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Sample records for gel electrophoresis matrix-assisted

  1. Cu2+-assisted two dimensional charge-mass double focusing gel electrophoresis and mass spectrometric analysis of histone variants.

    Science.gov (United States)

    Zhang, Wenyang; Tang, Xuemei; Ding, Mengjie; Zhong, Hongying

    2014-12-10

    Abundant isoforms and dynamic posttranslational modifications cause the separation and identification of histone variants to be experimentally challenging. To meet this need, we employ two-dimensional electrophoretic gel separation followed by mass spectrometric detection which takes advantage of the chelation of Cu(2+) with amino acid residues exposed on the surfaces of the histone proteins. Acid-extracted rat liver histones were first mixed with CuSO4 solution and then separated in one dimension with triton-acid-urea (TAU) gel electrophoresis and in a second dimension using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separations result from both the changes in charge and mass upon Cu(2+) chelation. Identities of each separated gel bands were obtained by using matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). It was found that the migration of H3 histone isoforms of rat liver is markedly affected by the use of Cu(2+) ions. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Supramolecular gel electrophoresis of large DNA fragments.

    Science.gov (United States)

    Tazawa, Shohei; Kobayashi, Kazuhiro; Oyoshi, Takanori; Yamanaka, Masamichi

    2017-10-01

    Pulsed-field gel electrophoresis is a frequent technique used to separate exceptionally large DNA fragments. In a typical continuous field electrophoresis, it is challenging to separate DNA fragments larger than 20 kbp because they migrate at a comparable rate. To overcome this challenge, it is necessary to develop a novel matrix for the electrophoresis. Here, we describe the electrophoresis of large DNA fragments up to 166 kbp using a supramolecular gel matrix and a typical continuous field electrophoresis system. C 3 -symmetric tris-urea self-assembled into a supramolecular hydrogel in tris-boric acid-EDTA buffer, a typical buffer for DNA electrophoresis, and the supramolecular hydrogel was used as a matrix for electrophoresis to separate large DNA fragments. Three types of DNA marker, the λ-Hind III digest (2 to 23 kbp), Lambda DNA-Mono Cut Mix (10 to 49 kbp), and Marker 7 GT (10 to 165 kbp), were analyzed in this study. Large DNA fragments of greater than 100 kbp showed distinct mobility using a typical continuous field electrophoresis system. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Sodium dodecyl sulfate-capillary gel electrophoresis of polyethylene glycolylated interferon alpha.

    Science.gov (United States)

    Na, Dong H; Park, Eun J; Youn, Yu S; Moon, Byung W; Jo, Yeong W; Lee, Sung H; Kim, Won-Bae; Sohn, Yeowon; Lee, Kang C

    2004-02-01

    Sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) using a hydrophilic replaceable polymer network matrix was applied to characterize the polyethylene glycol(PEG)ylated interferon alpha (PEG-IFN). The SDS-CGE method resulted in a clearer resolution in both the PEG-IFN species and the native IFN species. The distribution profile of PEGylation determined by SDS-CGE was consistent with that obtained by SDS-polyacrylamide gel electrophoresis (PAGE) with Coomassie blue or barium iodide staining. The result was also compared using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry. SDS-CGE was also useful for monitoring the PEGylation reaction to optimize the reaction conditions, such as reaction molar ratio. This study shows the potential of SDS-CGE as a new method for characterizing the PEGylated proteins with advantages of speed, minimal sample consumption and high resolution.

  4. Carbon nanotubes-assisted polyacrylamide gel electrophoresis for enhanced separation of human serum proteins and application in liverish diagnosis.

    Science.gov (United States)

    Jiang, Fubin; Wang, Yanan; Hu, Xinfang; Shao, Na; Na, Na; Delanghe, Joris R; Ouyang, Jin

    2010-11-01

    The application of pore-gradient polyacrylamide gel electrophoresis (PG-PAGE) incorporated with carbon nanotube modified by Triton X-100 and carboxylation so as to improve the separation of human serum proteins is reported. The novel PG-PAGE was made by adding water-soluble single-walled carbon nanotubes (CNTs) when preparing the polyacrylamide gel. Significant improvements in separation of complement C3 protein and haptoglobin (Hp) in human serum were achieved. It was estimated that the interactions between the hydrophilic groups on the proteins and the surface of the CNTs result in different adsorption kinetics of complement C3 and Hp subtype on the nanoparticles incorporated in the gel, thus enhancing the separation of the two proteins in serum. This new CNT matrix-assisted PG-PAGE method for enhanced separation of complement C3 and Hp in human serum was successfully applied to distinguish the samples from liverish patients and healthy people.

  5. Agarose gel electrophoresis for the separation of DNA fragments.

    Science.gov (United States)

    Lee, Pei Yun; Costumbrado, John; Hsu, Chih-Yuan; Kim, Yong Hoon

    2012-04-20

    Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb(1). Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits(2). During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel's molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight(3). The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along(4). The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation(5); 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: Understand the mechanism by which DNA fragments are separated within a gel matrix Understand how conformation of the DNA molecule will determine its mobility through a gel matrix Identify an agarose solution of appropriate

  6. Evaluation of wheat by polyacrylamide gel electrophoresis | Shuaib ...

    African Journals Online (AJOL)

    ... polyacrylamide gel electrophoresis (SDS-PAGE). Electrophorogram for each variety were scored and presence or absence of each band noted and was entered in a binary data matrix. Based on the data of SDS-PAGE gels cluster analysis was performed to check the variations among varieties. The overall result shows ...

  7. Decision peptide-driven: a free software tool for accurate protein quantification using gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry.

    Science.gov (United States)

    Santos, Hugo M; Reboiro-Jato, Miguel; Glez-Peña, Daniel; Nunes-Miranda, J D; Fdez-Riverola, Florentino; Carvallo, R; Capelo, J L

    2010-09-15

    The decision peptide-driven tool implements a software application for assisting the user in a protocol for accurate protein quantification based on the following steps: (1) protein separation through gel electrophoresis; (2) in-gel protein digestion; (3) direct and inverse (18)O-labeling and (4) matrix assisted laser desorption ionization time of flight mass spectrometry, MALDI analysis. The DPD software compares the MALDI results of the direct and inverse (18)O-labeling experiments and quickly identifies those peptides with paralleled loses in different sets of a typical proteomic workflow. Those peptides are used for subsequent accurate protein quantification. The interpretation of the MALDI data from direct and inverse labeling experiments is time-consuming requiring a significant amount of time to do all comparisons manually. The DPD software shortens and simplifies the searching of the peptides that must be used for quantification from a week to just some minutes. To do so, it takes as input several MALDI spectra and aids the researcher in an automatic mode (i) to compare data from direct and inverse (18)O-labeling experiments, calculating the corresponding ratios to determine those peptides with paralleled losses throughout different sets of experiments; and (ii) allow to use those peptides as internal standards for subsequent accurate protein quantification using (18)O-labeling. In this work the DPD software is presented and explained with the quantification of protein carbonic anhydrase. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  8. 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)

  9. Matrix-assisted laser desorption/ionization time of flight mass spectrometry peptide mass fingerprints and post source decay: a tool for the identification and analysis of phloem proteins from Cucurbita maxima Duch. separated by two-dimensional polyacrylamide gel electrophoresis.

    Science.gov (United States)

    Haebel, S; Kehr, J

    2001-08-01

    A combination of gel electrophoresis and mass spectrometry was used to analyze the soluble proteins from phloem sap of Cucurbita maxima Duch. Phloem proteins were separated using two-dimensional gel electrophoresis. Coomassie-stained spots were cut out and subjected to tryptic digestion. To identify proteins, peptide mass fingerprints were determined by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. In addition, MALDI-TOF post source decay measurements were used to obtain partial sequence information for the proteins. Results from both approaches were used for database searches. In this study, 17 proteins in the mass range 5-50 kDa were analyzed. Of these proteins six could be clearly identified, seven showed significant homologies to known plant proteins, and four were not significantly homologous to database entries. The present study suggests that the applied method is feasible for a large-scale analysis and identification of phloem proteins derived from different organs or from plants kept under various physiological conditions.

  10. A simple gel electrophoresis method for separating polyhedral gold nanoparticles

    Science.gov (United States)

    Kim, Suhee; Lee, Hye Jin

    2015-07-01

    In this paper, a simple approach to separate differently shaped and sized polyhedral gold nanoparticles (NPs) within colloidal solutions via gel electrophoresis is described. Gel running parameters for separating efficiently gold NPs including gel composition, added surfactant types and applied voltage were investigated. The plasmonic properties and physical structure of the separated NPs extracted from the gel matrix were then investigated using transmission electron microscopy (TEM) and UV-vis spectrophotometry respectively. Data analysis revealed that gel electrophoresis conditions of a 1.5 % agarose gel with 0.1 % sodium dodecyl sulfate (SDS) surfactant under an applied voltage of 100 V resulted in the selective isolation of ~ 50 nm polyhedral shaped gold nanoparticles. Further efforts are underway to apply the method to purify biomolecule-conjugated polyhedral Au NPs that can be readily used for NP-enhanced biosensing platforms.

  11. Gel electrophoresis of inorganic cations

    International Nuclear Information System (INIS)

    Schoenhofer, F.; Grass, F.

    1978-01-01

    In order to be able to separate the largest possible amounts of substance, polyacryl amide gel (PAA) and silica gel are used as carrier for the electrophoresis. Milligramme quantities can easily be separated on PAA gel plates. Electrophoretic ion focussing considerably improves it. Separations of Sr/Y and lanthanoids were carried out. The behaviour of the readily soluble complexing agent acids on silica gel thin layers was minutely investigated and an interpretation of the focussing effect was derived. The conditions for separating radionuclides were optimized. A further improved separation can be achieved by a time sequence combination of normal electrophoresis and ion focussing. Selective isolation methods are advantageous to determine radionuclide traces in environmental samples. The selective adsorption on preformed deposits was transferred to electrophoresis. After pre-investigations on silica gel layers, strontium and barium could also be retained on PAA gel and radium on strontium sulphate in PAA, whereas the disturbing calcium can easily pass through. Cesium can also be retained by prussian blue in the electrophoresis. (orig.) [de

  12. Conducting polymer electrodes for gel electrophoresis.

    Directory of Open Access Journals (Sweden)

    Katarina Bengtsson

    Full Text Available In nearly all cases, electrophoresis in gels is driven via the electrolysis of water at the electrodes, where the process consumes water and produces electrochemical by-products. We have previously demonstrated that π-conjugated polymers such as poly(3,4-ethylenedioxythiophene (PEDOT can be placed between traditional metal electrodes and an electrolyte to mitigate electrolysis in liquid (capillary electroosmosis/electrophoresis systems. In this report, we extend our previous result to gel electrophoresis, and show that electrodes containing PEDOT can be used with a commercial polyacrylamide gel electrophoresis system with minimal impact to the resulting gel image or the ionic transport measured during a separation.

  13. Conducting polymer electrodes for gel electrophoresis.

    Science.gov (United States)

    Bengtsson, Katarina; Nilsson, Sara; Robinson, Nathaniel D

    2014-01-01

    In nearly all cases, electrophoresis in gels is driven via the electrolysis of water at the electrodes, where the process consumes water and produces electrochemical by-products. We have previously demonstrated that π-conjugated polymers such as poly(3,4-ethylenedioxythiophene) (PEDOT) can be placed between traditional metal electrodes and an electrolyte to mitigate electrolysis in liquid (capillary electroosmosis/electrophoresis) systems. In this report, we extend our previous result to gel electrophoresis, and show that electrodes containing PEDOT can be used with a commercial polyacrylamide gel electrophoresis system with minimal impact to the resulting gel image or the ionic transport measured during a separation.

  14. Agarose gel electrophoresis and polyacrylamide gel electrophoresis for visualization of simple sequence repeats.

    Science.gov (United States)

    Anderson, James; Wright, Drew; Meksem, Khalid

    2013-01-01

    In the modern age of genetic research there is a constant search for ways to improve the efficiency of plant selection. The most recent technology that can result in a highly efficient means of selection and still be done at a low cost is through plant selection directed by simple sequence repeats (SSRs or microsatellites). The molecular markers are used to select for certain desirable plant traits without relying on ambiguous phenotypic data. The best way to detect these is the use of gel electrophoresis. Gel electrophoresis is a common technique in laboratory settings which is used to separate deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) by size. Loading DNA and RNA onto gels allows for visualization of the size of fragments through the separation of DNA and RNA fragments. This is achieved through the use of the charge in the particles. As the fragments separate, they form into distinct bands at set sizes. We describe the ability to visualize SSRs on slab gels of agarose and polyacrylamide gel electrophoresis.

  15. Comparison of non-electrophoresis grade with electrophoresis grade BIS in NIPAM polymer gel preparation

    Science.gov (United States)

    Khodadadi, Roghayeh; Khajeali, Azim; Farajollahi, Ali Reza; Hajalioghli, Parisa; Raeisi, Noorallah

    2015-01-01

    Introduction:The main objective of this study was to investigate the possibility of replacing electrophoresis cross-linker with non-electrophoresis N, N′-methylenebisacrylamide (BIS) in N-isopropyl acrylamide (NIPAM) polymer gel and its possible effect on dose response. Methods: NIPAM polymer gel was prepared from non-electrophoresis grade BIS and the relaxation rate (R2) was measured by MR imaging after exposing the gel to gamma radiation from Co-60 source. To compare the response of this gel with the one that contains electrophoresis grade BIS, two sets of NIPAM gel were prepared using electrophoresis and non-electrophoresis BIS and irradiated to different gamma doses. Results: It was found that the dose–response of NIPAM gel made from the non-electrophoresis grade BIS is coincident with that of electrophoresis grade BIS. Conclusion:Taken all, it can be concluded that the non-electrophoresis grade BIS not only is a suitable alternative for the electrophoresis grade BIS but also reduces the cost of gel due to its lower price. PMID:26457250

  16. The gel electrophoresis markup language (GelML) from the Proteomics Standards Initiative.

    Science.gov (United States)

    Gibson, Frank; Hoogland, Christine; Martinez-Bartolomé, Salvador; Medina-Aunon, J Alberto; Albar, Juan Pablo; Babnigg, Gyorgy; Wipat, Anil; Hermjakob, Henning; Almeida, Jonas S; Stanislaus, Romesh; Paton, Norman W; Jones, Andrew R

    2010-09-01

    The Human Proteome Organisation's Proteomics Standards Initiative has developed the GelML (gel electrophoresis markup language) data exchange format for representing gel electrophoresis experiments performed in proteomics investigations. The format closely follows the reporting guidelines for gel electrophoresis, which are part of the Minimum Information About a Proteomics Experiment (MIAPE) set of modules. GelML supports the capture of metadata (such as experimental protocols) and data (such as gel images) resulting from gel electrophoresis so that laboratories can be compliant with the MIAPE Gel Electrophoresis guidelines, while allowing such data sets to be exchanged or downloaded from public repositories. The format is sufficiently flexible to capture data from a broad range of experimental processes, and complements other PSI formats for MS data and the results of protein and peptide identifications to capture entire gel-based proteome workflows. GelML has resulted from the open standardisation process of PSI consisting of both public consultation and anonymous review of the specifications.

  17. Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution

    Science.gov (United States)

    Stellwagen, Nancy C.

    2009-01-01

    This review describes the electrophoresis of curved and normal DNA molecules in agarose gels, polyacrylamide gels and in free solution. These studies were undertaken to clarify why curved DNA molecules migrate anomalously slowly in polyacrylamide gels but not in agarose gels. Two milestone papers are cited, in which Ferguson plots were used to estimate the effective pore size of agarose and polyacrylamide gels. Subsequent studies on the effect of the electric field on agarose and polyacrylamide gel matrices, DNA interactions with the two gel matrices, and the effect of curvature on the free solution mobility of DNA are also described. The combined results suggest that the anomalously slow mobilities observed for curved DNA molecules in polyacrylamide gels are due primarily to preferential interactions of curved DNAs with the polyacrylamide gel matrix; the restrictive pore size of the matrix is of lesser importance. In free solution, DNA mobilities increase with increasing molecular mass until leveling off at a plateau value of (3.17 ± 0.01) × 10-4 cm2/Vs in 40 mM Tris-acetate-EDTA buffer at 20°C. Curved DNA molecules migrate anomalously slowly in free solution as well as in polyacrylamide gels, explaining why the Ferguson plots of curved and normal DNAs containing the same number of base pairs extrapolate to different mobilities at zero gel concentration. PMID:19517510

  18. Disc electrophoresis and related techniques of polyacrylamide gel electrophoresis

    National Research Council Canada - National Science Library

    Maurer, H. R

    1971-01-01

    ..., enzymes, antingens and radioactively labelled materials, and detailed treatments of micro disc electrophoresis, preparative polyacrylamide gel electrophoresis and many other techniques for special problems...

