its mechanism emerged. It now seems clear that the cell wall of Gram-positive microorganisms is responsible for retention of a crystal violet:iodine complex. In Gram-negative cells, the staining procedures damage the cell surface resulting in loss of dye complexes. Gram-positive microorganisms require a relatively thick cell wall, irrespective of composition, to retain the dye. Therefore, Gram-stainability is a function of the cell wall and is not related to chemistry of cell constituents. This review provides a chronology of the Gram stain and discusses its recently discovered mechanism.

  8. Standardization of the Romanowsky staining procedure: an overview.

    Science.gov (United States)

    Bentley, S A; Marshall, P N; Trobaugh, F E

    1980-01-01

    Commerically available Romanowsky blood stains are variable mixtures of thiazein dyes and brominated fluorescein derivatives with varying degrees of metallic salt contamination in a number of different solvent systems. There is a need for standardized Romanowsky stains of constant composition, which, when used in conjunction with a carefully controlled specimen preparation technique, should give consistent performance. Such a preparation system would be of great value to hematologists in general and would be essential to the validity of data obtained by the digital processing of blood cell images. It is possible to prepare standardized Romanowsky stains as mixtures of two or three dye components, namely, eosin Y, azure B and methylene blue, although azure B has only recently become commercially available at an acceptable degree of purity. The logistic problems of stain standardization are discussed.

  9. The effect of decalcifying solutions on hemosiderin staining.

    Science.gov (United States)

    Byard, Roger W; Bellis, Maria

    2010-09-01

    To determine whether routine decalcification may reduce the amount of stainable iron that is visible on tissue sections, samples of liver and lung tissue with excessive iron stores were placed in three standard decalcifying solutions (i) formic acid [33%], formaldehyde [4%], and NaCl [0.85%]; (ii) formic acid [30%], formaldehyde [4%], and water; and (iii) nitric acid [5%] for 24, 48, 72, and 96 h. After exposure to the decalcifying solutions, the tissues were stained with Perls stain. The slides were examined blind and the intensity of iron staining was scored semiquantitatively from 0 to 3+. The trend in all samples over the course of the experiment (96 h) was for reduction in the intensity of hemosiderin staining. As the amount of stainable hemosiderin in tissues may be significantly altered by decalcification, the absence of hemosiderin in tissues adjacent to a fracture site does not necessarily indicate that the injury was acute. © 2010 American Academy of Forensic Sciences.

  10. The value of intraoperative Gram stain in revision spine surgery.

    Science.gov (United States)

    Shifflett, Grant D; Nwachukwu, Benedict U; Bjerke-Kroll, Benjamin T; Kueper, Janina; Koltsov, Jayme B; Sama, Andrew A; Girardi, Federico P; Cammisa, Frank P; Hughes, Alexander P

    2015-10-01

    Intraoperative cultures and Gram stains are often obtained in cases of revision spine surgery even when clinical signs of infection are not present. The clinical utility and cost-effectiveness of this behavior remain unproven. The aim was to evaluate the clinical utility and cost-effectiveness of routine intraoperative Gram stains in revision spine surgery. This was a retrospective clinical review performed at an academic center in an urban setting. One hundred twenty-nine consecutive adult revision spine surgeries were performed. The outcome measures included intraoperative Gram stains. We retrospectively reviewed the records of 594 consecutive revision spine surgeries performed by four senior surgeons between 2008 and 2013 to identify patients who had operative cultures and Gram stains performed. All revision cases including cervical, thoracic, and lumbar fusion and non-fusion, with and without instrumentation were reviewed. One hundred twenty-nine (21.7%) patients had operative cultures obtained and were included in the study. The most common primary diagnosis code at the time of revision surgery was pseudarthrosis, which was present in 41.9% of cases (54 of 129). Infection was the primary diagnosis in 10.1% (13 of 129) of cases. Operative cultures were obtained in 129 of 595 (21.7%) cases, and 47.3% (61 of 129) were positive. Gram stains were performed in 98 of 129 (76.0%) cases and were positive in 5 of 98 (5.1%) cases. Overall, there was no correlation between revision diagnosis and whether or not a Gram stain was obtained (p=.697). Patients with a history of prior instrumentation were more likely to have a positive Gram stain (pGram staining was found to have a sensitivity of 10.9% (confidence interval [CI] 3.9%-23.6%) and specificity of 100% (CI 93.1%-100%). The positive and negative predictive values were 100% (CI 48.0%-100%) and 57.3% (CI 45.2%-66.2%), respectively. Kappa coefficient was calculated to be 0.1172 (CI 0.0194-0.2151). The cost per discrepant

  11. 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.

  12. NEONATAL OUTCOME IN MECONIUM STAINED DELIVERIES — A PROSPECTIVE STUDY

    OpenAIRE

    RAMAN, TS RAGHU; JAYAPRAKASH, DG

    1997-01-01

    This prospective study analyzes the neonatal outcome in deliveries complicated by meconium stained amniotic fluid. In a study of 1000 live born deliveries, meconium staining of amniotic fluid was seen in 50 (5%) deliveries. Out of these, 20 newborns (40%) developed classical signs of meconium aspiration syndrome and were managed according to a predetermined protocol. Multiparity, term deliveries, use of sedatives in mother, intrauterine growth retardation and prolonged labour were some of the...