  19. Pulsed-field gel electrophoresis of bacterial chromosomes.

    Science.gov (United States)

    Mawer, Julia S P; Leach, David R F

    2013-01-01

    The separation of fragments of DNA by agarose gel electrophoresis is integral to laboratory life. Nevertheless, standard agarose gel electrophoresis cannot resolve fragments bigger than 50 kb. Pulsed-field gel electrophoresis is a technique that has been developed to overcome the limitations of standard agarose gel electrophoresis. Entire linear eukaryotic chromosomes, or large fragments of a chromosome that have been generated by the action of rare-cutting restriction endonucleases, can be separated using this technique. As a result, pulsed-field gel electrophoresis has many applications, from karyotype analysis of microbial genomes, to the analysis of chromosomal strand breaks and their repair intermediates, to the study of DNA replication and the identification of origins of replication. This chapter presents a detailed protocol for the preparation of Escherichia coli chromosomal DNA that has been embedded in agarose plugs, digested with the rare-cutting endonuclease NotI, and separated by contour-clamped homogeneous field electrophoresis. The principles in this protocol can be applied to the separation of all fragments of DNA whose size range is between 40 kb and 1 Mb.

  20. DNA gel electrophoresis: the reptation model(s).

    Science.gov (United States)

    Slater, Gary W

    2009-06-01

    DNA gel electrophoresis has been the most important experimental tool to separate DNA fragments for several decades. The introduction of PFGE in the 1980s and capillary gel electrophoresis in the 1990s made it possible to study, map and sequence entire genomes. Explaining how very large DNA molecules move in a gel and why PFGE is needed to separate them has been an active field of research ever since the launch of the journal Electrophoresis. This article presents a personal and historical overview of the development of the theory of gel electrophoresis, focusing on the reptation model, the band broadening mechanisms, and finally the factors that limit the read length and the resolution of electrophoresis-based sequencing systems. I conclude with a short discussion of some of the questions that remain unanswered.

  1. Improved gel electrophoresis matrix for hydrophobic protein separation and identification.

    Science.gov (United States)

    Tokarski, Caroline; Fillet, Marianne; Rolando, Christian

    2011-03-01

    We propose an improved acrylamide gel for the separation of hydrophobic proteins. The separation strategy is based on the incorporation of N-alkylated and N,N'-dialkylated acrylamide monomers in the gel composition in order to increase hydrophobic interactions between the gel matrix and the membrane proteins. Focusing on the most efficient monomer, N,N'-dimethylacrylamide, the potentiality of the new matrix was evaluated on membrane proteins of the human colon HCT-116 cell line. Protein analysis was performed using an adapted analytical strategy based on FT-ICR tandem mass spectrometry. As a result of this comparative study, including advanced reproducibility experiments, more hydrophobic proteins were identified in the new gel (average GRAVY: -0.085) than in the classical gel (average GRAVY: -0.411). Highly hydrophobic peptides were identified reaching a GRAVY value up to 1.450, therefore indicating their probable locations in the membrane. Focusing on predicted transmembrane domains, it can be pointed out that 27 proteins were identified in the hydrophobic gel containing up to 11 transmembrane domains; in the classical gel, only 5 proteins containing 1 transmembrane domain were successfully identified. For example, multiple ionic channels and receptors were characterized in the hydrophobic gel such as the sodium/potassium channel and the glutamate or the transferrin receptors whereas they are traditionally detected using specific enrichment techniques such as immunoprecipitation. In total, membrane proteins identified in the classical gel are well documented in the literature, while most of the membrane proteins only identified on the hydrophobic gel have rarely or never been described using a proteomic-based approach. 2010 Elsevier Inc. All rights reserved.

  2. Conducting Polymer Electrodes for Gel Electrophoresis

    OpenAIRE

    Bengtsson, Katarina; Nilsson, Sara; Robinson, Nathaniel D

    2014-01-01

    In nearly all cases, electrophoresis in gels is driven via the electrolysis of water at the electrodes, where the process consumes water and produces electrochemical by-products. We have previously demonstrated that p-conjugated polymers such as poly(3,4-ethylenedioxythiophene) (PEDOT) can be placed between traditional metal electrodes and an electrolyte to mitigate electrolysis in liquid (capillary electroosmosis/electrophoresis) systems. In this report, we extend our previous result to gel ...

  3. Stacking gels: A method for maximising output for pulsed-field gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Heng See

    2009-01-01

    Full Text Available Pulsed field gel electrophoresis (PFGE, the gold standard of molecular typing methods, has a major disadvantage of an unusually long electrophoretic time. From the original protocol of 6 days, it was modified to 3 days and subsequently to a single day. We describe the procedure of stacking five to six gels one on top of another in order to increase and maximize the output in a shorter time without compromising the resolution and reproducibility. All the variables that affect pulsed field gels during electrophoresis were taken into consideration. We firstly optimized the parameters to be used and secondly determined whether stacking of five to six gels had any effect on the molecular separation during electrophoresis in comparison with a single gel run. DNA preparation, restriction, electrophoresis, staining and gel documentation was carried out based on previously published methods. Gels were analysed using BioNumerics and dice coefficient and unweighted pair group methods were used to generate dendrograms based on 1.5% tolerance values. Identical band profiles and band resolution-separation were seen in the PFGE patterns with single gel and multiple stacking gels. Cluster analysis further strengthened the fact that results from stacking gels were reproducible and comparable with a single gel run. This method of stacking gels saves time and maximizes the output at the same time. The run time for a single gel was about 28 hours, but with six stacked gels the run time was 54 hours compared with 28 x 6 = 168 hours if they were run separately as single gels thus saving time of 67.86%. Beside the big factor of saving time, stacking gels save resources (electricity, reagents, water, chemicals and working time by increasing the sample throughput in a shorter time without compromising on quality of data. But optimization of working parameters is vital depending on the PFGE system used.

  4. DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.

    Energy Technology Data Exchange (ETDEWEB)

    SUTHERLAND,B.M.; BENNETT,P.V.; SUTHERLAND, J.C.

    2004-03-24

    Physical and chemical agents in the environment, those used in clinical applications, or encountered during recreational exposures to sunlight, induce damages in DNA. Understanding the biological impact of these agents requires quantitation of the levels of such damages in laboratory test systems as well as in field or clinical samples. Alkaline gel electrophoresis provides a sensitive (down to {approx} a few lesions/5Mb), rapid method of direct quantitation of a wide variety of DNA damages in nanogram quantities of non-radioactive DNAs from laboratory, field, or clinical specimens, including higher plants and animals. This method stems from velocity sedimentation studies of DNA populations, and from the simple methods of agarose gel electrophoresis. Our laboratories have developed quantitative agarose gel methods, analytical descriptions of DNA migration during electrophoresis on agarose gels (1-6), and electronic imaging for accurate determinations of DNA mass (7-9). Although all these components improve sensitivity and throughput of large numbers of samples (7,8,10), a simple version using only standard molecular biology equipment allows routine analysis of DNA damages at moderate frequencies. We present here a description of the methods, as well as a brief description of the underlying principles, required for a simplified approach to quantitation of DNA damages by alkaline gel electrophoresis.

  5. Identification of proteins in a human pleural exudate using two-dimensional preparative liquid-phase electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry.

    Science.gov (United States)

    Nilsson, C L; Puchades, M; Westman, A; Blennow, K; Davidsson, P

    1999-01-01

    Pleural effusion may occur in patients suffering from physical trauma or systemic disorders such as infection, inflammation, or cancer. In order to investigate proteins in a pleural exudate from a patient with severe pneumonia, we used a strategy that combined preparative two-dimensional liquid-phase electrophoresis (2-D LPE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Western blotting. Preparative 2-D LPE is based on the same principles as analytical 2-D gel electrophoresis, except that the proteins remain in liquid phase during the entire procedure. In the first dimension, liquid-phase isoelectric focusing allows for the enrichment of proteins in liquid fractions. In the Rotofor cell, large volumes (up to 55 mL) and protein amounts (up to 1-2 g) can be loaded. Several low abundance proteins, cystatin C, haptoglobin, transthyretin, beta2-microglobulin, and transferrin, were detected after liquid-phase isoelectric focusing, through Western blotting analysis, in a pleural exudate (by definition, >25 g/L total protein). Direct MALDI-TOF-MS analysis of proteins in a Rotofor fraction is demonstrated as well. MALDI-TOF-MS analysis of a tryptic digest of a continuous elution sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fraction confirmed the presence of cystatin C. By applying 2-D LPE, MALDI-TOF-MS, and Western blotting to the analysis of this pleural exudate, we were able to confirm the identity of proteins of potential diagnostic value. Our findings serve to illustrate the usefulness of this combination of methods in the analysis of pathological fluids.

  6. Increase in local protein concentration by field-inversion gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Paulus Aran

    2007-09-01

    Full Text Available Abstract Background Proteins that migrate through cross-linked polyacrylamide gels (PAGs under the influence of a constant electric field experience negative factors, such as diffusion and non-specific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein separation efficiency. Enhancement of protein separation efficiency was investigated by implementing pulsed field-inversion gel electrophoresis (FIGE. Results Separation of model protein species and large protein complexes was compared between FIGE and constant field electrophoresis (CFE in different percentages of PAGs. Band intensities of proteins in FIGE with appropriate ratios of forward and backward pulse times were superior to CFE despite longer running times. These results revealed an increase in band intensity per defined gel volume. A biphasic protein relative mobility shift was observed in percentages of PAGs up to 14%. However, the effect of FIGE on protein separation was stochastic at higher PAG percentage. Rat liver lysates subjected to FIGE in the second-dimension separation of two-dimensional polyarcylamide gel electrophoresis (2D PAGE showed a 20% increase in the number of discernible spots compared with CFE. Nine common spots from both FIGE and CFE were selected for peptide sequencing by mass spectrometry (MS, which revealed higher final ion scores of all nine protein spots from FIGE. Native protein complexes ranging from 800 kDa to larger than 2000 kDa became apparent using FIGE compared with CFE. Conclusion The present investigation suggests that FIGE under appropriate conditions improves protein separation efficiency during PAGE as a result of increased local protein concentration. FIGE can be implemented with minimal additional instrumentation in any laboratory setting. Despite the tradeoff of longer running times, FIGE can be a powerful protein

  7. Increase in local protein concentration by field-inversion gel electrophoresis.

    Science.gov (United States)

    Tsai, Henghang; Low, Teck Yew; Freeby, Steve; Paulus, Aran; Ramnarayanan, Kalpana; Cheng, Chung-Pui Paul; Leung, Hon-Chiu Eastwood

    2007-09-26

    Proteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and non-specific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein separation efficiency. Enhancement of protein separation efficiency was investigated by implementing pulsed field-inversion gel electrophoresis (FIGE). Separation of model protein species and large protein complexes was compared between FIGE and constant field electrophoresis (CFE) in different percentages of PAGs. Band intensities of proteins in FIGE with appropriate ratios of forward and backward pulse times were superior to CFE despite longer running times. These results revealed an increase in band intensity per defined gel volume. A biphasic protein relative mobility shift was observed in percentages of PAGs up to 14%. However, the effect of FIGE on protein separation was stochastic at higher PAG percentage. Rat liver lysates subjected to FIGE in the second-dimension separation of two-dimensional polyarcylamide gel electrophoresis (2D PAGE) showed a 20% increase in the number of discernible spots compared with CFE. Nine common spots from both FIGE and CFE were selected for peptide sequencing by mass spectrometry (MS), which revealed higher final ion scores of all nine protein spots from FIGE. Native protein complexes ranging from 800 kDa to larger than 2000 kDa became apparent using FIGE compared with CFE. The present investigation suggests that FIGE under appropriate conditions improves protein separation efficiency during PAGE as a result of increased local protein concentration. FIGE can be implemented with minimal additional instrumentation in any laboratory setting. Despite the tradeoff of longer running times, FIGE can be a powerful protein separation tool.

  8. Serum protein concentrations from clinically healthy horses determined by agarose gel electrophoresis.

    Science.gov (United States)

    Riond, Barbara; Wenger-Riggenbach, Bettina; Hofmann-Lehmann, Regina; Lutz, Hans

    2009-03-01

    Serum protein electrophoresis is a useful screening test in equine laboratory medicine. The method can provide valuable information about changes in the concentrations of albumin and alpha-, beta-, and gamma-globulins and thereby help characterize dysproteinemias in equine patients. Reference values for horses using agarose gel as a support medium have not been reported. The purpose of this study was to establish reference intervals for serum protein concentrations in adult horses using agarose gel electrophoresis and to assess differences between warm-blooded and heavy draught horses. In addition, the precision of electrophoresis for determining fraction percentages and the detection limit were determined. Blood samples were obtained from 126 clinically healthy horses, including 105 Thoroughbreds and 21 heavy draught horses of both sexes and ranging from 2 to 20 years of age. The total protein concentration was determined by an automated biuret method. Serum protein electrophoresis was performed using a semi-automated agarose gel electrophoresis system. Coefficients of variation (CVs) were calculated for within-run and within-assay precision. Data from warm-blooded and draught horses were compared using the Mann-Whitney U test. Within-run and within-assay CVs were draught horses and so combined reference intervals (2.5-97.5%) were calculated for total protein (51.0-72.0 g/L), albumin (29.6-38.5 g/L), alpha(1)-globulin (1.9-3.1 g/L), alpha(2)-globulin (5.3-8.7 g/L), beta(1)-globulin (2.8-7.3g/L), beta(2)-globulin (2.2-6.0 g/L), and gamma-globulin (5.8-12.7 g/L) concentrations, and albumin/globulin ratio (0.93-1.65). Using agarose gel as the supporting matrix for serum protein electrophoresis in horses resulted in excellent resolution and accurate results that facilitated standardization into 6 protein fractions.

  9. Automatic DNA Diagnosis for 1D Gel Electrophoresis Images using Bio-image Processing Technique.

    Science.gov (United States)

    Intarapanich, Apichart; Kaewkamnerd, Saowaluck; Shaw, Philip J; Ukosakit, Kittipat; Tragoonrung, Somvong; Tongsima, Sissades

    2015-01-01

    gel electrophoresis images, called GELect, which was written in Java and made available through the imageJ framework. With a novel automated image processing workflow, the tool can accurately segment lanes from a gel matrix, intelligently extract distorted and even doublet bands that are difficult to identify by existing image processing tools. Consequently, genotyping from DNA gel electrophoresis can be performed automatically allowing users to efficiently conduct large scale DNA fingerprinting via DNA gel electrophoresis. The software is freely available from http://www.biotec.or.th/gi/tools/gelect.

  10. Automatic DNA Diagnosis for 1D Gel Electrophoresis Images using Bio-image Processing Technique

    Science.gov (United States)

    2015-01-01

    automated genotyping tool from DNA gel electrophoresis images, called GELect, which was written in Java and made available through the imageJ framework. With a novel automated image processing workflow, the tool can accurately segment lanes from a gel matrix, intelligently extract distorted and even doublet bands that are difficult to identify by existing image processing tools. Consequently, genotyping from DNA gel electrophoresis can be performed automatically allowing users to efficiently conduct large scale DNA fingerprinting via DNA gel electrophoresis. The software is freely available from http://www.biotec.or.th/gi/tools/gelect. PMID:26681167

  11. Colloid molecular weight estimation by gel chromatography/acrylamide gel electrophoresis

    International Nuclear Information System (INIS)

    Liberatore, F.A.; Dearborn, C.; Nigam, S.; Poon, C.; Camin, L.; Liteplo, M.