  13. News from the Biological Stain Commission no. 15

    DEFF Research Database (Denmark)

    Lyon, H O; Horobin, R W

    2014-01-01

    In the 15(th) issue of News from the Biological Stain Commission (BSC), under the heading of Regulatory affairs, the Biological Stain Commission's International Affairs Committee presents information from the plenary meetings of the International Standards Organization ISO/TC 212 Clinical laborat...... laboratory testing and in vitro diagnostic test systems held on August 22-24, 2012 in Berlin, Germany. An additional discussion of the use of food dyes in India also is included....

  14. An improved method for staining cell colonies in clonogenic assays.

    Science.gov (United States)

    Guda, Kishore; Natale, Leanna; Markowitz, Sanford D

    2007-06-01

    Clonogenic assay is a widely used experimental approach to test for the effects of drugs/genes on the growth and proliferative characteristics of cells in vitro. Accurate quantitation of treatment effects in clonogeneic assays depends on the ability to visualize and count cell colonies precisely. We report a novel method (referred as ETeB) for staining cell colonies grown on plastic and specially coated substrates like collagen. Using colon cancer cell lines grown on plastic and collagen, we compared the colony staining efficiencies of the widely used methylene blue, and Ethidium bromide (ETeB) stains. Results show that the ETeB protocol works well on plastic and is extremely effective for staining colonies on collagen when compared to methylene blue. The key features and advantages of ETeB technique are; (a) reduction in background for colonies grown on collagen and possibly other substrates, (b) the whole procedure takes less than a minute, (c) no post-stain washing step is required which eliminates colony losses for cell lines that are loosely adherent, (d) colony visualization and counting can be done immediately following the staining procedure using a standard UV illuminator and software, and (e) the method works across a wide variety of cell lines. The simplicity and robustness of this procedure should warrant its usage in both small and large-scale clonogenic experiments.

  15. 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 .

  16. 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.

  17. 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.

  18. Evaluación de las pruebas dot blot y aglutinación de látex para el diagnóstico de cisticercosis en Perú

    Directory of Open Access Journals (Sweden)

    Eduardo Miranda-Ulloa

    Full Text Available Con el objetivo de evaluar las pruebas dot blot y aglutinación de látex para la detección de cisticercosis humana con antígeno de líquido de cisticerco de Taenia solium, se usaron 125 sueros humanos, de los cuales 60 procedían de personas con cisticercosis confirmada por Western Blot, 45 de personas con otras enfermedades parasitarias y 20 de personas aparentemente sanas. La concentración óptima del antígeno para impregnar las tiras dot blot fue de 0,01 ug/uL, y para impregnar las partículas de látex fue de 0,092 ug/uL. Para la prueba dot blot se encontró una sensibilidad del 100% y especificidad del 87,7%; para la aglutinación de látex una sensibilidad del 93,3% y especificidad del 89,2%. Ambas pruebas podrían ser de utilidad y factibles de implementar como alternativas de diagnóstico serológico en laboratorios de áreas endémicas del Perú

  19. The influence of Romanowsky-Giemsa type stains on nuclear and cytoplasmic features of cytological specimens.

    Science.gov (United States)

    Schulte, E; Wittekind, D

    1989-04-01

    The aim of the present study was to compare the staining pattern of the standard azure B-eosin Y stain with commercial May-Grünwald-Giemsa (MGG) stains on cytological specimens by means of high resolution image analysis. Several cytological specimens (blood smears, abdominal serous effusions, bronchial scrape material) were air dried, methanol fixed and stained with the standard azure B-eosin Y stain and with commercial May-Grünwald-Giemsa stains. Integrated optical density (IOD) and colour intensities of cell nuclei and cytoplasm were measured with the IBAS 2000 image analyser. Commercial MGG stains gave much higher coefficients of variation for all parameters than the standard stain. Reproducibility of cell nuclei segmentation versus cytoplasm was significantly better for the standard stain. Contamination of the standard stain with methylene blue partly copied the staining pattern of commercial stains. The standard azure B-eosin Y stain is recommended for high resolution image analysis (HRIA) of cytological samples.

  20. Leishman-Giemsa Cocktail - Is it an Effective Stain for Air Dried Cytology Smears.

    Science.gov (United States)

    Doddagowda, Shilpa Manigatta; Shashidhar, Hemalatha Anantharamaiah; Prasad, Chinaiah Subramanyam Babu Rajendra

    2017-03-01

    Air dried cytology smears are stained routinely with Romanowsky stains so that the relative cell size, nuclear size, cytoplasmic details, smear background elements and intercellular matrix components are better appreciated. A variety of modified Romanowsky stains are used in cytology. Leishman-Giemsa (LG) cocktail is one of the new staining techniques which can be used for staining the air dried cytology smears. To evaluate the quality of staining of LG cocktail on air dried smears and to compare the quality of staining of LG cocktail with May Grunwald Giemsa (MGG) which is the most commonly used stain in cytology. The present prospective comparative study was carried out with 100 cases and two extra smears were prepared for each case and stained with MGG and LG cocktail stains. The stained slides were blinded and were evaluated for the staining characteristics of the nucleus, cytoplasm and background staining. Based on this, scoring was done by two pathologists independently. Quality Index (QI) was calculated by dividing the scores obtained with the total score possible. LG cocktail stained slides were excellent in cytoplasmic staining, granularity, nuclear morphology, background material staining and overall staining characteristics. QI of LG cocktail was 0.8 while that of MGG was 0.59. Staining of air dried smears by LG cocktail has a good QI. It is also cheaper, requires short duration for staining as compared to MGG. Hence, LG cocktail can be an effective replacement for MGG for staining the air dried cytology smears.

  1. The standard Romanowsky-Giemsa stain in histology.

    Science.gov (United States)

    Wittekind, D; Schulte, E; Schmidt, G; Frank, G

    1991-01-01

    A new and technically simple Romanowsky-Giemsa (RG) stain is proposed as a standardized technique for use in histology. An RG stock solution (pure azure B 7.5 g/l, eosin Y as eosinic acid 1.2 g/l in dimethylsulfoxide) is diluted to form the working solution with HEPES-buffer, pH 6. Staining time is 30-90 min after formol-calcium solution (or 2-4 hr after formaldehyde-organic acid mixtures). The resulting overstained sections are to be differentiated. A tannic acid-acetic acid combination in an isopropanol-water mixture was found to give optimum results within 100 sec. Subsequent dehydration is in isopropanol only. The staining pattern obtained is polychrome. The distribution of colors in detail is influenced by the modes of pre- and posttreatment. Of practical interest is the development of green and greenish blue colors on collagen fibrils which contrast strongly against the pink of sarcoplasm. For this and other reasons, this RG stain version seems suitable to replace the trichrome Gomori-type trichrome stains under appropriate processing conditions.

  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. 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.

  5. 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.

  6. 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.

  7. A staining protocol for identifying secondary compounds in Myrtaceae1

    Science.gov (United States)

    Retamales, Hernan A.; Scharaschkin, Tanya

    2014-01-01

    • Premise of the study: Here we propose a staining protocol using toluidine blue (TBO) and ruthenium red to reliably identify secondary compounds in the leaves of some species of Myrtaceae. • Methods and Results: Leaves of 10 species representing 10 different genera of Myrtaceae were processed and stained using five different combinations of ruthenium red and TBO. Optimal staining conditions were determined as 1 min of ruthenium red (0.05% aqueous) and 45 s of TBO (0.1% aqueous). Secondary compounds clearly identified under this treatment include mucilage in the mesophyll, polyphenols in the cuticle, lignin in fibers and xylem, tannins and carboxylated polysaccharides in the epidermis, and pectic substances in the primary cell walls. • Conclusions: Potential applications of this protocol include systematic, phytochemical, and ecological investigations in Myrtaceae. It might be applicable to other plant families rich in secondary compounds and could be used as a preliminary screening method for extraction of these elements. PMID:25309840

  8. A staining protocol for identifying secondary compounds in Myrtaceae.

    Science.gov (United States)

    Retamales, Hernan A; Scharaschkin, Tanya

    2014-10-01

    Here we propose a staining protocol using toluidine blue (TBO) and ruthenium red to reliably identify secondary compounds in the leaves of some species of Myrtaceae. • Leaves of 10 species representing 10 different genera of Myrtaceae were processed and stained using five different combinations of ruthenium red and TBO. Optimal staining conditions were determined as 1 min of ruthenium red (0.05% aqueous) and 45 s of TBO (0.1% aqueous). Secondary compounds clearly identified under this treatment include mucilage in the mesophyll, polyphenols in the cuticle, lignin in fibers and xylem, tannins and carboxylated polysaccharides in the epidermis, and pectic substances in the primary cell walls. • Potential applications of this protocol include systematic, phytochemical, and ecological investigations in Myrtaceae. It might be applicable to other plant families rich in secondary compounds and could be used as a preliminary screening method for extraction of these elements.

  9. Sperm viability staining in ecology and evolution: potential pitfalls

    DEFF Research Database (Denmark)

    Holman, Luke

    2009-01-01

    The causes and consequences of variation in sperm quality, survival and ageing are active areas of research in ecology and evolution. In order to address these topics, many recent studies have measured sperm viability using fluorescent staining. Although sperm viability staining has produced...... a number of interesting results, it has some potential pitfalls that have rarely been discussed. In the present paper, I review the major findings of ecology and evolution studies employing sperm viability staining and outline the method's principle limitations. The key problem is that the viability assay...... may itself kill sperm, which is likely to confound many common experimental designs in addition to producing artificially low estimates of sperm viability. I further suggest that sperm number should be routinely measured in sperm viability studies, as it may be an important but overlooked source...