    1984-01-01

    Size or molecular weight (MW) estimation of radiolabeled collides in aqueous solutions has long been a problem. The authors have prepared several minimicroaggregated albumin colloids (mμAA) by heat denaturation of stannous-containing HSA solutions at pH 7.0, 7.5, and 8.5). The resulting colloids were labeled with Tc-99m and compared with Au-198 colloid and Tc-99m-antimony sulfide colloid (Tc-99m-Sb/sub 2/S3) by gel chromatography and gel electrophoresis. Tc-99mm-mμAA aggregated at pH 7.0 and the Au-198 colloid appeared in the external void volume of a BioRad A5.0 agarose column indicating an apparent MW of > 5 x 10/sup 6/ daltons. The pH7.5 Tc-99m-mμAA, migrated within the filtration range of the column as did a small fraction of Tc-99m-Sb/sub 2/S/sub 3/, suggesting that the MW is between 6 x 10/sup 4/ - 5 x 10/sup 6/ daltons. The Tc-99m-mμAA, aggregated at pH 8.5, had an apparent MW on gel filtration similar to that of untreated albumin, MW 6.6 x 10-/sup 4/ daltons. The mobilities of the colloids, on acrylamide disc gel electrophoresis, were consistent with the results on gel chromatography. The largest colloids, Au-198 colloid and pH 7.0 Tc-99m-mμAA, barely entered the separating gel; intermediate sized colloids, a small fraction of Tc-99m-Sb/sub 2/S/sub 3/ and pH 7.5 Tc-99m-mμAA migrated farther into the separating gel; while pH 8.5 Tc-99m-mμAA had mobility approaching that of untreated albumin. Lymphoscintigraphy studies using these colloids in animals showed the predicted, particle size-related differences in migration and clearance. The authors conclude that gel chromatography and gel electrophoresis are useful methods for estimating the apparent size of the colloidal particles

  12. Development in electrophoresis: instrumentation for two-dimensional gel electrophoresis of protein separation and application of capillary electrophoresis in micro-bioanalysis

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Aoshuang [Iowa State Univ., Ames, IA (United States)

    2008-01-01

    This dissertation begins with a general introduction of topics related to this work. The following chapters contain three scientific manuscripts, each presented in a separate chapter with accompanying tables, figures, and literature citations. The final chapter summarizes the work and provides some prospective on this work. This introduction starts with a brief treatment of the basic principles of electrophoresis separation, followed by a discussion of gel electrophoresis and particularly polyacrylamide gel electrophoresis for protein separation, a summary of common capillary electrophoresis separation modes, and a brief treatment of micro-bioanalysis application of capillary electrophoresis, and ends with an overview of protein conformation and dynamics.

  13. Images of gel electrophoresis - RGP caps | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us RGP caps Images of gel electrophoresis Data detail Data name Images of gel electrophoresis D...OI 10.18908/lsdba.nbdc00318-05-002 Description of data contents Detailed information and images of gel electrophoresis... of each marker. Data file File name: rgp_caps_electrophoresis_image.zip File URL: ftp://ftp.biosc...iencedbc.jp/archive/rgp-caps/LATEST/rgp_caps_electrophoresis_image.zip File size:... 28.7 MB Simple search URL - Data acquisition method Gel electrophoresis Data analysis method STS markers :

  14. Analysis of phloem protein patterns from different organs of Cucurbita maxima Duch. by matrix-assisted laser desorption/ionization time of flight mass spectroscopy combined with sodium dodecyl sulfate polyacrylamide gel electrophoresis.

    Science.gov (United States)

    Kehr, J; Haebel, S; Blechschmidt-Schneider, S; Willmitzer, L; Steup, M; Fisahn, J

    1999-02-01

    Sieve tubes mediate the long-distance transport of nutrients and signals between source and sink organs of plants. To detect mobile phloem proteins that are differentially distributed in source and sink organs of Cucurbita maxima, we used both one-dimensional gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Both techniques revealed that phloem protein patterns depend on the sampling site: whilst several proteins were consistently observed in all phloem samples studied others appeared to occur in a organ-specific manner. For a characterization and identification of distinct phloem polypeptides, two approaches were chosen. First, protein bands resolved by SDS-PAGE were eluted from the polyacrylamide gel and the masses of the proteins were then determined by MALDI-TOF MS. Second, proteins resolved by SDS-PAGE were subjected to proteolytic degradation and the resulting peptides were analyzed by MALDI-TOF MS: the masses of the proteolytic peptides were used for a database search. By the latter approach, three mobile phloem compounds were identified as the phloem-specific protein PP2 (D.E. Bostwick et al., 1992, The Plant Cell 4, 1539-1548) a chymotrypsin and an aspartic proteinase inhibitor. None of the other polypeptides studied corresponded to any of the protein sequences present in the database. Furthermore, MALDI-TOF MS analyses indicated that some of the mobile phloem proteins occur in a covalently modified form and that the extent of the modification depends upon the plant organ.

  15. Inexpensive and Safe DNA Gel Electrophoresis Using Household Materials

    Science.gov (United States)

    Ens, S.; Olson, A. B.; Dudley, C.; Ross, N. D., III; Siddiqi, A. A.; Umoh, K. M.; Schneegurt, M. A.

    2012-01-01

    Gel electrophoresis is the single most important molecular biology technique and it is central to life sciences research, but it is often too expensive for the secondary science classroom or homeschoolers. A simple safe low-cost procedure is described here that uses household materials to construct and run DNA gel electrophoresis. Plastic…

  16. Expression of Raf kinase inhibitor protein in human hepatoma tissues by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight methods.

    Science.gov (United States)

    Tsao, D A; Shiau, Y F; Tseng, C S; Chang, H R

    2016-01-01

    Hepatocellular carcinoma (HCC) is the most common malignant liver tumor. To reduce the mortality and improve the effectiveness of therapy, it is important to search for changes in tumor-specific biomarkers whose function may involve in disease progression and which may be useful as potential therapeutic targets. Materials and Mehtods: In this study, we use two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to observe proteome alterations of 12 tissue pairs isolated from HCC patients: Normal and tumorous tissue. Comparing the tissue types with each other, 40 protein spots corresponding to fifteen differentially expressed between normal and cancer part of HCC patients. Raf kinase inhibitor protein (RKIP), an inhibitor of Raf-mediated activation of mitogen-activated protein kinase/extracellular signal-regulated kinase, may play an important role in cancer metastasis and cell proliferation and migration of human hepatoma cells. RKIP may be considered as a marker for HCC, because its expression level changes considerably in HCC compared with normal tissue. In addition, we used the methods of Western blotting and real time-polymerase chain reaction to analysis the protein expression and gene expression of RKIP. The result showed RKIP protein and gene expression in tumor part liver tissues of HCC patient is lower than peritumorous non-neoplastic liver tissue of the corresponding HCC samples. These results strongly suggest that RKIP may be considered to be a marker for HCC and RKIP are down-regulated in liver cancer cell.

  17. Ceramic protective coatings applied by sol-gel or electrophoresis

    International Nuclear Information System (INIS)

    Stoch, A.

    1993-01-01

    Sol-gel and electrophoresis are the complementary techniques which may be used for obtaining the ceramic coatings. The composition of such a coatings depends on the composition of electrophoresis bath or sol solution. Thermal treatment is used for densifying the coating and promoting the adherence of coating to the substrate. In presented work silica, silica-alumina or alumina coatings are applied by sol-gel dip coating procedure on steel, aluminium or ceramic substrates. Electrophoresis is employed for obtaining zirconia, alumina or hydroxyapatite coatings on stainless steel. (author). 7 refs

  18. Gel Electrophoresis--The Easy Way for Students

    Science.gov (United States)

    VanRooy, Wilhelmina; Sultana, Khalida

    2010-01-01

    This article describes a simple, inexpensive, easy to conduct gel-electrophoresis activity using food dyes. It is an alternative to the more expensive counterparts which require agarose gel, DNA samples, purchased chamber and Tris-borate-EDTA buffer. We suggest some learning activities for senior biology students along with comments on several…

  19. Development of bufferless gel electrophoresis chip for easy preparation and rapid DNA separation.

    Science.gov (United States)

    Oleksandrov, Sergiy; Aman, Abdurazak; Lim, Wanyoung; Kim, Younghee; Bae, Nam Ho; Lee, Kyoung G; Lee, Seok Jae; Park, Sungsu

    2018-02-01

    This work presents a handy, fast, and compact bufferless gel electrophoresis chip (BGEC), which consists of precast agarose gel confined in a disposable plastic body with electrodes. It does not require large volumes of buffer to fill reservoirs, or the process of immersing the gel in the buffer. It withstands voltages up to 28.4 V/cm, thereby allowing DNA separation within 10 min with a similar separation capability to the standard gel electrophoresis. The results suggest that our BGEC is highly suitable for in situ gel electrophoresis in forensic, epidemiological settings and crime scenes where standard gel electrophoresis equipment cannot be brought in while quick results are needed. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Gel Electrophoresis on a Budget to Dye for

    Science.gov (United States)

    Yu, Julie H.

    2010-01-01

    Gel electrophoresis is one of the most important tools used in molecular biology and has facilitated the entire field of genetic engineering by enabling the separation of nucleic acids and proteins. However, commercial electrophoresis kits can cost up to $800 for each setup, which is cost prohibitive for most classroom budgets. This article…

  1. Mutation screening of the TP53 gene by temporal temperature gradient gel electrophoresis.

    Science.gov (United States)

    Sørlie, Therese; Johnsen, Hilde; Vu, Phuong; Lind, Guro Elisabeth; Lothe, Ragnhild; Børresen-Dale, Anne-Lise

    2005-01-01

    A protocol for detection of mutations in the TP53 gene using temporal temperature gradient gel electrophoresis (TTGE) is described. TTGE is a mutation detection technique that separates DNA fragments differing by single base pairs according to their melting properties in a denaturing gel. It is based on constant denaturing conditions in the gel combined with a temperature gradient during the electrophoretic run. This method combines some of the advantages of the related techniques denaturing gradient gel electrophoresis (DGGE) and constant denaturant gel electrophoresis (CDGE) and eliminates some of the problems. The result is a rapid and sensitive screening technique that is robust and easily set up in smaller laboratory environments.

  2. Matching Two-dimensional Gel Electrophoresis' Spots

    DEFF Research Database (Denmark)

    Dos Anjos, António; AL-Tam, Faroq; Shahbazkia, Hamid Reza

    2012-01-01

    This paper describes an approach for matching Two-Dimensional Electrophoresis (2-DE) gels' spots, involving the use of image registration. The number of false positive matches produced by the proposed approach is small, when compared to academic and commercial state-of-the-art approaches. This ar...

  3. Pulsed field gel electrophoresis a practical guide

    CERN Document Server

    Birren, Bruce

    1993-01-01

    Pulsed Field Gel Electrophoresis: A Practical Guide is the first laboratory manual to describe the theory and practice of this technique. Based on the authors' experience developing pulsed field gel instruments and teaching procedures, this book provides everything a researcher or student needs to know in order to understand and carry out pulsed field gel experiments. Clear, well-tested protocols assume only that users have a basic familiarity with molecular biology. Thorough coverage of useful data, theory, and applications ensures that this book is also a lasting resource for more adv

  4. Purification of Peptide Components including Melittin from Bee Venom using gel filtration chromatography and propionic acid/urea polyacrylamide gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Young Chon Choi

    2006-06-01

    Full Text Available Objectives : This study was conducted to carry out Purification of Melittin and other peptide components from Bee Venom using gel filtration chromatography and propionic acid/urea polyacrylamide gel electrophoresis Methods : Melittin and other peptide components were separated from bee venom by using gel filtration chromatography on Sephadex G-50 column in 0.05M ammonium acetate buffer. Results : Melittin and other peptide components were separated from bee venom by using gel filtration chromatography on Sephadex G-50 column in 0.05M ammonium acetate buffer. The fractions obtained from gel filtration chromatography was analyzed by using SDS-PAGE and propionic acid/urea polyacrylamide gel electrophoresis. The melittin obtained from the gel filtration contained residual amount of phospholipase A2 and a protein with molecular weight of 6,000. The contaminating proteins were removed by the second gel filtration chromatography. Conclusion : Gel filtration chromatography and propionic acid/urea polyacrylamide gel electrophoresis are useful to separate peptide components including melittin from bee venom.

  5. 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.

  6. Using Gel Electrophoresis To Illustrate Protein Diversity and Isoelectric Point.

    Science.gov (United States)

    Browning, Mark; Vanable, Joseph

    2002-01-01

    Demonstrates the differences in protein structures by focusing on isoelectric point with an experiment that is observable under certain pH levels in gel electrophoresis. Explains the electrophoresis procedure and reports results of the experiments. (YDS)

  7. Modification of gel architecture and TBE/TAE buffer composition to minimize heating during agarose gel electrophoresis.

    Science.gov (United States)

    Sanderson, Brian A; Araki, Naoko; Lilley, Jennifer L; Guerrero, Gilberto; Lewis, L Kevin

    2014-06-01

    Agarose gel electrophoresis of DNA and RNA is routinely performed using buffers containing either Tris, acetate, and EDTA (TAE) or Tris, borate, and EDTA (TBE). Gels are run at a low, constant voltage (∼10 V/cm) to minimize current and asymmetric heating effects, which can induce band artifacts and poor resolution. In this study, alterations of gel structure and conductive media composition were analyzed to identify factors causing higher electrical currents during horizontal slab gel electrophoresis. Current was reduced when thinner gels and smaller chamber buffer volumes were used, but was not influenced by agarose concentration or the presence of ethidium bromide. Current was strongly dependent on the amount and type of EDTA used and on the concentrations of the major acid-base components of each buffer. Interestingly, resolution and the mobilities of circular versus linear plasmid DNAs were also affected by the chemical form and amount of EDTA. With appropriate modifications to gel structure and buffer constituents, electrophoresis could be performed at high voltages (20-25 V/cm), reducing run times by up to 3-fold. The most striking improvements were observed with small DNAs and RNAs (10-100 bp): high voltages and short run times produced sharper bands and higher resolution. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Diamond cubic phase of monoolein and water as an amphiphilic matrix for electrophoresis of oligonucleotides.

    Science.gov (United States)

    Carlsson, Nils; Winge, Ann-Sofie; Engström, Sven; Akerman, Björn

    2005-10-06

    We used a cubic liquid crystal formed by the nonionic monoglyceride monoolein and water as a porous matrix for the electrophoresis of oligonucleotides. The diamond cubic phase is thermodynamically stable when in contact with a water-rich phase, which we exploit to run the electrophoresis in the useful submarine mode. Oligonucleotides are separated according to size and secondary structure by migration through the space-filling aqueous nanometer pores of the regular liquid crystal, but the comparatively slow migration means the cubic phase will not be a replacement for the conventional DNA gels. However, our demonstration that the cubic phase can be used in submarine electrophoresis opens up the possibility for a new matrix for electrophoresis of amphiphilic molecules. From this perspective, the results on the oligonucleotides show that water-soluble particles of nanometer size, typical for the hydrophilic parts of membrane-bound proteins, may be a useful separation motif. A charged contamination in the commercial sample of monoolein, most likely oleic acid that arises from its hydrolysis, restricts useful buffer conditions to a pH below 5.6.

  9. Mutation screening of the TP53 gene by temporal temperature gel electrophoresis (TTGE).

    Science.gov (United States)

    Sørlie, Therese; Johnsen, Hilde; Vu, Phuong; Lind, Guro Elisabeth; Lothe, Ragnhild; Børresen-Dale, Anne-Lise

    2014-01-01

    A protocol for detection of mutations in the TP53 gene using temporal temperature gradient electrophoresis (TTGE) is described. TTGE is a mutation detection technique that separates DNA fragments differing by single base pairs according to their melting properties in a denaturing gel. It is based on constant denaturing conditions in the gel combined with a temperature gradient during the electrophoretic run. This method combines some of the advantages of the related techniques, denaturing gradient gel electrophoresis and constant denaturant gel electrophoresis, and eliminates some of the problems. The result is a rapid and sensitive screening technique which is robust and easily set up in smaller laboratory environments.

  10. Nonradioactive telomerase activity assay by microchip electrophoresis: privileges to the classical gel electrophoresis assay.

    Science.gov (United States)

    Zhelev, Zhivko; Bakalova, Rumiana; Ewis, Ashraf; Ohba, Hideki; Ishikawa, Mitsuru; Baba, Yoshinobu

    2005-08-01

    The present study accents on the privileges of microchip-based electrophoresis to the conventional gel electrophoresis in separation of telomerase repeat amplification protocol/polymerase chain reaction (PCR) ladder products obtained in telomerase-catalyzed reaction in cancer cells. We try to clarify the interpretation of the results obtained by both electrophoretic procedures and to avoid misinterpretation as a result of PCR-dependent artefacts.

  11. Separation and identification of Musa acuminate Colla (banana) leaf proteins by two-dimensional gel electrophoresis and mass spectrometry.