  10. Direct gram stain and urease test to detect Helicobacter pylori.

    Science.gov (United States)

    Van Horn, K G; Dworkin, B M

    1990-01-01

    Antral biopsies were obtained by gastrointestinal endoscopy on 143 adult patients with dyspeptic symptoms of gastritis or peptic ulcer disease. A direct Gram stain and a direct urease test were performed on each biopsy in addition to culture. Forty-three biopsies (30%) were considered positive for Helicobacter pylori based on culture or histologic examination, or both. Thirty-one biopsies (72% sensitivity) were positive for both direct tests, whereas 95 of 100 negative cultures were negative for both tests. Thirty-eight of the 43 positive biopsies were Gram stain positive (sensitivity, 88%; specificity, 100%). The direct urease test alone was positive at 4 hr for 29 biopsies (sensitivity, 67%; specificity, 100%) and at 24 hr for 38 biopsies (sensitivity, 74%; specificity, 95%). Rapid presumptive diagnosis of H. pylori in antral biopsies was obtained when at least one direct test, Gram stain or urease, was positive.

  11. Stain-free histopathology by programmable supercontinuum pulses

    Science.gov (United States)

    Tu, Haohua; Liu, Yuan; Turchinovich, Dmitry; Marjanovic, Marina; Lyngsø, Jens K.; Lægsgaard, Jesper; Chaney, Eric J.; Zhao, Youbo; You, Sixian; Wilson, William L.; Xu, Bingwei; Dantus, Marcos; Boppart, Stephen A.

    2016-08-01

    The preparation, staining, visualization and interpretation of histological images of tissue is well accepted as the gold standard process for the diagnosis of disease. These methods have a long history of development, and are used ubiquitously in pathology, despite being highly time- and labour-intensive. Here, we introduce a unique optical imaging platform and methodology for label-free multimodal multiphoton microscopy that uses a novel photonic-crystal fibre source to generate tailored chemical contrast based on programmable supercontinuum pulses. We demonstrate the collection of optical signatures of the tumour microenvironment, including evidence of mesoscopic biological organization, tumour cell migration and (lymph-) angiogenesis collected directly from fresh ex vivo mammary tissue. Acquisition of these optical signatures and other cellular or extracellular features, which are largely absent from histologically processed and stained tissue, combined with an adaptable platform for optical alignment-free programmable-contrast imaging, offers the potential to translate stain-free molecular histopathology into routine clinical use.

  12. Vital staining of coronal dentin in monkey teeth.

    Science.gov (United States)

    Tronstad, L

    1978-04-01

    Vital staining of monkey incisor teeth with the incisal dentin exposed to the oral environment by attrition was carried out, with the use of a number of dyes (pH and redox indicators). There was a distinct staining of the coronal dentin, regardless of which dye was introduced into the pulpal cavity. The exposed dentin was stained like the unaffected dentin, with the exception of a narrow centrally located zone that extended from the tip of the original pulp horn to the incisal edge of the tooth. The suggestion is that this zone is not unstained because of exposure of the dentin to the oral environment, but because it coincides with an area of the tissue where the pulpal ends of the dentinal tubules are blocked by atubular hard tissue normally laid down in the pulp horn of incisor teeth.

  13. Chromosome-specific staining to detect genetic rearrangements

    Energy Technology Data Exchange (ETDEWEB)

    Gray, Joe W.; Pinkel, Daniel; Tkachuk, Douglas; Westbrook, Carol

    2013-04-09

    Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyzes. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods and reagents are provided for the detection of genetic rearrangements. Probes and test kits are provided for use in detecting genetic rearrangements, particularly for use in tumor cytogenetics, in the detection of disease related loci, specifically cancer, such as chronic myelogenous leukemia (CML) and for biological dosimetry. Methods and reagents are described for cytogenetic research, for the differentiation of cytogenetically similar but genetically different diseases, and for many prognostic and diagnostic applications.

  14. Intrapartum amnioinfusion for meconium-stained liquor in developing countries.

    Science.gov (United States)

    Moodley, J; Matchaba, P; Payne, A J

    1998-01-01

    Intrapartum amnioinfusion (AI) has been reported to decrease perinatal mortality and morbidity in women with meconium-stained liquor. Such work has not previously been performed at King Edward VIII Hospital (KEH), in a developing country, where the incidence of meconium-stained liquor is said to be extremely high. To establish whether AI during the intrapartum period for meconium-stained liquor decreases Caesarean section rates for fetal distress and decreases perinatal morbidity. Informed consent was obtained from patients in labour who were 3-8 cm dilated, with meconium-staining of the liquor, grades I to III inclusive, and who had a normal cardiotocograph on presentation at term. Sixty patients were included in the trial; 30 had AI. The control group was managed by standard methods. The study group had an amnioinfusion of 0.9% normal saline at 15 ml/min under continuous cardiotocographic monitoring, until a volume of 11 was completed. This was repeated if delivery did not occur within 4 h. The mean pH of umbilical arterial blood was significantly higher in the AI group (7.30 versus 7.23; P = 0.0029). In addition fewer patients in this group developed hypoxic ischaemic encephalopathy (0 versus 2 controls) or meconium aspiration syndrome (1 versus 4 controls). This was not statistically significant. Caesarean section for fetal distress was performed on fewer patients in the AI group (3 versus 7 controls), although this was not statistically significant. These results demonstrate that amnioinfusion is an effective technique for improving the perinatal outcome of pregnancies complicated by meconium-stained liquor in labour. The decrease in Caesarean sections for fetal distress, though not statistically significant in this study, has clinical relevance. Furthermore, this study suggests that amnioinfusion is cost effective in a busy, high-risk labour ward unit and consequently should become standard practice in the management of meconium-stained liquor in labour.

  15. Optical Monte Carlo modeling of a true portwine stain anatomy

    Science.gov (United States)

    Barton, Jennifer K.; Pfefer, T. Joshua; Welch, Ashley J.; Smithies, Derek J.; Nelson, Jerry; van Gemert, Martin J.

    1998-04-01

    A unique Monte Carlo program capable of accommodating an arbitrarily complex geometry was used to determine the energy deposition in a true port wine stain anatomy. Serial histologic sections taken from a biopsy of a dark red, laser therapy resistant stain were digitized and used to create the program input for simulation at wavelengths of 532 and 585 nm. At both wavelengths, the greatest energy deposition occurred in the superficial blood vessels, and subsequently decreased with depth as the laser beam was attenuated. However, more energy was deposited in the epidermis and superficial blood vessels at 532 nm than at 585 nm.

  16. DETECTION OF TISSUE MYCOBACTERIUM TUBERCULOSIS BY DIFFERENTIATING IMMUNOPEROXIDASE STAINING

    Directory of Open Access Journals (Sweden)

    A. P. Lysenko

    2014-01-01

    Full Text Available Staining impression smears from organ and tissues with peroxidase conjugated antibodies to Mycobacterium tuberculosis complex antigens, followed by visualization with diaminobenzidine and Kinyoun stains, ensured the painting of acid-resistant Mycobacterium tuberculosis forms to rubin red, acid-susceptible ones to brown, and tissue cells and microorganisms of other species to blue. Typical bacilli were absent in the lymph nodes of patients and animals with latent infection, but acid-resistant (rubin-red granular forms were encountered in the granulomatous masses. Brown fat cells containing mycobacterial antigens, as well as acid-susceptible granular, reticular, fungoid, and rod-like forms were also found in considerable quantities.

  17. Evaluation Method of Accumulated Cooking Oil Stains in Room

    OpenAIRE

    五十嵐, 由利子; 中村, 和吉; 萬羽, 郁子; Igarashi, Yuriko; Nakamura, Kazuyoshi; Banba, Ikuko

    2005-01-01

    The diffusion of the oil mist while cooking affects the entire room, leaving stains on the ceiling and walls. The validity of measuring the color difference of stains was examined by installing teflon plates in the kitchen and the adjacent space with a view to assessing the oil diffusion. Four houses were designated for the examination. In each house, four teflon plates (20×40mm) were installed on the ceiling and walls of the kitchen and the space adjacent to it. Then, one plate was removed a...

  18. One-day detection of PCR amplified Chlamydia trachomatis DNA in clinical samples: ELISA versus Southern blot hybridisation.

    OpenAIRE

    Roymans, R T; Onland, G; Postma, B H

    1996-01-01

    AIMS: To compare ELISA and Southern blot hybridisation for the detection of PCR amplified Chlamydia trachomatis DNA extracted from clinical samples; to assess the value of the ELISA method in a clinical setting. METHODS: DNA was extracted from urogenital samples of 508 patients, purified and amplified using C trachomatis specific primers, one of which was endlabelled with biotin. Amplification products were detected by Streptavidin biotin based ELISA and non-radioactive Southern blotting. RES...

  19. EFFECTS OF THE GRAM STAIN ON MICROSPHERES FROM THERMAL POLYAMINO ACIDS1

    Science.gov (United States)

    Fox, Sidney W.; Yuyama, Shuhei

    1963-01-01

    Fox, Sidney W. (The Florida State University, Tallahassee) and Shuhei Yuyama. Effects of the Gram stain on microspheres from thermal polyamino acids. J. Bacteriol. 85:279–283. 1963.—Microspheres produced from acid proteinoid accept the Gram stain. The stain is negative, but microspheres produced from mixtures containing a sufficient proportion of lysine proteinoid stain positive. Microspheres produced from mixtures containing the appropriate proportions contain individuals which stain positive and others which stain negative. Images PMID:13959050

  20. Comparative assessment of seller's staining test (SST) and direct ...

    African Journals Online (AJOL)

    Conclusion:The relatively low sensitivity of the SST observed in this study calls for its replacement with the dFAT for accurate ... seller's staining test (SST) and direct fluorescent antibody test for rapid and accurate laboratory diagnosis of rabies. Afri Health Sci. 2016 ... lack of laboratory equipment and reliable reagents16,17.