    Science.gov (United States)

    Lu, Y; Qi, Y X; Zhang, H; Zhang, H Q; Pu, J J; Xie, Y X

    2013-12-19

    To establish a proteomic reference map of Musa acuminate Colla (banana) leaf, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 44 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. Three spots that were not identified by MALDI-TOF MS analysis were identified by searching against the NCBInr, SwissProt, and expressed sequence tag (EST) databases. We identified 41 unique proteins. The majority of the identified leaf proteins were found to be involved in energy metabolism. The results indicate that 2D-PAGE is a sensitive and powerful technique for the separation and identification of Musa leaf proteins. A summary of the identified proteins and their putative functions is discussed.

  12. Analysis of Two-Dimensional Electrophoresis Gel Images

    DEFF Research Database (Denmark)

    Pedersen, Lars

    2002-01-01

    This thesis describes and proposes solutions to some of the currently most important problems in pattern recognition and image analysis of two-dimensional gel electrophoresis (2DGE) images. 2DGE is the leading technique to separate individual proteins in biological samples with many biological...

  13. Gel versus capillary electrophoresis genotyping for categorizing treatment outcomes in two anti-malarial trials in Uganda

    Directory of Open Access Journals (Sweden)

    Hubbard Alan E

    2010-01-01

    Full Text Available Abstract Background Molecular genotyping is performed in anti-malarial trials to determine whether recurrent parasitaemia after therapy represents a recrudescence (treatment failure or new infection. The use of capillary instead of agarose gel electrophoresis for genotyping offers technical advantages, but it is unclear whether capillary electrophoresis will result in improved classification of anti-malarial treatment outcomes. Methods Samples were genotyped using both gel and capillary electrophoresis from randomized trials of artemether-lumefantrine (AL vs. dihydroartemisinin-piperaquine (DP performed in two areas of Uganda: Kanungu, where transmission is moderate, and Apac, where transmission is very high. Both gel and capillary methods evaluated polymorphic regions of the merozoite surface protein 1 and 2 and glutamine rich protein genes. Results Capillary electrophoresis detected more alleles and provided higher discriminatory power than agarose gel electrophoresis at both study sites. There was only moderate agreement between classification of outcomes with the two methods in Kanungu (kappa = 0.66 and poor agreement in Apac (kappa = 0.24. Overall efficacy results were similar when using gel vs. capillary methods in Kanungu (42-day risk of treatment failure for AL: 6.9% vs. 5.5%, p = 0.4; DP 2.4% vs. 2.9%, p = 0.5. However, the measured risk of recrudescence was significantly higher when using gel vs. capillary electrophoresis in Apac (risk of treatment failure for AL: 17.0% vs. 10.7%, p = 0.02; DP: 8.5% vs. 3.4%, p = 0.03. Risk differences between AL and DP were not significantly different whether gel or capillary methods were used. Conclusions Genotyping with gel electrophoresis overestimates the risk of recrudescence in anti-malarial trials performed in areas of high transmission intensity. Capillary electrophoresis provides more accurate outcomes for such trials and should be performed when possible. In areas of moderate transmission

  14. Gel versus capillary electrophoresis genotyping for categorizing treatment outcomes in two anti-malarial trials in Uganda.

    Science.gov (United States)

    Gupta, Vinay; Dorsey, Grant; Hubbard, Alan E; Rosenthal, Philip J; Greenhouse, Bryan

    2010-01-15

    Molecular genotyping is performed in anti-malarial trials to determine whether recurrent parasitaemia after therapy represents a recrudescence (treatment failure) or new infection. The use of capillary instead of agarose gel electrophoresis for genotyping offers technical advantages, but it is unclear whether capillary electrophoresis will result in improved classification of anti-malarial treatment outcomes. Samples were genotyped using both gel and capillary electrophoresis from randomized trials of artemether-lumefantrine (AL) vs. dihydroartemisinin-piperaquine (DP) performed in two areas of Uganda: Kanungu, where transmission is moderate, and Apac, where transmission is very high. Both gel and capillary methods evaluated polymorphic regions of the merozoite surface protein 1 and 2 and glutamine rich protein genes. Capillary electrophoresis detected more alleles and provided higher discriminatory power than agarose gel electrophoresis at both study sites. There was only moderate agreement between classification of outcomes with the two methods in Kanungu (kappa = 0.66) and poor agreement in Apac (kappa = 0.24). Overall efficacy results were similar when using gel vs. capillary methods in Kanungu (42-day risk of treatment failure for AL: 6.9% vs. 5.5%, p = 0.4; DP 2.4% vs. 2.9%, p = 0.5). However, the measured risk of recrudescence was significantly higher when using gel vs. capillary electrophoresis in Apac (risk of treatment failure for AL: 17.0% vs. 10.7%, p = 0.02; DP: 8.5% vs. 3.4%, p = 0.03). Risk differences between AL and DP were not significantly different whether gel or capillary methods were used. Genotyping with gel electrophoresis overestimates the risk of recrudescence in anti-malarial trials performed in areas of high transmission intensity. Capillary electrophoresis provides more accurate outcomes for such trials and should be performed when possible. In areas of moderate transmission, gel electrophoresis appears adequate to estimate comparative

  15. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS ...

    African Journals Online (AJOL)

    Four strains of eri, Samia cynthia ricini Lepidoptera: Saturniidae that can be identified morphologically and maintained at North East Institute of Science and Technology, Jorhat were characterized based on their protein profile by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and DNA by random ...

  16. High lane density slab-gel electrophoresis using micromachined instrumentation.

    Science.gov (United States)

    Papautsky, I; Mohanty, S; Weiss, R; Frazier, A B

    2001-10-01

    In this paper, micromachined pipette arrays (MPAs) and microcombs were studied as a means of enabling high lane density gel electrophoresis. The MPA provide a miniaturized format to interface sub-microliter volumes of samples between macroscale sample preparation formats and microscale biochemical analysis systems. The microcombs provide a means of creating sample loading wells in the gel material on the same center-to-center spacing as the MPAs. Together, the two micromachined instruments provide an alternative to current combs and pipetting technologies used for creating sample loading wells and sample delivery in gel electrophoresis systems. Using three designs for the microcomb-MPA pair, center-to-center spacings of 1.0 mm, 500 microm, and 250 microm are studied. The results demonstrate an approximate 10-fold increase in lane density and a 10-fold reduction in sample size from 5 microL to 500 pL. As a result, the number of theoretical plates has increased 2.5-fold, while system resolution has increased 1.5-fold over the conventional agarose gel systems. An examination of changes in resolution across the width of individual separation lanes in both systems revealed dependence in the case of the conventional gels and no dependence for the gels loaded with the micromachined instrumentation.

  17. A method for easily customizable gradient gel electrophoresis.

    Science.gov (United States)

    Miller, Andrew J; Roman, Brandon; Norstrom, Eric

    2016-09-15

    Gradient polyacrylamide gel electrophoresis is a powerful tool for the resolution of polypeptides by relative mobility. Here, we present a simplified method for generating polyacrylamide gradient gels for routine analysis without the need for specialized mixing equipment. The method allows for easily customizable gradients which can be optimized for specific polypeptide resolution requirements. Moreover, the method eliminates the possibility of buffer cross contamination in mixing equipment, and the time and resources saved with this method in place of traditional gradient mixing, or the purchase of pre-cast gels, are noteworthy given the frequency with which many labs use gradient gel SDS-PAGE. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Capillary gel electrophoresis for rapid, high resolution DNA sequencing.

    OpenAIRE

    Swerdlow, H; Gesteland, R

    1990-01-01

    Capillary gel electrophoresis has been demonstrated for the separation and detection of DNA sequencing samples. Enzymatic dideoxy nucleotide chain termination was employed, using fluorescently tagged oligonucleotide primers and laser based on-column detection (limit of detection is 6,000 molecules per peak). Capillary gel separations were shown to be three times faster, with better resolution (2.4 x), and higher separation efficiency (5.4 x) than a conventional automated slab gel DNA sequenci...

  19. Two-dimensional polyacrylamide gel electrophoresis of intracellular proteins

    International Nuclear Information System (INIS)

    Ojima, N.; Sakamoto, T.; Yamashita, M.

    1996-01-01

    Since two-dimensional electrophoresis was established by O'Farrell for analysis of intracellular proteins of Escherichia coli, it has been applied to separation of proteins of animal cells and tissues, and especially to identification of stress proteins. Using this technique, proteins are separated by isoelectric focusing containing 8 m urea in the first dimension and by SDS-PAGE in the second dimension. The gels are stained with Coomassie Blue R-250 dye, followed by silver staining. In the case of radio-labeled proteins, the gels are dried and then autoradiographed. In order to identify a specific protein separated by two-dimensional electrophoresis, a technique determining the N-terminal amino acid sequence of the protein has been developed recently. After the proteins in the gel were electrotransferred to a polyvinylidene difluoride membrane, the membrane was stained for protein with Commassie Blue and a stained membrane fragment was applied to a protein sequencer. Our recent studies demonstrated that fish cells newly synthesized various proteins in response to heat shock, cold nd osmotic stresses. For example, when cellular proteins extracted from cold-treated rainbow trout cells were subjected to two-dimensional gel electrophoresis, the 70 kDa protein was found to be synthesized during the cold-treatment. N-Terminal sequence analysis showed that the cold-inducible protein was a homolog of mammalian valosin-containing protein and yeast cell division cycle gene product CDC48p. Furthermore, the sequence data were useful for preparing PCR primers and a rabbit antibody against a synthetic peptide to analyze a role for the protein in the function of trout cells and mechanisms for regulation

  20. Photopatterned free-standing polyacrylamide gels for microfluidic protein electrophoresis.

    Science.gov (United States)

    Duncombe, Todd A; Herr, Amy E

    2013-06-07

    Designed for compatibility with slab-gel polyacrylamide gel electrophoresis (PAGE) reagents and instruments, we detail development of free-standing polyacrylamide gel (fsPAG) microstructures supporting electrophoretic performance rivalling that of microfluidic platforms. For the protein electrophoresis study described here, fsPAGE lanes are comprised of a sample reservoir and contiguous separation gel. No enclosed microfluidic channels are employed. The fsPAG devices (120 μm tall) are directly photopatterned atop of and covalently attached to planar polymer or glass surfaces. Leveraging the fast prototype-test cycle - significantly faster than mold based fabrication techniques - we optimize the fsPAG architecture to minimize injection dispersion for rapid (prototyping of the fsPAGE provides researchers a powerful tool for developing custom analytical assays. We highlight the utility of assay customization by fabricating a polyacrylamide gel with a spatial pore-size distribution and demonstrate the resulting enhancement in separation performance over a uniform gel. Further, we up-scale from a unit separation to an array of 96 concurrent fsPAGE assays in 10 min run time driven by one electrode pair. The fsPAG array layout matches that of a 96-well plate to facilitate integration of the planar free standing gel array with multi-channel pipettes while remaining compatible with conventional slab-gel PAGE reagents, such as staining for label-free protein detection. Notably, the entire fsPAGE workflow from fabrication, to operation, and readout uses readily available materials and instruments - making this technique highly accessible.

  1. Gel-eletroforese no diagnóstico da varíola Gel-electrophoresis in the smallpox diagnosis

    Directory of Open Access Journals (Sweden)

    Julio A. Mesquita

    1972-01-01

    Full Text Available O emprego de gel-eletroforese no diagnóstico da varíola, demonstrou ser ao menos trinta vezes (30X mais sensível que o teste de agar-gel, nas condições descritas (tabela I. Doze (12 espécimes, cujos testes convencionais de inoculação em ovos embrionados e de agar-gel resultaram positivos, foram testados em suas diluições originais congeladas por mais de um ano, sendo seis deles revelados por gel-eletroforese enquanto nenhum o foi por agar-gel (tabela II. Trinta e três (33 amostras isoladas no laboratório, foram testadas com material colhido de membrana cório-alantóica da primeira inoculação para o diagnóstico, conservado em glicerina 50%, resultando 15 positivas em gel-eletroforese e apenas 3 em agar-gel (tabela II. Os últimos 60 espécimes recebidos para diagnóstico, através a Campanha de Erradicação da Varíola, também resultaram negativos em gel-eletroforese, que não mostrou falsos-positivos nas condições descritas.The test of gel-electrophoresis applied to the pox virus group showed to be at least thirth times (30X more sensitive than agar-gel test on the described conditions (Table I. Twelve specimens, which were positives form Smallpox in the conventional tests of egg inoculation and agar-gel difusion test, have been screened in their original dilutions frozen for more than 1 year and six of them were still detectable by gel-eletrophoresis, while by agar-gel test any of them was positive (Table II. Thirty three Smallpox isolates have been tested with material from first egg inoculation (chorioallantoic membranes which have been stored in glycerin 50%, at - 15ºC. Fifteen of them were still positive by gel-electrophoresis and only 3 by agar-gel (Table II. The last 60 specimens received for diagnosis from Smallpox Erradication Campaign (CEV, were negatives by both tests. The gel-electrophoresis, did not show false-positives on described conditions.

  2. Proteomic study of muscle sarcoplasmic proteins using AUT-PAGE/SDS-PAGE as two-dimensional gel electrophoresis.

    Science.gov (United States)

    Picariello, Gianluca; De Martino, Alessandra; Mamone, Gianfranco; Ferranti, Pasquale; Addeo, Francesco; Faccia, Michele; Spagnamusso, Salvatore; Di Luccia, Aldo

    2006-03-20

    In the present study, an alternative procedure for two-dimensional (2D) electrophoretic analysis in proteomic investigation of the most represented basic muscle water-soluble proteins is suggested. Our method consists of Acetic acid-Urea-Triton polyacrylamide gel (AUT-PAGE) analysis in the first dimension and standard sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) in the second dimension. Although standard two-dimensional Immobilized pH Gradient-Sodium Dodecyl-Sulphate (2D IPG-SDS) gel electrophoresis has been successfully used to study these proteins, most of the water-soluble proteins are spread on the alkaline part of the 2D map and are poorly focused. Furthermore, the similarity in their molecular weights impairs resolution of the classical approach. The addition of Triton X-100, a non-ionic detergent, into the gel induces a differential electrophoretic mobility of proteins as a result of the formation of mixed micelles between the detergent and the hydrophobic moieties of polypeptides, separating basic proteins with a criterion similar to reversed phase chromatography based on their hydrophobicity. The acid pH induces positive net charges, increasing with the isoelectric point of proteins, thus allowing enhanced resolution in the separation. By using 2D AUT-PAGE/SDS electrophoresis approach to separate water-soluble proteins from fresh pork and from dry-cured products, we could spread proteins over a greater area, achieving a greater resolution than that obtained by IPG in the pH range 3-10 and 6-11. Sarcoplasmic proteins undergoing proteolysis during the ripening of products were identified by Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-ToF) mass spectrometry peptide mass fingerprinting in a easier and more effective way. Two-dimensional AUT-PAGE/SDS electrophoresis has allowed to simplify separation of sarcoplasmic protein mixtures making this technique suitable in the defining of quality of dry-cured pork products by immediate

  3. Analysis of rRNA gene methylation in Arabidopsis thaliana by CHEF-Conventional 2D gel electrophoresis

    Science.gov (United States)

    Mohannath, Gireesha; Pikaard, Craig S.

    2017-01-01

    Summary Contour-clamped homogenous electric field (CHEF) gel electrophoresis, a variant of Pulsed-field gel electrophoresis (PFGE), is a powerful technique for resolving large fragments of DNA (10 kb to 9 Mb). CHEF has many applications including the physical mapping of chromosomes, artificial chromosomes and sub-chromosomal DNA fragments, etc. Here we describe the use of CHEF and two-dimensional gel electrophoresis to analyze rRNA gene methylation patterns within the two ~ 4 million base pair nucleolus organizer regions (NORs) of Arabidopsis thaliana. The method involves CHEF gel electrophoresis of agarose-embedded DNA following restriction endonuclease digestion to cut the NORs into large but resolvable segments, followed by digestion with methylation-sensitive restriction endonucleases and conventional (or CHEF) gel electrophoresis, in a second dimension. Resulting products are then detected by Southern blotting or PCR analyses capable of discriminating rRNA gene subtypes. PMID:27576719

  4. Rapid DNA sequencing by horizontal ultrathin gel electrophoresis.

    OpenAIRE

    Brumley, R L; Smith, L M

    1991-01-01

    A horizontal polyacrylamide gel electrophoresis apparatus has been developed that decreases the time required to separate the DNA fragments produced in enzymatic sequencing reactions. The configuration of this apparatus and the use of circulating coolant directly under the glass plates result in heat exchange that is approximately nine times more efficient than passive thermal transfer methods commonly used. Bubble-free gels as thin as 25 microns can be routinely cast on this device. The appl...