  1. Interlaboratory variability of Ki67 staining in breast cancer.

    Science.gov (United States)

    Focke, Cornelia M; Bürger, Horst; van Diest, Paul J; Finsterbusch, Kai; Gläser, Doreen; Korsching, Eberhard; Decker, Thomas

    2017-10-01

    Postanalytic issues of Ki67 assessment in breast cancers like counting method standardisation and interrater bias have been subject of various studies, but little is known about analytic variability of Ki67 staining between pathology labs. Our aim was to study interlaboratory variability of Ki67 staining in breast cancer using tissue microarrays (TMAs) and central assessment to minimise preanalytic and postanalytic influences. Thirty European pathology labs stained serial slides of a TMA set of breast cancer tissues with Ki67 according to their routine in-house protocol. The Ki67-labelling index (Ki67-LI) of 70 matched samples was centrally assessed by one observer who counted all cancer cells per sample. We then tested for differences between the labs in Ki67-LI medians by analysing variance on ranks and in proportions of tumours classified as luminal A after dichotomising oestrogen receptor-positive cancers into cancers showing low (Ki67-LI using Cochran's Q. Substantial differences between the 30 labs were indicated for median Ki67-LI (0.65%-33.0%, p cancers classified as luminal A (17%-57%, p Ki67 staining of breast cancer tissue was found between 30 routine pathology labs. Clinical use of the Ki67-LI for therapeutic decisions should be considered only fully aware of lab-specific reference values. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Comparative assessment of seller's staining test (SST) and direct ...

    African Journals Online (AJOL)

    Background: Rabies causes 55, 000 annual human deaths globally and about 10,000 people are exposed annually in Nigeria. Diagnosis of animal rabies in most African countries has been by direct microscopic examination. In Nigeria, the Seller's stain test (SST) was employed until 2009. Before then, both SST and dFAT ...

  3. The gram stain smear: A screening test for genital mycoplasmas ...

    African Journals Online (AJOL)

    Result of 168 vaginal specimens from women examined for genital mycoplasmas showed that more of these organisms were isolated from specimens whose Gram stain smears were devoid of Gram positive bacilli (GPB) (43%) as against those whose smears contain GPB (22.1%). This result was found to be statistically ...

  4. Gram's Stain Does Not Cross the Bacterial Cytoplasmic Membrane.

    Science.gov (United States)

    Wilhelm, Michael J; Sheffield, Joel B; Sharifian Gh, Mohammad; Wu, Yajing; Spahr, Christian; Gonella, Grazia; Xu, Bolei; Dai, Hai-Lung

    2015-07-17

    For well over a century, Hans Christian Gram's famous staining protocol has been the standard go-to diagnostic for characterizing unknown bacteria. Despite continuous and ubiquitous use, we now demonstrate that the current understanding of the molecular mechanism for this differential stain is largely incorrect. Using the fully complementary time-resolved methods: second-harmonic light-scattering and bright-field transmission microscopy, we present a real-time and membrane specific quantitative characterization of the bacterial uptake of crystal-violet (CV), the dye used in Gram's protocol. Our observations contradict the currently accepted mechanism which depicts that, for both Gram-negative and Gram-positive bacteria, CV readily traverses the peptidoglycan mesh (PM) and cytoplasmic membrane (CM) before equilibrating within the cytosol. We find that not only is CV unable to traverse the CM but, on the time-scale of the Gram-stain procedure, CV is kinetically trapped within the PM. Our results indicate that CV, rather than dyes which rapidly traverse the PM, is uniquely suited as the Gram stain.

  5. A comparision of modified and standard papanicolaou staining ...

    African Journals Online (AJOL)

    Objective: To compare modified and standard Papanicolaou (Pap) staining methods in the assessment of the cervical smears. Design: A descriptive cross sectional study. setting: Kenyatta National Hospital. Subjects: One hundred and sixty two women who were eligible for a pap smear and met the inclusion criteria.

  6. News from the Biological Stain Commission No. 14

    DEFF Research Database (Denmark)

    Lyon, Hans O

    2013-01-01

    In the 14(th) issue of News from the Biological Stain Commission (BSC) the BSC's International Affairs Committee presents information from the meetings of ISO/TC 212/WG 3, In vitro diagnostic products, and from the final plenary meeting of ISO/TC 212, Clinical laboratory testing and in vitro...

  7. News from the Biological Stain Commission No. 5

    DEFF Research Database (Denmark)

    Lyon, H O; Dapson, R W

    2009-01-01

    In this fifth issue of News from the Biological Stain Commission (BSC), under the heading of Regulatory Affairs, the BSC's International Affairs Committee provides more information from the meeting of the International Standards Organization ISO/TC 212 Committee that took place on June 2-4, 2008...