  5. Seed Biology of Medicinal Plants (IX) : The Relationship of Corydalis Species Derived by Gel Electrophoresis

    OpenAIRE

    米田, 該典; 加賀, 順二; 那須, 正夫; KAISUKE, YONEDA; JUNJI, KAGA; MASAO, NASU; 大阪大学薬学部; 大阪大学薬学部; 大阪大学薬学部; Faculty of Pharmaceutical Sciences, Osaka University; Faculty of Pharmaceutical Sciences, Osaka University; Faculty of Pharmaceutical Sciences, Osaka University

    1987-01-01

    The saline soluble protein fraction of seeds of the Corydalis species (Papaveraceae) in Japan was examined by polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. The esterase zymogram suggested that C. pallida, C. pallida var. tenuis, C. heterocarpa var. japonica and C. speciosa, having yellow flowers and no tuber, are closely related to each other. Electrophoresis and SDS-electrophoresis patterns also coincided with the result of the esterase zymogram. They also su...

  6. Fractionation of SWNT/nucleic acid complexes by agarose gel electrophoresis

    International Nuclear Information System (INIS)

    Vetcher, Alexandre A; Srinivasan, Srimeenakshi; Vetcher, Ivan A; Abramov, Semen M; Kozlov, Mikhail; Baughman, Ray H; Levene, Stephen D

    2006-01-01

    We show that aqueous dispersions of single-walled carbon nanotubes (SWNTs), prepared with the aid of nucleic acids (NAs) such as RNA or DNA, can be separated into fractions using agarose gel electrophoresis. In a DC electric field, SWNT/NA complexes migrate in the gel in the direction of positive potential to form well-defined bands. Raman spectroscopy as a function of band position shows that nanotubes having different spectroscopic properties possess different electrophoretic mobilities. The migration patterns for SWNT/RNA and SWNT/DNA complexes differ. Parallel elution of the SWNT/NA complexes from the gel during electrophoresis and subsequent characterization by AFM reveals differences in nanotube diameter, length and curvature. The results suggest that fractionation of nanotubes can be achieved by this procedure. We discuss factors affecting the mobility of the nanotube complexes and propose analytical applications of this technique

  7. Fractionation of SWNT/nucleic acid complexes by agarose gel electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Vetcher, Alexandre A [Institute of Biomedical Sciences and Technology and Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX 75083 (United States); Srinivasan, Srimeenakshi [Institute of Biomedical Sciences and Technology and Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX 75083 (United States); Vetcher, Ivan A [Institute of Biomedical Sciences and Technology and Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX 75083 (United States); Abramov, Semen M [NanoTech Institute, University of Texas at Dallas, Richardson, TX 75083 (United States); Kozlov, Mikhail [NanoTech Institute, University of Texas at Dallas, Richardson, TX 75083 (United States); Baughman, Ray H [NanoTech Institute, University of Texas at Dallas, Richardson, TX 75083 (United States); Levene, Stephen D [Institute of Biomedical Sciences and Technology and Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX 75083 (United States)

    2006-08-28

    We show that aqueous dispersions of single-walled carbon nanotubes (SWNTs), prepared with the aid of nucleic acids (NAs) such as RNA or DNA, can be separated into fractions using agarose gel electrophoresis. In a DC electric field, SWNT/NA complexes migrate in the gel in the direction of positive potential to form well-defined bands. Raman spectroscopy as a function of band position shows that nanotubes having different spectroscopic properties possess different electrophoretic mobilities. The migration patterns for SWNT/RNA and SWNT/DNA complexes differ. Parallel elution of the SWNT/NA complexes from the gel during electrophoresis and subsequent characterization by AFM reveals differences in nanotube diameter, length and curvature. The results suggest that fractionation of nanotubes can be achieved by this procedure. We discuss factors affecting the mobility of the nanotube complexes and propose analytical applications of this technique.

  8. Explorative data analysis of two-dimensional electrophoresis gels

    DEFF Research Database (Denmark)

    Schultz, J.; Gottlieb, D.M.; Petersen, Marianne Kjerstine

    2004-01-01

    of gels is presented. First, an approach is demonstrated in which no prior knowledge of the separated proteins is used. Alignment of the gels followed by a simple transformation of data makes it possible to analyze the gels in an automated explorative manner by principal component analysis, to determine......Methods for classification of two-dimensional (2-DE) electrophoresis gels based on multivariate data analysis are demonstrated. Two-dimensional gels of ten wheat varieties are analyzed and it is demonstrated how to classify the wheat varieties in two qualities and a method for initial screening...... if the gels should be further analyzed. A more detailed approach is done by analyzing spot volume lists by principal components analysis and partial least square regression. The use of spot volume data offers a mean to investigate the spot pattern and link the classified protein patterns to distinct spots...

  9. Evaluation of agarose gel electrophoresis for characterization of silver nanoparticles in industrial products.

    Science.gov (United States)

    Jimenez, Maria S; Luque-Alled, Jose M; Gomez, Teresa; Castillo, Juan R

    2016-05-01

    Agarose gel electrophoresis (AGE) has been used extensively for characterization of pure nanomaterials or mixtures of pure nanomaterials. We have evaluated the use of AGE for characterization of Ag nanoparticles (NPs) in an industrial product (described as strong antiseptic). Influence of different stabilizing agents (PEG, SDS, and sodium dodecylbenzenesulfonate), buffers (TBE and Tris Glycine), and functionalizing agents (mercaptosuccinic acid (TMA) and proteins) has been investigated for the characterization of AgNPs in the industrial product using different sizes-AgNPs standards. The use of 1% SDS, 0.1% TMA, and Tris Glycine in gel, electrophoresis buffer and loading buffer led to the different sizes-AgNPs standards moved according to their size/charge ratio (obtaining a linear relationship between apparent mobility and mean diameter). After using SDS and TMA, the behavior of the AgNPs in the industrial product (containing a casein matrix) was completely different, being not possible their size characterization. However we demonstrated that AGE with LA-ICP-MS detection is an alternative method to confirm the protein corona formation between the industrial product and two proteins (BSA and transferrin) maintaining NPs-protein binding (what is not possible using SDS-PAGE). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Evaluation of denaturing gradient gel electrophoresis (DGGE) used ...

    African Journals Online (AJOL)

    Denaturing gradient gel electrophoresis (DGGE) is a powerful method used to study structure of bacterial communities, without cultivation, based on the diversity of the genes coding for ribosomal RNA. However, the results are strongly dependent on the respective target region of the used primer systems. Therefore, three ...

  11. Speciation of protein-bound trace elements by gel electrophoresis and atomic spectrometry.

    Science.gov (United States)

    Ma, Renli; McLeod, Cameron W; Tomlinson, Kerry; Poole, Robert K

    2004-08-01

    The metabolism of trace elements, in particular their binding to proteins in biological systems is of great importance in biochemical, toxicological, and pharmacological studies. As a result there has been a sustained interest over the last two decades in the speciation of protein-bound metals. Various analytical approaches have been employed, combining efficient separation of metalloproteins by liquid chromatography or electrophoresis with high-sensitivity elemental detection. Slab-gel electrophoresis (GE) is a key platform for high-resolution protein separation, and has been combined with autoradiography and various atomic spectrometric techniques for in-gel determination of protein-bound metals. Recently, the combination of GE with state-of-the-art inductively coupled plasma-mass spectrometry (ICP-MS), particularly when linked to laser ablation (LA) for direct gel interrogation, has opened up new opportunities for rapid characterization of metalloproteins. The use of GE and atomic spectrometry for the speciation of protein-bound trace elements is reviewed in this paper. Technical requirements for gel electrophoresis/atomic spectrometric measurement are considered in terms of method compatibilities, detection capability and potential usefulness. The literature is also surveyed to illustrate current status and future trends. Copyright 2004 Wiley-VCH Verlag GmbH and Co.

  12. Detection of human DNA polymorphisms with a simplified denaturing gradient gel electrophoresis technique.

    OpenAIRE

    Noll, W W; Collins, M

    1987-01-01

    Single base pair differences between otherwise identical DNA molecules can result in altered melting behavior detectable by denaturing gradient gel electrophoresis. We have developed a simplified procedure for using denaturing gradient gel electrophoresis to detect base pair changes in genomic DNA. Genomic DNA is digested with restriction enzymes and hybridized in solution to labeled single-stranded probe DNA. The excess probe is then hybridized to complementary phage M13 template DNA, and th...

  13. Preparation of Barley Storage Protein, Hordein, for Analytical Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

    DEFF Research Database (Denmark)

    Doll, Hans; Andersen, Bente

    1981-01-01

    The extraction, reduction, and alkylation of barley hordein for routine electrophoresis in sodium dodecyl sulfate-polyacrylamide gels were studied to set up a simple preparation procedure giving well-resolved bands in the electrophoresis gel. Hordein was extracted from single crushed seeds or flour...... by aqueous 50% propan-2-ol containing a Tris-borate buffer, pH 8.6. The presence of the buffer facilitates the consecutive complete reduction of the extracted protein in the alcohol. Reduction and alkylation in the buffer containing propan-2-ol give sharper bands in the electrophoresis than reduction...

  14. Pulsed-field gel electrophoresis typing of Staphylococcus aureus isolates

    Science.gov (United States)

    Pulsed-field gel electrophoresis (PFGE) is the most applied and effective genetic typing method for epidemiological studies and investigation of foodborne outbreaks caused by different pathogens, including Staphylococcus aureus. The technique relies on analysis of large DNA fragments generated by th...

  15. Ocular Proteomics with Emphasis on Two-Dimensional Gel Electrophoresis and Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Honoré Bent

    2010-01-01

    Full Text Available Abstract The intention of this review is to provide an overview of current methodologies employed in the rapidly developing field of ocular proteomics with emphasis on sample preparation, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE and mass spectrometry (MS. Appropriate sample preparation for the diverse range of cells and tissues of the eye is essential to ensure reliable results. Current methods of protein staining for 2D-PAGE, protein labelling for two-dimensional difference gel electrophoresis, gel-based expression analysis and protein identification by MS are summarised. The uses of gel-free MS-based strategies (MuDPIT, iTRAQ, ICAT and SILAC are also discussed. Proteomic technologies promise to shed new light onto ocular disease processes that could lead to the discovery of strong novel biomarkers and therapeutic targets useful in many ophthalmic conditions.

  16. Analyzing import intermediates of mitochondrial proteins by blue native gel electrophoresis.

    Science.gov (United States)

    Waizenegger, Thomas; Rapaport, Doron

    2007-01-01

    Blue native gel electrophoresis (BNGE) is a powerful tool for analyzing native protein complexes from biological membranes as well as water-soluble proteins. It can be used for determining relative molecular masses of protein complexes and their subunit composition and for the detection of subcomplexes. We describe the analysis by BNGE of in vitro import reactions composed of radiolabeled precursor proteins and isolated mitochondria. Such an analysis is a powerful tool to follow import intermediates and to study assembly of protein complexes. Analysis of import reactions by BNGE provides information on the molecular mass of the complex with which the imported precursor is associated. In addition, components of such a complex can be identified by incubating the mitochondrial lysate with either soluble antibodies or antibodies coupled to protein A matrix. The binding of soluble antibodies to specific complexes results in an observed shift in their apparent molecular mass (antibody shift). Alternatively, addition of matrix-bound antibodies followed by removal of the matrix from the mixture will result in depletion of the specific complex from the mitochondrial lysate (antibody depletion). The experimental details of these techniques are described.

  17. Performance comparison of capillary and agarose gel electrophoresis for the identification and characterization of monoclonal immunoglobulins.

    Science.gov (United States)

    McCudden, Christopher R; Mathews, Stephanie P; Hainsworth, Shirley A; Chapman, John F; Hammett-Stabler, Catherine A; Willis, Monte S; Grenache, David G

    2008-03-01

    The objective of this study was to compare gel- and capillary-based serum protein electrophoresis methods to identify and characterize monoclonal immunoglobulins (M proteins). Five reviewers interpreted 149 consecutively ordered serum specimens following agarose gel electrophoresis (AGE), capillary electrophoresis (CE), immunofixation electrophoresis (IFE), and subtraction immunotyping (IT). As a screening test for detecting M proteins, AGE and CE displayed similar sensitivity (91% and 92%, respectively). CE was less specific (74%) than AGE (81%). An analysis of interinterpreter agreement revealed that interpretations were more consistent using gel-based methods than capillary-based methods, with 80% of the gel interpretations being in complete (5/5) agreement compared with 67% of the capillary interpretations. After implementing the capillary-based methods, the number of tests per reportable result increased (from 1.58 to 1.73). CE is an analytically suitable alternative to AGE, but laboratories implementing it will need to continue IFE testing to characterize all M proteins detected by CE.

  18. Detection of human DNA polymorphisms with a simplified denaturing gradient gel electrophoresis technique

    International Nuclear Information System (INIS)

    Noll, W.W.; Collins, M.

    1987-01-01

    Single base pair differences between otherwise identical DNA molecules can result in altered melting behavior detectable by denaturing gradient gel electrophoresis. The authors have developed a simplified procedure for using denaturing gradient gel electrophoresis to detect base pair changes in genomic DNA. Genomic DNA is digested with restriction enzymes and hybridized in solution to labeled single-stranded probe DNA. The excess probe is then hybridized to complementary phage M13 template DNA, and the reaction mixture is electrophoresed on a denaturing gradient gel. Only the genomic DNA probe hybrids migrate into the gel. Differences in hybrid mobility on the gel indicate base pair changes in the genomic DNA. They have used this technique to identify two polymorphic sites within a 1.2-kilobase region of human chromosome 20. This approach should greatly facilitate the identification of DNA polymorphisms useful for gene linkage studies and the diagnosis of genetic diseases

  19. Two-dimensional gel electrophoresis analysis of different parts of ...

    African Journals Online (AJOL)

    Two-dimensional gel electrophoresis analysis of different parts of Panax quinquefolius L. root. ... From these results it was concluded that proteomic analysis method was an effective way to identify the different parts of quinquefolius L. root. These findings may contribute to further understanding of the physiological ...

  20. Lesion measurement in non-radioactive DNA by quantitative gel electrophoresis

    International Nuclear Information System (INIS)

    Sutherland, J.C.; Chen, Chun Zhang; Emrick, A.; Hacham, H; Monteleone, D.; Ribeiro, E.; Trunk, J.; Sutherland, B.M.

    1989-01-01

    The gel electrophoresis method developed during the past ten years in our laboratories makes possible the quantitation of UV induced pyrimidine dimers, gamma ray induced single- and double-strand breaks and many other types of lesions in nanogram quantities of DNA. The DNA does not have to be labeled with radionuclides or of a particular conformation, thus facilitating the use of the method in measuring damage levels and repair rates in the DNA of intact organisms -- including man. The gel method can quantitate any lesion in DNA that either is, or can be converted to a single- or double-strand break. The formation of a strand break produces two shorter DNA molecules for each molecule that existed before the treatment that produced the break. Determining the number of breaks, and hence the number of lesions, becomes a matter of comparing the average lengths of molecules in samples differing only in lesion-induced breaks. This requires that we determine the distribution of mass of DNA on a gel as a function of its distance of migration and also the dispersion function of its distance of migration and also the dispersion function (the relationship between molecular length and distance of migration) in the gel electrophoresis system. 40 refs., 5 figs

  1. The single-cell gel electrophoresis assay to determine apoptosis ...

    African Journals Online (AJOL)

    When the frequency of appearance of apoptotic cells following was observed over a period of time, there was a significant increase in appearance of apoptosis when using single cell gel electrophoresis assay. The present report demonstrates that the characteristic pattern of apoptotic comets detected by the comet assay ...

  2. Blood grouping based on PCR methods and agarose gel electrophoresis.

    Science.gov (United States)

    Sell, Ana Maria; Visentainer, Jeane Eliete Laguila

    2015-01-01

    The study of erythrocyte antigens continues to be an intense field of research, particularly after the development of molecular testing methods. More than 300 specificities have been described by the International Society for Blood Transfusion as belonging to 33 blood group systems. The polymerase chain reaction (PCR) is a central tool for red blood cells (RBC) genotyping. PCR and agarose gel electrophoresis are low cost, easy, and versatile in vitro methods for amplifying defined target DNA (RBC polymorphic region). Multiplex-PCR, AS-PCR (Specific Allele Polymerase Chain Reaction), and RFLP-PCR (Restriction Fragment Length Polymorphism-Polymerase Chain Reaction) techniques are usually to identify RBC polymorphisms. Furthermore, it is an easy methodology to implement. This chapter describes the PCR methodology and agarose gel electrophoresis to identify the polymorphisms of the Kell, Duffy, Kidd, and MNS blood group systems.

  3. The application of single cell gel electrophoresis or comet assay to human monitoring studies

    Directory of Open Access Journals (Sweden)

    Valverde Mahara

    1999-01-01

    Full Text Available Objective. In the search of new human genotoxic biomarkers, the single cell gel electrophoresis assay has been proposed as a sensible alternative. Material and methods. This technique detects principally single strand breaks as well as alkali-labile and repair-retarded sites. Results. Herein we present our experience using the single cell gel electrophoresis assay in human population studies, both occupationally and environmentally exposed. Conclusions. We discuss the assay feasibility as a genotoxic biomarker.

  4. Optimal processing for gel electrophoresis images: Applying Monte Carlo Tree Search in GelApp.

    Science.gov (United States)

    Nguyen, Phi-Vu; Ghezal, Ali; Hsueh, Ya-Chih; Boudier, Thomas; Gan, Samuel Ken-En; Lee, Hwee Kuan

    2016-08-01

    In biomedical research, gel band size estimation in electrophoresis analysis is a routine process. To facilitate and automate this process, numerous software have been released, notably the GelApp mobile app. However, the band detection accuracy is limited due to a band detection algorithm that cannot adapt to the variations in input images. To address this, we used the Monte Carlo Tree Search with Upper Confidence Bound (MCTS-UCB) method to efficiently search for optimal image processing pipelines for the band detection task, thereby improving the segmentation algorithm. Incorporating this into GelApp, we report a significant enhancement of gel band detection accuracy by 55.9 ± 2.0% for protein polyacrylamide gels, and 35.9 ± 2.5% for DNA SYBR green agarose gels. This implementation is a proof-of-concept in demonstrating MCTS-UCB as a strategy to optimize general image segmentation. The improved version of GelApp-GelApp 2.0-is freely available on both Google Play Store (for Android platform), and Apple App Store (for iOS platform). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Gel Electrophoresis and Fluorescamine Methods for the Detection of ...

    African Journals Online (AJOL)

    For the fluorescamine method, clarification was achieved by isoelectric precipitation and precipitation with acid to obtain pH 4.6 and 6% TCA soluble extracts respectively. Non-clarified samples were used for gel electrophoresis. Both methods confirmed that raw milk and milk processed at 85/15s were the most proteolysed, ...

  6. Defining carbohydrate binding of glucan phosphatases via Affinity gel electrophoresis

    DEFF Research Database (Denmark)

    Auger, Kyle; Raththagala, Madushi; Wilkens, Casper

    2016-01-01

    was to determine a technique to measure carbohydrate binding quickly and efficiently. We established a protocol to reproducibly and quantitatively measure the binding of the enzymes to glucans utilizing Affinity Gel Electrophoresis (AGE). The results show that the various glucan phosphatases possess differing...

  7. Improving precision in gel electrophoresis by stepwisely decreasing variance components.

    Science.gov (United States)

    Schröder, Simone; Brandmüller, Asita; Deng, Xi; Ahmed, Aftab; Wätzig, Hermann

    2009-10-15

    Many methods have been developed in order to increase selectivity and sensitivity in proteome research. However, gel electrophoresis (GE) which is one of the major techniques in this area, is still known for its often unsatisfactory precision. Percental relative standard deviations (RSD%) up to 60% have been reported. In this case the improvement of precision and sensitivity is absolutely essential, particularly for the quality control of biopharmaceuticals. Our work reflects the remarkable and completely irregular changes of the background signal from gel to gel. This irregularity was identified as one of the governing error sources. These background changes can be strongly reduced by using a signal detection in the near-infrared (NIR) range. This particular detection method provides the most sensitive approach for conventional CCB (Colloidal Coomassie Blue) stained gels, which is reflected in a total error of just 5% (RSD%). In order to further investigate variance components in GE, an experimental Plackett-Burman screening design was performed. The influence of seven potential factors on the precision was investigated using 10 proteins with different properties analyzed by NIR detection. The results emphasized the individuality of the proteins. Completely different factors were identified to be significant for each protein. However, out of seven investigated parameters, just four showed a significant effect on some proteins, namely the parameters of: destaining time, staining temperature, changes of detergent additives (SDS and LDS) in the sample buffer, and the age of the gels. As a result, precision can only be improved individually for each protein or protein classes. Further understanding of the unique properties of proteins should enable us to improve the precision in gel electrophoresis.

  8. Strain identification in Rhizobium by starch gel electrophoresis of isoenzymes

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen; Nielsen, G.

    1985-01-01

    Sonieated extracts of rhizobia, especiaUy Rhizobium leguminosarum from pea and vetch, were run in horizontal starch gel electrophoresis in the cold. The rhizobia were grown on agar on a slime suppressing substrate of tryptone-yeast extract-CaCl2 with small amounts of mannitol, sorbitol...

  9. [Establishment of two-dimensional differential gel electrophoresis using cerebrospinal fluid from neurocysticercosis patients].

    Science.gov (United States)

    Li, Jing-Yi; Tian, Xiao-Jun; Huang, Yong; Yang, Yan-Jun; Ma, Qiao-Rong; Xue, Yan-Ping

    2008-06-30

    To establish the method of two-dimensional differential gel electrophoresis and obtain high resolution 2D images from cerebrospinal fluid (CSF) of patients with neurocysticercosis. CSF samples were collected from four patients diagnosed as neurocysticercosis clinically and by ELISA, computed tomography (CT) or magnetic resonance imaging (MRI), and from four healthy subjects without neurological disorders. The CSF samples were precipitated with cold acetone, then pooled by equal amount as patients and controls. The internal standard comprised equal amounts of proteins extracted from both groups. Internal standard, and proteins from the two groups were labeled prior to electrophoresis with spectrally resolvable fluorescent dyes, cyanein dye2 (Cy2), Cy3 and Cy5. Sodium dodecylsulfonate polyacrylamide gel chromatography (SDS-PAGE) and two-dimensional differential in-gel electrophoresis (2-D DIGE) of labeled samples were then run. The differential expressed proteins showed in the images of SDS-PAGE and 2-D DIGE gels scanned with 488 nm, 532 nm and 633 nm wavelength laser were analyzed by ImageQuant and DeCyde 5.0 respectively. Spot detection and quantification was performed for the differential in-gel analysis (DIA) module of DeCyder. Biological variation analysis (BVA) module of DeCyder was matched gel 1 and gel 2 images to provide data on differential protein expression levels between the two groups. The ImageQuant result displayed that the CSF protein was compatible with the dye, and the difference of protein amount was revealed by the difference of fluorescence intensity. DIA indicated that there were 896 and 894 protein dots on gel 1 and gel 2 respectively, and 90% of them were matched each other. BVA showed that there were 55 protein spots with different expressional level between neurocysticercosis and control groups. Protein spots with two-fold increase or decrease were 47 and 8 respectively in neurocysticercosis patients compared with healthy controls. The

  10. Gel versus capillary electrophoresis genotyping for categorizing treatment outcomes in two anti-malarial trials in Uganda

    OpenAIRE

    Hubbard Alan E; Dorsey Grant; Gupta Vinay; Rosenthal Philip J; Greenhouse Bryan

    2010-01-01

    Abstract Background Molecular genotyping is performed in anti-malarial trials to determine whether recurrent parasitaemia after therapy represents a recrudescence (treatment failure) or new infection. The use of capillary instead of agarose gel electrophoresis for genotyping offers technical advantages, but it is unclear whether capillary electrophoresis will result in improved classification of anti-malarial treatment outcomes. Methods Samples were genotyped using both gel and capillary elec...

  11. Pulsed-field Gel Electrophoresis for Salmonella Infection Surveillance, Texas, USA, 2007

    Centers for Disease Control (CDC) Podcasts

    This podcast describes monitoring of the use of pulsed-field gel electrophoresis for Salmonella surveillance in Houston, Texas. CDC microbiologist Peter Gerner-Smidt discusses the importance of the PulseNet national database in surveillance of food-borne infections.

  12. A Robust Identification of the Protein Standard Bands in Two-Dimensional Electrophoresis Gel Images

    Directory of Open Access Journals (Sweden)

    Serackis Artūras

    2017-12-01

    Full Text Available The aim of the investigation presented in this paper was to develop a software-based assistant for the protein analysis workflow. The prior characterization of the unknown protein in two-dimensional electrophoresis gel images is performed according to the molecular weight and isoelectric point of each protein spot estimated from the gel image before further sequence analysis by mass spectrometry. The paper presents a method for automatic and robust identification of the protein standard band in a two-dimensional gel image. In addition, the method introduces the identification of the positions of the markers, prepared by using pre-selected proteins with known molecular mass. The robustness of the method was achieved by using special validation rules in the proposed original algorithms. In addition, a self-organizing map-based decision support algorithm is proposed, which takes Gabor coefficients as image features and searches for the differences in preselected vertical image bars. The experimental investigation proved the good performance of the new algorithms included into the proposed method. The detection of the protein standard markers works without modification of algorithm parameters on two-dimensional gel images obtained by using different staining and destaining procedures, which results in different average levels of intensity in the images.

  13. Diffusive transfer to membranes as an effective interface between gel electrophoresis and mass spectrometry

    Science.gov (United States)

    Ogorzalek Loo, Rachel R.; Mitchell, Charles; Stevenson, Tracy I.; Loo, Joseph A.; Andrews, Philip C.

    1997-12-01

    Diffusive transfer was examined as a blotting method to transfer proteins from polyacrylamide gels to membranes for ultraviolet matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The method is well-suited for transfers from isoelectric focusing (IEF) gels. Spectra have been obtained for 11 pmol of 66 kDa albumin loaded onto an IEF gel and subsequently blotted to polyethylene. Similarly, masses of intact carbonic anhydrase and hemoglobin were obtained from 14 and 20 pmol loadings. This methodology is also compatible with blotting high molecular weight proteins, as seen for 6 pmol of the 150 kDa monoclonal antibody anti-[beta]-galactosidase transferred to Goretex. Polypropylene, Teflon, Nafion and polyvinylidene difluoride (PVDF) also produced good spectra following diffusive transfer. Only analysis from PVDF required that the membrane be kept wet prior to application of matrix. Considerations in mass accuracy for analysis from large-area membranes with continuous extraction and delayed extraction were explored, as were remedies for surface charging. Vapor phase CNBr cleavage was applied to membrane-bound samples for peptide mapping.

  14. Alkaline gel electrophoresis assay to detect DNA strand breaks and repair mechanisms in Escherichia coli

    International Nuclear Information System (INIS)

    Mattos, Jose Carlos Pelielo de; Motta, Ellen Serri da; Oliveira, Marcia Betania Nunes de; Dantas, Flavio Jose da Silva; Araujo, Adriano Caldeira de

    2008-01-01

    Reactive oxygen species (ROS) can induce lesions in different cellular targets, including DNA. Stannous chloride (SnCl 2 ) is a ROS generator, leading to lethality in Escherichia coli (E. coli), with the base excision repair (BER) mechanism playing a role in this process. Many techniques have been developed to detect genotoxicity, as comet assay, in eukaryotic cells, and plasmid DNA agarose gel electrophoresis. In this study, an adaptation of the alkaline gel electrophoresis method was carried out to ascertain the induction of strand breaks by SnCl 2 in bacterial DNA, from E. coli BER mutants, and its repair pathway. Results obtained show that SnCl 2 was able to induce DNA strand breaks in all strains tested. Moreover, endonuclease IV and exonuclease III play a role in DNA repair. On the whole, data has shown that the alkaline gel electrophoresis assay could be used both for studying DNA strand breaks induction and for associated repair mechanisms. (author)

  15. Alkaline gel electrophoresis assay to detect DNA strand breaks and repair mechanisms in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Mattos, Jose Carlos Pelielo de; Motta, Ellen Serri da; Oliveira, Marcia Betania Nunes de; Dantas, Flavio Jose da Silva; Araujo, Adriano Caldeira de [Universidade do Estado do Rio de Janeiro (UERJ), RJ (Brazil). Dept. de Biofisica e Biometria. Lab. de Radio e Fotobiologia]. E-mail: jcmattos@uerj.br

    2008-12-15

    Reactive oxygen species (ROS) can induce lesions in different cellular targets, including DNA. Stannous chloride (SnCl{sub 2}) is a ROS generator, leading to lethality in Escherichia coli (E. coli), with the base excision repair (BER) mechanism playing a role in this process. Many techniques have been developed to detect genotoxicity, as comet assay, in eukaryotic cells, and plasmid DNA agarose gel electrophoresis. In this study, an adaptation of the alkaline gel electrophoresis method was carried out to ascertain the induction of strand breaks by SnCl{sub 2} in bacterial DNA, from E. coli BER mutants, and its repair pathway. Results obtained show that SnCl{sub 2} was able to induce DNA strand breaks in all strains tested. Moreover, endonuclease IV and exonuclease III play a role in DNA repair. On the whole, data has shown that the alkaline gel electrophoresis assay could be used both for studying DNA strand breaks induction and for associated repair mechanisms. (author)

  16. Product-selective blot: a technique for measuring enzyme activities in large numbers of samples and in native electrophoresis gels

    International Nuclear Information System (INIS)

    Thompson, G.A.; Davies, H.M.; McDonald, N.

    1985-01-01

    A method termed product-selective blotting has been developed for screening large numbers of samples for enzyme activity. The technique is particularly well suited to detection of enzymes in native electrophoresis gels. The principle of the method was demonstrated by blotting samples from glutaminase or glutamate synthase reactions into an agarose gel embedded with ion-exchange resin under conditions favoring binding of product (glutamate) over substrates and other substances in the reaction mixture. After washes to remove these unbound substances, the product was measured using either fluorometric staining or radiometric techniques. Glutaminase activity in native electrophoresis gels was visualized by a related procedure in which substrates and products from reactions run in the electrophoresis gel were blotted directly into a resin-containing image gel. Considering the selective-binding materials available for use in the image gel, along with the possible detection systems, this method has potentially broad application

  17. Agar/gelatin bilayer gel matrix fabricated by simple thermo-responsive sol-gel transition method.

    Science.gov (United States)

    Wang, Yifeng; Dong, Meng; Guo, Mengmeng; Wang, Xia; Zhou, Jing; Lei, Jian; Guo, Chuanhang; Qin, Chaoran

    2017-08-01

    We present a simple and environmentally-friendly method to generate an agar/gelatin bilayer gel matrix for further biomedical applications. In this method, the thermally responsive sol-gel transitions of agar and gelatin combined with the different transition temperatures are exquisitely employed to fabricate the agar/gelatin bilayer gel matrix and achieve separate loading for various materials (e.g., drugs, fluorescent materials, and nanoparticles). Importantly, the resulting bilayer gel matrix provides two different biopolymer environments (a polysaccharide environment vs a protein environment) with a well-defined border, which allows the loaded materials in different layers to retain their original properties (e.g., magnetism and fluorescence) and reduce mutual interference. In addition, the loaded materials in the bilayer gel matrix exhibit an interesting release behavior under the control of thermal stimuli. Consequently, the resulting agar/gelatin bilayer gel matrix is a promising candidate for biomedical applications in drug delivery, controlled release, fluorescence labeling, and bio-imaging. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. MALDI MS peptide mapping performance by in-gel digestion on a probe with prestructured sample supports

    DEFF Research Database (Denmark)

    Klenø, Tina Guldberg; Andreasen, Christian Maaløv; Kjeldal, Helle Ørsted

    2004-01-01

    Matrix-assisted laser desorption/ionization (tandem) mass spectrometry (MALDI MS) is widely used in protein chemistry and proteomics research for the identification and characterization of proteins isolated by polyacrylamide gel electrophoresis. In an effort to minimize sample handling and increa......-probe digestion protocol combined with MALDI tandem mass spectrometry provides a robust platform for proteomics research, including protein identification and determination of posttranslational modifications....

  19. Studies of transferin polymorphism in Swedish cattle using agarose gel electrophoresis

    International Nuclear Information System (INIS)

    Liberg, P.; Carlstroem, G.