  8. Comparison of Giemsa Staining, Intraperitoneal Injection and Oral

    Directory of Open Access Journals (Sweden)

    Sajad Rashidi

    2014-02-01

    Full Text Available Background: Toxoplasma gondii is one of the most common protozoan parasites in humans and animals in all countries of the world. The aim of this study was to detect Toxoplasma parasite in the brain of wild rats in Tehran using smear preparation, Giemsa staining, Intraperitoneal injection and oral administration to Souri mice. Materials and Methods: Forty rats were collected from different areas of Tehran. Smears were prepared from rat brains on glass slides and stained using Giemsa. In the second method, a cell suspension was prepared from rat brain and was given orally and injected intraperitoneally into Souri mice. In peritoneal method, peritoneum of the mice was examined for parasites. In oral method, the titer of Toxoplasma antibody in sera of Souri mice was determined using Toxoplasma IgG antibody kit and anti-mouse conjugate of Sigma company. Results: All results were negative in Giemsa staining method. In the second method, the results were negative and no parasites were observed in peritoneum of Souri mice. In oral administration method, after ingestion of suspensions by Souri mice and measuring the IgG titer, 50% of them showed a positive titer after one month. Conclusion: In detection of Toxoplasma gondii, the method of smear preparation on glass slides followed by Giemsa staining, and intraperitoneal injection of brain suspensions to Souri mice are of less value in comparison with oral administration of suspensions and determining the titer of IgG in sera of Souri mice.

  9. a comparison of modified and standard papanicolaou staining ...

    African Journals Online (AJOL)

    2011-07-07

    Jul 7, 2011 ... Corporation, Hollywood, Florida Gynecologic. Seminars in Surgical Oncology 1999; 16: 217–221. Gupta, S., Chachra, K. L., Bhadola, P. 7. et al. Modified. Papanicolaou staining protocol with minimum alcohol use: a cost-cutting measure for resource limited. Division of Cytopathology, Institute of Cytology ...

  10. Microscopic analysis of MTT stained boar sperm cells

    African Journals Online (AJOL)

    tulyasys

    2015-06-08

    Jun 8, 2015 ... Microscopic analysis of MTT stained boar sperm cells. B.M. van den Berg*. Barex Biochemical Products, Seb. Centenweg 45, 1602ML Enkhuizen, The Netherlands. Abstract. The ability of sperm cells to develop colored formazan by reduction of MTT was used earlier to develop a spectrophotometric.

  11. Amalgam stained dentin: a proper substrate for bonding resin composite?

    NARCIS (Netherlands)

    Scholtanus, J.D.

    2016-01-01

    Nowadays the use of dental amalgam is mostly abandoned and substituted by tooth colored resin composites that can be bonded to teeth tissues by adhesive techniques. The aim of this thesis was to find out whether dark stained dentin, as often observed after removal of amalgam restorations and

  12. The Stained Glass Paintings of Nigeria's Prime Artists, YCA Grillo

    African Journals Online (AJOL)

    Nneka Umera-Okeke

    Abstract. Many lamps same Light' investigates the place of agency in the transmutation of indigenous imageries in the art works of the pictorial turn. Through an investigation that entailed an empirical analysis of the works of two Nigerian prime stained glass artists, Yusuf Grillo and David Dale, this study established that in ...

  13. Borax methylene blue: a spectroscopic and staining study.

    Science.gov (United States)

    Donaldson, P T; Russo, A; Reynolds, C; Lillie, R D

    1978-07-01

    Borax methylene blue is quite stable at room temperatures of 22-25 C. At 30 C polychroming is slow; during 50 days in a water bath at this temperature the absorption peak moves from 665 to 656 nm. At 35 C, the absorption peak reaches 660 nm in 7 days, 654 nm in 14. At 60 C polychroming is rapid, the absorption peak reaching 640-620 nm in 3 days. When the pH of the borax methylene blue solutions, normally about 9.0, is adjusted to pH 6.5, the absorption peak remains at 665 nm even when incubated at 60 C for extended periods. When used as a blood stain 0.4 ml borax methylene blue (1% methylene blue in 1% borax), 4 ml acetone, 2 ml borax-acid phosphate buffer to bring the solution to pH 6.5, and distilled water to make 40 ml, with 0.2 ml 1% eosin added just before using, an excellent Nocht-Giemsa type stain is achieved after 30 minutes staining. The material plasmodia P. falciparum, P. vivax, and P. berghei stain moderate blue with dark red chromatin and green to black pigment granules. The study confirms Malachowski's 1891 results and explains Gautier's 1896-98 failure to duplicate it.

  14. Christendom's Narratives and the Stained Glass Designs of Yusuf ...

    African Journals Online (AJOL)

    This paper attempts a recast of Christendom's narratives in the stained glass designs of Yusuf Cameron Adebayo Grillo as the distinctive overarching mechanism of the evangelisation paradigm of the post Vatican II Church. It, therefore, draws attention to the delimitation of time frames in the history of the art form. Using the ...