    1976-01-01

    The polymorphic transferrin picture in the sera from 894 Swedish cattle was investigated with an agarose gel electrophoresis technique. The serum transferrin bands in the electrophoresis pattern were first identified by labelling with 59 Fe. Six existing phenotypes based on the alleles Tf(supA), Tf(supD) and Tf(supE) could be detected. The frequencies of transferrin types and transferrin alleles are presented, and it is concluded that there are great differences in the frequencis between the Swedish Red and White and the Swedish Friesian. (author)

  20. An XML standard for the dissemination of annotated 2D gel electrophoresis data complemented with mass spectrometry results

    Directory of Open Access Journals (Sweden)

    Arthur John

    2004-01-01

    Full Text Available Abstract Background Many proteomics initiatives require a seamless bioinformatics integration of a range of analytical steps between sample collection and systems modeling immediately assessable to the participants involved in the process. Proteomics profiling by 2D gel electrophoresis to the putative identification of differentially expressed proteins by comparison of mass spectrometry results with reference databases, includes many components of sample processing, not just analysis and interpretation, are regularly revisited and updated. In order for such updates and dissemination of data, a suitable data structure is needed. However, there are no such data structures currently available for the storing of data for multiple gels generated through a single proteomic experiments in a single XML file. This paper proposes a data structure based on XML standards to fill the void that exists between data generated by proteomics experiments and storing of data. Results In order to address the resulting procedural fluidity we have adopted and implemented a data model centered on the concept of annotated gel (AG as the format for delivery and management of 2D Gel electrophoresis results. An eXtensible Markup Language (XML schema is proposed to manage, analyze and disseminate annotated 2D Gel electrophoresis results. The structure of AG objects is formally represented using XML, resulting in the definition of the AGML syntax presented here. Conclusion The proposed schema accommodates data on the electrophoresis results as well as the mass-spectrometry analysis of selected gel spots. A web-based software library is being developed to handle data storage, analysis and graphic representation. Computational tools described will be made available at http://bioinformatics.musc.edu/agml. Our development of AGML provides a simple data structure for storing 2D gel electrophoresis data.

  1. An XML standard for the dissemination of annotated 2D gel electrophoresis data complemented with mass spectrometry results.

    Science.gov (United States)

    Stanislaus, Romesh; Jiang, Liu Hong; Swartz, Martha; Arthur, John; Almeida, Jonas S

    2004-01-29

    Many proteomics initiatives require a seamless bioinformatics integration of a range of analytical steps between sample collection and systems modeling immediately assessable to the participants involved in the process. Proteomics profiling by 2D gel electrophoresis to the putative identification of differentially expressed proteins by comparison of mass spectrometry results with reference databases, includes many components of sample processing, not just analysis and interpretation, are regularly revisited and updated. In order for such updates and dissemination of data, a suitable data structure is needed. However, there are no such data structures currently available for the storing of data for multiple gels generated through a single proteomic experiments in a single XML file. This paper proposes a data structure based on XML standards to fill the void that exists between data generated by proteomics experiments and storing of data. In order to address the resulting procedural fluidity we have adopted and implemented a data model centered on the concept of annotated gel (AG) as the format for delivery and management of 2D Gel electrophoresis results. An eXtensible Markup Language (XML) schema is proposed to manage, analyze and disseminate annotated 2D Gel electrophoresis results. The structure of AG objects is formally represented using XML, resulting in the definition of the AGML syntax presented here. The proposed schema accommodates data on the electrophoresis results as well as the mass-spectrometry analysis of selected gel spots. A web-based software library is being developed to handle data storage, analysis and graphic representation. Computational tools described will be made available at http://bioinformatics.musc.edu/agml. Our development of AGML provides a simple data structure for storing 2D gel electrophoresis data.

  2. Salmonella enterica pulsed-field gel electrophoresis clusters, Minnesota, USA, 2001-2007.

    Science.gov (United States)

    Rounds, Joshua M; Hedberg, Craig W; Meyer, Stephanie; Boxrud, David J; Smith, Kirk E

    2010-11-01

    We determined characteristics of Salmonella enterica pulsed-field gel electrophoresis clusters that predict their being solved (i.e., that result in identification of a confirmed outbreak). Clusters were investigated by the Minnesota Department of Health by using a dynamic iterative model. During 2001-2007, a total of 43 (12.5%) of 344 clusters were solved. Clusters of ≥4 isolates were more likely to be solved than clusters of 2 isolates. Clusters in which the first 3 case isolates were received at the Minnesota Department of Health within 7 days were more likely to be solved than were clusters in which the first 3 case isolates were received over a period >14 days. If resources do not permit investigation of all S. enterica pulsed-field gel electrophoresis clusters, investigation of clusters of ≥4 cases and clusters in which the first 3 case isolates were received at a public health laboratory within 7 days may improve outbreak investigations.

  3. Proteomic profile in glomeruli of type-2 diabetic KKAy mice using 2-dimensional differential gel electrophoresis.

    Science.gov (United States)

    Liu, Xiaodan; Yang, Gang; Fan, Qiuling; Wang, Lining

    2014-12-17

    Diabetic nephropathy (DN) is a leading cause of end-stage renal disease. To search for glomerular proteins associated with early-stage DN, glomeruli of spontaneous type 2 diabetic KKAy mice were analyzed by 2-dimensional differential gel electrophoresis (2D-DIGE). Glomeruli of 20-week spontaneous type 2 diabetic KKAy mice and age-matched C57BL/6 mice were isolated by kidney perfusion with magnetic beads. Proteomic profiles of glomeruli were investigated by using 2D-DIGE and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Western blot analysis was used to confirm the results of proteomics. Immunohistochemical and semi-quantitative analysis were used to confirm the differential expression of prohibitin and annexin A2 in glomeruli. We identified 19 differentially expressed proteins - 17 proteins were significantly up-regulated and 2 proteins were significantly down-regulated in glomeruli of diabetic KKAy mice. Among them, prohibitin and annexin A2 were up-regulated and Western blot analysis validated the same result in proteomics. Immunohistochemical analysis also revealed up-regulation of prohibitin and annexin A2 in glomeruli of KKAy mice. Our findings suggest that prohibitin and annexin A2 may be associated with early-stage DN. Further functional research might help to reveal the pathogenesis of DN.

  4. Electrophoresis gel image processing and analysis using the KODAK 1D software.

    Science.gov (United States)

    Pizzonia, J

    2001-06-01

    The present article reports on the performance of the KODAK 1D Image Analysis Software for the acquisition of information from electrophoresis experiments and highlights the utility of several mathematical functions for subsequent image processing, analysis, and presentation. Digital images of Coomassie-stained polyacrylamide protein gels containing molecular weight standards and ethidium bromide stained agarose gels containing DNA mass standards are acquired using the KODAK Electrophoresis Documentation and Analysis System 290 (EDAS 290). The KODAK 1D software is used to optimize lane and band identification using features such as isomolecular weight lines. Mathematical functions for mass standard representation are presented, and two methods for estimation of unknown band mass are compared. Given the progressive transition of electrophoresis data acquisition and daily reporting in peer-reviewed journals to digital formats ranging from 8-bit systems such as EDAS 290 to more expensive 16-bit systems, the utility of algorithms such as Gaussian modeling, which can correct geometric aberrations such as clipping due to signal saturation common at lower bit depth levels, is discussed. Finally, image-processing tools that can facilitate image preparation for presentation are demonstrated.

  5. Trapping and breaking of in vivo nicked DNA during pulsed-field gel electrophoresis

    Science.gov (United States)

    Khan, Sharik R.; Kuzminov, Andrei

    2013-01-01

    Pulsed field gel electrophoresis (PFGE) offers a high-resolution approach to quantify chromosomal fragmentation in bacteria, measured as percent of chromosomal DNA entering the gel. The degree of separation in PFG depends upon the size of DNA, as well as various conditions of electrophoresis, such as electric field strength (FS), time of electrophoresis, switch time and buffer composition. Here we describe a new parameter, the structural integrity of the sample DNA itself, that influences its migration through PFGs. We show that sub-chromosomal fragments containing both spontaneous and DNA damage-induced nicks are prone to breakage during PFGE. Such breakage at single strand interruptions results in artefactual decrease in molecular weight of linear DNA making accurate determination of the number of double strand breaks difficult. While breakage of nicked sub-chromosomal fragments is FS-independent, some high molecular weight sub-chromosomal fragments are also trapped within wells under the standard PFGE conditions. This trapping can be minimized by lowering the field strength and increasing the time of electrophoresis. We discuss how breakage of nicked DNA may be mechanistically linked to trapping. Our results suggest how to optimize conditions for PFGE when quantifying chromosomal fragmentation induced by DNA damage. PMID:23770235

  6. A Novel Strategy for Characterization of Glycosylated Proteins Separated by Gel Electrophoresis

    DEFF Research Database (Denmark)

    Larsen, Martin; Skottrup, Peter; Enghild, Jan J.

    2005-01-01

    . We present a new technique, which allows full characterization of low amounts of glycoproteins separated by gel electrophoresis. The method takes advantage of sequential specific and non-specific enzymatic treatment, followed by selective purification and characterization of the glycopeptides using...

  7. Determination of molecular weight of silk fibroin by non-gel sieving capillary electrophoresis.

    Science.gov (United States)

    Wei, Wei; Zhang, Yaopeng; Shao, Huili; Hu, Xuechao

    2010-01-01

    A simple non-gel sieving capillary electrophoresis (NGSCE) method was established to determine the MW of silk fibroin using CE. The background electrolyte with a pH of 8.8 was based on three components: polyethylene glycol, tris(hydroxymethyl)aminomethane, and sodium dodecyl sulfate (SDS). NGSCE showed a good linear relationship with satisfactory reproducibility between the migration time and the MW of standard proteins. It was found that the regenerated silk fibroin had an MW around 83 kDa with a wide MW distribution (MWD). This absolute value is lower than the result obtained from SDS-polyacrylamide gel electrophoresis due to the different principles of the methods, but their similar MWD shapes indicated that NGSCE could be a feasible, highly sensitive, rapid method for determination of the MW of silk fibroin.

  8. Determination of size distribution of small DNA fragments by polyacrylamide gel electrophoresis

    International Nuclear Information System (INIS)

    Lau How Mooi

    1998-01-01

    Size distribution determination of DNA fragments can be normally determined by the agarose gel electrophoresis, including the normal DNA banding pattern analysis. However this method is only good for large DNA, such as the DNA of the size of kilo base pairs to mega base pairs range. DNA of size less than kilo base pairs is difficult to be quantified by the agarose gel method. Polyacrylamide gel electrophoresis however can be used to measure the quantity of DNA fragments of size less than kilo base pairs in length, down to less than ten base pairs. This method is good for determining the quantity of the smaller size DNA, single stranded polymers or even some proteins, if the known standards are available. In this report detail description of the method of preparing the polyacrylamide gel, and the experimental set up is discussed. Possible uses of this method, and the comparison with the standard sizes of DNA is also shown. This method is used to determine the distribution of the amount of the fragmented DNA after the Calf-thymus DNA has been exposed to various types of radiation and of different doses. The standards were used to determine the sizes of the fragmented Calf-thymus DNA. The higher the dose the higher is the amount of the smaller size DNA measured

  9. New Approach for Segmentation and Quantification of Two-Dimensional Gel Electrophoresis Images

    DEFF Research Database (Denmark)

    Anjo, Antonio dos; Laurell Blom Møller, Anders; Ersbøll, Bjarne Kjær

    2011-01-01

    Motivation: Detection of protein spots in two-dimensional gel electrophoresis images (2-DE) is a very complex task and current approaches addressing this problem still suffer from significant shortcomings. When quantifying a spot, most of the current software applications include a lot of backgro...

  10. Investigation of two different anoxia models by 2-dimensional gel electrophoresis

    DEFF Research Database (Denmark)

    Wulff, Tune; Jessen, Flemming; Hoffmann, Else Kay

    2006-01-01

    anoxia obtained by NaN3 is a widely used model for simulating anoxia (Ossum et al., 2004). The effects of anoxia were studied by protein expression analysis using 2-dimensional gel electrophoresis followed by MS/MS. In this way we were able to separate more than 1500 protein spots with an apparent range...

  11. Coupling Sodium Dodecyl Sulfate–Capillary Polyacrylamide Gel Electrophoresis with MALDI-TOF-MS via a PTFE Membrane

    Science.gov (United States)

    Lu, Joann J.; Zhu, Zaifang; Wang, Wei; Liu, Shaorong

    2011-01-01

    Sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) is a fundamental analytical technique for proteomic research, and SDS–capillary gel electrophoresis (CGE) is its miniaturized version. Compared to conventional slab-gel electrophoresis, SDS-CGE has many advantages such as increased separation efficiency, reduced separation time and automated operation. SDS-CGE is not widely accepted in proteomic research primarily due to the difficulties in identifying the well-resolved proteins. MALDI–TOF–MS is an outstanding platform for protein identifications. Coupling the two would solve the problem but is extremely challenging because the MS detector has no access to the SDS-CGE resolved proteins and the SDS interferes with MS detection. In this work we introduce an approach to address these issues. We discover that poly(tetrafluoroethylene) (PTFE) membranes are excellent materials for collecting SDS-CGE separated proteins. We demonstrate that we can wash off the SDS bound to the collected proteins and identify these proteins on-membrane with MALDI-TOF-MS. We also show that we can immunoblot and Coomassie-stain the proteins collected on these membranes. PMID:21309548

  12. Characterization of Seed Storage Proteins from Chickpea Using 2D Electrophoresis Coupled with Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Pramod Kumar Singh

    2016-01-01

    Full Text Available Proteomic analysis was employed to map the seed storage protein network in landrace and cultivated chickpea accessions. Protein extracts were separated by two-dimensional gel electrophoresis (2D-GE across a broad range 3.0–10.0 immobilized pH gradient (IPG strips. Comparative elucidation of differentially expressed proteins between two diverse geographically originated chickpea accessions was carried out using 2D-GE coupled with mass spectrometry. A total of 600 protein spots were detected in these accessions. In-gel protein expression patterns revealed three protein spots as upregulated and three other as downregulated. Using trypsin in-gel digestion, these differentially expressed proteins were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS which showed 45% amino acid homology of chickpea seed storage proteins with Arabidopsis thaliana.

  13. Mapping and identification of interferon gamma-regulated HeLa cell proteins separated by immobilized pH gradient two-dimensional gel electrophoresis

    DEFF Research Database (Denmark)

    Shaw, A.; Larsen, M.; Roepstorff, P.

    1999-01-01

    magnitude of IFN-gamma responsive genes has been reported previously. Our goal is to identify and map IFN-gamma-regulated HeLa cell proteins to the two-dimensional polyacrylamide gel electrophoresis with the immobilized pH gradient (IPG) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) system...

  14. A spot-matching method using cumulative frequency matrix in 2D gel images

    Science.gov (United States)

    Han, Chan-Myeong; Park, Joon-Ho; Chang, Chu-Seok; Ryoo, Myung-Chun

    2014-01-01

    A new method for spot matching in two-dimensional gel electrophoresis images using a cumulative frequency matrix is proposed. The method improves on the weak points of the previous method called ‘spot matching by topological patterns of neighbour spots’. It accumulates the frequencies of neighbour spot pairs produced through the entire matching process and determines spot pairs one by one in order of higher frequency. Spot matching by frequencies of neighbour spot pairs shows a fairly better performance. However, it can give researchers a hint for whether the matching results can be trustworthy or not, which can save researchers a lot of effort for verification of the results. PMID:26019609

  15. Genetic heterogeneity of Campylobacter concisus determined by pulsed field gel electrophoresis-based macrorestriction profiling

    DEFF Research Database (Denmark)

    Matsheka, M.I.; Elisha, B.G.; Lastovica, A.L.

    2002-01-01

    1 for pulsed field gel electrophoresis-based genotyping. Subsequently, 53 strains of C concisus, principally from cases of diarrhoea in children, were examined. Fifty-one distinct patterns were obtained, indicating the high discriminatory potential of the method. Patterns comprised between one...... comprised of several genomospecies. The pulsed field gel electrophoresis typing method described here has considerable potential for molecular epidemiological studies of C concisus and may be a useful adjunctive method for helping to resolve key taxonomic issues for this species....... and 14 restriction fragments, with type and reference strains of two well-defined genomospecies of oral and faecal origin containing six and 12 fragments respectively. Our results show that C concisus is genetically diverse and suggest the species as currently defined to be a taxonomic continuum...