  15. A generally applicable sequential alkaline phosphatase immunohistochemical double staining

    NARCIS (Netherlands)

    van der Loos, Chris M.; Teeling, Peter

    2008-01-01

    A universal type of sequential double alkaline phosphatase immunohistochemical staining is described that can be used for formalin-fixed, paraffin-embedded and cryostat tissue sections from human and mouse origin. It consists of two alkaline phosphatase detection systems including enzymatic

  16. Color and dichroism of silver-stained glasses

    International Nuclear Information System (INIS)

    Molina, Gloria; Murcia, Sonia; Molera, Judit; Roldan, Clodoaldo; Crespo, Daniel; Pradell, Trinitat

    2013-01-01

    Yellow decorations in glasses have been produced since the beginning of the fourteenth century by incorporating metallic silver nanoparticles into the glass (from a few to some tens of nanometers). The optical response of the glass-particles composite is determined by the surface plasmon resonance absorption and scattering of the nanometric metallic particles. Generally, the same color is perceived in reflection and in transmission although dichroic effects are occasionally observed. As silver-stained glasses were designed to be observed in transmission, tuning the transmission color from yellow to red was of technological interest. The relationship between the color observed both in transmission and reflection and the composition and nanostructure of regular (yellow) and dichroic (yellow and red) silver stains from the Renaissance (late fifteenth and sixteenth century, respectively) is related to the presence of a layer (of about 10–20 μm thick) of metallic silver nanoparticles (from few to 100 nm in size). The correlation between the colors observed and the silver stain nanostructure is studied with particular emphasis on the origin of the dichroic behavior. The optical response is computed and compared to the experimental data. Differences in the synthesis parameters responsible for the colors and for the dichroic behavior of the silver stain glasses are proposed. This is essential for the replication of the glass pieces which are required as replacements in the restoration/conservation of the windows but is also of broader interest

  17. [Pnemocystis jiroveci pneumonia: Comparison between conventional PCR and staining techniques].

    Science.gov (United States)

    Kaouech, E; Kallel, K; Anane, S; Belhadj, S; Abdellatif, S; Mnif, K; Ben Othmane, T; Ben Lakhal, S; Kilani, B; Ben Châabane, T; Chaker, E

    2009-07-01

    Diagnosis of pneumocystis pneumonia is usually based on clinical features and X-rays photography and confirmed in the laboratory by visualisation of Pneumocystis organisms in stained preparations of respiratory specimens using several techniques (Gomori-Grocott, May-Grünwald Giemsa, bleu de toluidine O). Actually, PCR has considerably increased sensitivity of detection of Pneumocystis. The aim of this study is to compare conventional PCR results to those of staining techniques (Gomori-Grocott, May-Grünwald Giemsa) in addition to the X-ray and clinical findings in order to evaluate the contribution of each method. Sixty-four respiratory specimens were collected from 54 immuno-compromised patients with clinical symptoms of pulmonary infection. We diagnosed pneumocystis pneumonia in 16 patients according to staining techniques and/or typical clinical and radiological findings and/or response to treatment. Of the 15 patients, 14 were positive by PCR and only five were positive by direct examination, yielding a sensitivity and specificity of 93.3 and 87.1% for PCR and 33.3 and 100% for staining techniques. Conventional PCR provides a sensitive and objective method for the detection Pneumocystis jiroveci from less invasive sample.

  18. Black stain and dental caries in Filipino schoolchildren.

    NARCIS (Netherlands)

    Heinrich-Weltzien, R.; Monse, B.; Palenstein Helderman, W.H. van

    2009-01-01

    Black stain is defined as dark pigmented exogenous substance in lines or dots parallel to the gingival margin and firmly adherent to the enamel at the cervical third of the tooth crowns in the primary and permanent dentition. OBJECTIVES: This study was conducted to assess the prevalence of black

  19. Meconium-stained amniotic fluid – what is the evidence ...

    African Journals Online (AJOL)

    Opinions regarding the significance of meconium-stained liquor detected during labour have varied although there is consensus that meconium aspirated into the lungs of the neonate may lead to meconium aspiration syndrome. The efficacy of various interventions designed to prevent meconium aspiration syndrome are ...

  20. Image analysis of dye stained patterns in soils

    Science.gov (United States)

    Bogner, Christina; Trancón y Widemann, Baltasar; Lange, Holger

    2013-04-01

    Quality of surface water and groundwater is directly affected by flow processes in the unsaturated zone. In general, it is difficult to measure or model water flow. Indeed, parametrization of hydrological models is problematic and often no unique solution exists. To visualise flow patterns in soils directly dye tracer studies can be done. These experiments provide images of stained soil profiles and their evaluation demands knowledge in hydrology as well as in image analysis and statistics. First, these photographs are converted to binary images classifying the pixels in dye stained and non-stained ones. Then, some feature extraction is necessary to discern relevant hydrological information. In our study we propose to use several index functions to extract different (ideally complementary) features. We associate each image row with a feature vector (i.e. a certain number of image function values) and use these features to cluster the image rows to identify similar image areas. Because images of stained profiles might have different reasonable clusterings, we calculate multiple consensus clusterings. An expert can explore these different solutions and base his/her interpretation of predominant flow mechanisms on quantitative (objective) criteria. The complete workflow from reading-in binary images to final clusterings has been implemented in the free R system, a language and environment for statistical computing. The calculation of image indices is part of our own package Indigo, manipulation of binary images, clustering and visualization of results are done using either build-in facilities in R, additional R packages or the LATEX system.