  16. Progress in two-dimensional polyacrylamide gel electrophoresis and application in radiation research

    International Nuclear Information System (INIS)

    Wang Zhidong; Chen Xiaohua

    2003-01-01

    Two-dimensional polyacrylamide gel electrophoresis is the key separation technique in proteomics research, which is designed by protein character: molecular weight and PI. Some progress has been made in disease mechanism detection, tumor indicator research and drug development. This technique also has some potential application in radiation research

  17. An effective placental cotyledons proteins extraction method for 2D gel electrophoresis.

    Science.gov (United States)

    Tan, Niu J; Daim, Leona D J; Jamil, Amilia A M; Mohtarrudin, Norhafizah; Thilakavathy, Karuppiah

    2017-03-01

    Effective protein extraction is essential especially in producing a well-resolved proteome on 2D gels. A well-resolved placental cotyledon proteome, with good reproducibility, have allowed researchers to study the proteins underlying the physiology and pathophysiology of pregnancy. The aim of this study is to determine the best protein extraction protocol for the extraction of protein from placental cotyledons tissues for a two-dimensional gel electrophoresis (2D-GE). Based on widely used protein extraction strategies, 12 different extraction methodologies were carefully selected, which included one chemical extraction, two mechanical extraction coupled protein precipitations, and nine chemical extraction coupled protein precipitations. Extracted proteins were resolved in a one-dimensional gel electrophoresis and 2D-GE; then, it was compared with set criteria: extraction efficacy, protein resolution, reproducibility, and recovery efficiency. Our results revealed that a better profile was obtained by chemical extraction in comparison to mechanical extraction. We further compared chemical extraction coupled protein precipitation methodologies, where the DNase/lithium chloride-dense sucrose homogenization coupled dichloromethane-methanol precipitation (DNase/LiCl-DSH-D/MPE) method showed good protein extraction efficiency. This, however, was carried out with the best protein resolution and proteome reproducibility on 2D-gels. DNase/LiCl-DSH-D/MPE was efficient in the extraction of proteins from placental cotyledons tissues. In addition, this methodology could hypothetically allow the protein extraction of any tissue that contains highly abundant lipid and glycogen. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Technical improvement to prevent DNA degradation of Leptospira spp. in pulsed field gel electrophoresis.

    Science.gov (United States)

    Ribeiro, R L; Machry, L; Brazil, J M V; Ramos, T M V; Avelar, K E S; Pereira, M M

    2009-08-01

    Leptospirosis is a public health problem. Infection with pathogenic Leptospira occurs by exposure to many environments and is traditionally associated with occupational risk activities. Pulsed-field gel electrophoresis was used to investigate the epidemiological relatedness among Leptospira isolates. However, analysis by PFGE yielded inconclusive data as a result of extensive DNA degradation. This degradation can be significantly reduced by the inclusion of thiourea in the electrophoresis buffer, improving the analysis of DNA banding patterns.

  19. EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Chevreux, Sylviane; Roudeau, Stephane; Deves, Guillaume; Ortega, Richard [Laboratoire de Chimie Nucleaire Analytique et Bioenvironnementale, CNRS UMR5084, Universite Bordeaux 1, Chemin du Solarium, F-33175 Gradignan cedex (France); Solari, Pier Lorenzo [Synchrotron SOLEIL, L' Orme des Merisiers, BP 48, F-91192 Gif-sur-Yvette cedex, Saint-Aubin (France); Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis, E-mail: ortega@cenbg.in2p3.f [FAME, ESRF, 6 rue Jules Horowitz, BP220, F-38043 Grenoble cedex (France)

    2009-11-15

    Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

  20. EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

    Science.gov (United States)

    Chevreux, Sylviane; Solari, Pier Lorenzo; Roudeau, Stéphane; Deves, Guillaume; Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis; Ortega, Richard

    2009-11-01

    Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

  1. EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

    International Nuclear Information System (INIS)

    Chevreux, Sylviane; Roudeau, Stephane; Deves, Guillaume; Ortega, Richard; Solari, Pier Lorenzo; Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis

    2009-01-01

    Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

  2. Pulsed-field Gel Electrophoresis for Salmonella Infection Surveillance, Texas, USA, 2007

    Centers for Disease Control (CDC) Podcasts

    2010-06-14

    This podcast describes monitoring of the use of pulsed-field gel electrophoresis for Salmonella surveillance in Houston, Texas. CDC microbiologist Peter Gerner-Smidt discusses the importance of the PulseNet national database in surveillance of food-borne infections.  Created: 6/14/2010 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 6/14/2010.

  3. Performing Isoelectric Focusing and Simultaneous Fractionation of Proteins on A Rotary Valve Followed by Sodium Dodecyl – Polyacrylamide Gel Electrophoresis

    Science.gov (United States)

    Wang, Wei; Lu, Joann J.; Gu, Congying; Zhou, Lei; Liu, Shaorong

    2013-01-01

    In this technical note, we design and fabricate a novel rotary valve and demonstrate its feasibility for performing isoelectric focusing and simultaneous fractionation of proteins, followed by sodium dodecyl – polyacrylamide gel electrophoresis. The valve has two positions. In one position, the valve routes a series of capillary loops together into a single capillary tube where capillary isoelectric focusing (CIEF) is performed. By switching the valve to another position, the CIEF-resolved proteins in all capillary loops are isolated simultaneously, and samples in the loops are removed and collected in vials. After the collected samples are briefly processed, they are separated via sodium dodecyl – polyacrylamide gel electrophoresis (SDS-PAGE, the 2nd-D separation) on either a capillary gel electrophoresis instrument or a slab-gel system. The detailed valve configuration is illustrated, and the experimental conditions and operation protocols are discussed. PMID:23819755

  4. Extracting information from two-dimensional electrophoresis gels by partial least squares regression

    DEFF Research Database (Denmark)

    Jessen, Flemming; Lametsch, R.; Bendixen, E.

    2002-01-01

    of all proteins/spots in the gels. In the present study it is demonstrated how information can be extracted by multivariate data analysis. The strategy is based on partial least squares regression followed by variable selection to find proteins that individually or in combination with other proteins vary......Two-dimensional gel electrophoresis (2-DE) produces large amounts of data and extraction of relevant information from these data demands a cautious and time consuming process of spot pattern matching between gels. The classical approach of data analysis is to detect protein markers that appear...... or disappear depending on the experimental conditions. Such biomarkers are found by comparing the relative volumes of individual spots in the individual gels. Multivariate statistical analysis and modelling of 2-DE data for comparison and classification is an alternative approach utilising the combination...

  5. Optimized sample preparation for two-dimensional gel electrophoresis of soluble proteins from chicken bursa of Fabricius

    Directory of Open Access Journals (Sweden)

    Zheng Xiaojuan

    2009-10-01

    Full Text Available Abstract Background Two-dimensional gel electrophoresis (2-DE is a powerful method to study protein expression and function in living organisms and diseases. This technique, however, has not been applied to avian bursa of Fabricius (BF, a central immune organ. Here, optimized 2-DE sample preparation methodologies were constructed for the chicken BF tissue. Using the optimized protocol, we performed further 2-DE analysis on a soluble protein extract from the BF of chickens infected with virulent avibirnavirus. To demonstrate the quality of the extracted proteins, several differentially expressed protein spots selected were cut from 2-DE gels and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS. Results An extraction buffer containing 7 M urea, 2 M thiourea, 2% (w/v 3-[(3-cholamidopropyl-dimethylammonio]-1-propanesulfonate (CHAPS, 50 mM dithiothreitol (DTT, 0.2% Bio-Lyte 3/10, 1 mM phenylmethylsulfonyl fluoride (PMSF, 20 U/ml Deoxyribonuclease I (DNase I, and 0.25 mg/ml Ribonuclease A (RNase A, combined with sonication and vortex, yielded the best 2-DE data. Relative to non-frozen immobilized pH gradient (IPG strips, frozen IPG strips did not result in significant changes in the 2-DE patterns after isoelectric focusing (IEF. When the optimized protocol was used to analyze the spleen and thymus, as well as avibirnavirus-infected bursa, high quality 2-DE protein expression profiles were obtained. 2-DE maps of BF of chickens infected with virulent avibirnavirus were visibly different and many differentially expressed proteins were found. Conclusion These results showed that method C, in concert extraction buffer IV, was the most favorable for preparing samples for IEF and subsequent protein separation and yielded the best quality 2-DE patterns. The optimized protocol is a useful sample preparation method for comparative proteomics analysis of chicken BF tissues.

  6. Two-dimensional polyacrylamide gel analysis of Plodia interpunctella granulosis virus

    International Nuclear Information System (INIS)

    Russell, D.L.; Consigli, R.A.

    1986-01-01

    The structural polypeptides of purified Plodia interpunctella granulosis virus were analyzed by three different two-dimensional gel systems. Isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis allowed resolution of 53 acidic polypeptides in the enveloped nucleocapsid of the virus ranging in molecular weight from 97,300 to 8000. Nine of these polypeptides were shown to be glycoproteins by the technique of radiolabeled lectin blotting. Separation of the granulin in this system allowed resolution of five species, all of which have identical tryptic peptide maps. This matrix protein was demonstrated to be a phosphoglycoprotein by radiolabeled lectin blotting and acid phosphatase dephosphorylation. Nonequilibrium pH gel electrophoresis followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis allowed resolution of the major basic protein of the virus, VP12, from a more acidic protein of the same molecular weight. Tryptic peptide analysis demonstrated that these two proteins were indeed different and acid urea gels followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis allowed localization of the acidic protein to the envelope and the basic protein to the nucleocapsid of the virus. Finally, probing of the separated envelope nucleocapsid proteins in both the isoelectric focusing and nonequilibrium pH gel electrophoresis two-dimensional systems after transfer to nitrocellulose with iodinated, purified viral proteins allowed further insight into reactions which may be important in the maintenance of the virion structure

  7. Immunochemical characterization of Mycobacterium leprae antigens by the SDS-polyacrylamide gel electrophoresis immunoperoxidase technique (SGIP) using patients' sera

    NARCIS (Netherlands)

    Klatser, P. R.; van Rens, M. M.; Eggelte, T. A.

    1984-01-01

    In this study the SDS-polyacrylamide gel electrophoresis immunoperoxidase (SGIP) assay was used for characterizing the antigenic components of Mycobacterium leprae using patients' sera. This technique involved the separation of mycobacterial sonicates on SDS-polyacrylamide gels, longitudinal

  8. pI-Control in comparative fluorescence gel electrophoresis (CoFGE) using amphoteric azo dyes

    Czech Academy of Sciences Publication Activity Database

    Hanneken, M.; Šlais, Karel; König, S.

    2015-01-01

    Roč. 3, JUN (2015), s. 221-228 ISSN 2352-3409 Institutional support: RVO:68081715 Keywords : 2D-PAGE * CoFGE * Gel electrophoresis Subject RIV: CB - Analytical Chemistry, Separation http://www.sciencedirect.com/science/article/pii/S2352340915000414

  9. Serum protein fractionation using supported molecular matrix electrophoresis.

    Science.gov (United States)

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

    2013-08-01

    Supported molecular matrix electrophoresis (SMME), in which a hydrophilic polymer such as PVA serves as a support within a porous PVDF membrane, was recently developed. This method is similar to cellulose acetate membrane electrophoresis but differs in the compatibility to glycan analysis of the separated bands. In this report, we describe the first instance of the application of SMME to human serum fractionation, and demonstrate the differences with serum fractionation by cellulose acetate membrane electrophoresis. The SMME membrane exhibited almost no EOF during electrophoresis, unlike the cellulose acetate membrane, but afforded comparative results for serum fractionation. The visualization of each fraction was achieved by conventional staining with dye such as Direct Blue-71, and objective quantification was obtained by densitometry after inducing membrane transparency with 1-nonene. Immunostaining was also achieved. Moreover, mass spectrometric analysis of both N-linked and O-linked glycans from the separated bands was demonstrated. Serum fractionation and glycan profiling of each fraction using SMME will enable novel insights into the relationships between various glycosylation profiles and disease states. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Quantitative profiling of O-glycans by electrospray ionization- and matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry after in-gel derivatization with isotope-coded 1-phenyl-3-methyl-5-pyrazolone

    International Nuclear Information System (INIS)

    Sić, Siniša; Maier, Norbert M.; Rizzi, Andreas M.

    2016-01-01

    The potential and benefits of isotope-coded labeling in the context of MS-based glycan profiling are evaluated focusing on the analysis of O-glycans. For this purpose, a derivatization strategy using d_0/d_5-1-phenyl-3-methyl-5-pyrazolone (PMP) is employed, allowing O-glycan release and derivatization to be achieved in one single step. The paper demonstrates that this release and derivatization reaction can be carried out also in-gel with only marginal loss in sensitivity compared to in-solution derivatization. Such an effective in-gel reaction allows one to extend this release/labeling method also to glycoprotein/glycoform samples pre-separated by gel-electrophoresis without the need of extracting the proteins/digested peptides from the gel. With highly O-glycosylated proteins (e.g. mucins) LODs in the range of 0.4 μg glycoprotein (100 fmol) loaded onto the electrophoresis gel can be attained, with minor glycosylated proteins (like IgAs, FVII, FIX) the LODs were in the range of 80–100 μg (250 pmol–1.5 nmol) glycoprotein loaded onto the gel. As second aspect, the potential of isotope coded labeling as internal standardization strategy for the reliable determination of quantitative glycan profiles via MALDI-MS is investigated. Towards this goal, a number of established and emerging MALDI matrices were tested for PMP-glycan quantitation, and their performance is compared with that of ESI-based measurements. The crystalline matrix 2,6-dihydroxyacetophenone (DHAP) and the ionic liquid matrix N,N-diisopropyl-ethyl-ammonium 2,4,6-trihydroxyacetophenone (DIEA-THAP) showed potential for MALDI-based quantitation of PMP-labeled O-glycans. We also provide a comprehensive overview on the performance of MS-based glycan quantitation approaches by comparing sensitivity, LOD, accuracy and repeatability data obtained with RP-HPLC-ESI-MS, stand-alone nano-ESI-MS with a spray-nozzle chip, and MALDI-MS. Finally, the suitability of the isotope-coded PMP labeling strategy for

  11. Quantitative profiling of O-glycans by electrospray ionization- and matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry after in-gel derivatization with isotope-coded 1-phenyl-3-methyl-5-pyrazolone

    Energy Technology Data Exchange (ETDEWEB)

    Sić, Siniša; Maier, Norbert M.; Rizzi, Andreas M., E-mail: Andreas.Rizzi@univie.ac.at

    2016-09-07

    The potential and benefits of isotope-coded labeling in the context of MS-based glycan profiling are evaluated focusing on the analysis of O-glycans. For this purpose, a derivatization strategy using d{sub 0}/d{sub 5}-1-phenyl-3-methyl-5-pyrazolone (PMP) is employed, allowing O-glycan release and derivatization to be achieved in one single step. The paper demonstrates that this release and derivatization reaction can be carried out also in-gel with only marginal loss in sensitivity compared to in-solution derivatization. Such an effective in-gel reaction allows one to extend this release/labeling method also to glycoprotein/glycoform samples pre-separated by gel-electrophoresis without the need of extracting the proteins/digested peptides from the gel. With highly O-glycosylated proteins (e.g. mucins) LODs in the range of 0.4 μg glycoprotein (100 fmol) loaded onto the electrophoresis gel can be attained, with minor glycosylated proteins (like IgAs, FVII, FIX) the LODs were in the range of 80–100 μg (250 pmol–1.5 nmol) glycoprotein loaded onto the gel. As second aspect, the potential of isotope coded labeling as internal standardization strategy for the reliable determination of quantitative glycan profiles via MALDI-MS is investigated. Towards this goal, a number of established and emerging MALDI matrices were tested for PMP-glycan quantitation, and their performance is compared with that of ESI-based measurements. The crystalline matrix 2,6-dihydroxyacetophenone (DHAP) and the ionic liquid matrix N,N-diisopropyl-ethyl-ammonium 2,4,6-trihydroxyacetophenone (DIEA-THAP) showed potential for MALDI-based quantitation of PMP-labeled O-glycans. We also provide a comprehensive overview on the performance of MS-based glycan quantitation approaches by comparing sensitivity, LOD, accuracy and repeatability data obtained with RP-HPLC-ESI-MS, stand-alone nano-ESI-MS with a spray-nozzle chip, and MALDI-MS. Finally, the suitability of the isotope-coded PMP labeling

  12. Gel-aided sample preparation (GASP)--a simplified method for gel-assisted proteomic sample generation from protein extracts and intact cells.

    Science.gov (United States)

    Fischer, Roman; Kessler, Benedikt M

    2015-04-01

    We describe a "gel-assisted" proteomic sample preparation method for MS analysis. Solubilized protein extracts or intact cells are copolymerized with acrylamide, facilitating denaturation, reduction, quantitative cysteine alkylation, and matrix formation. Gel-aided sample preparation has been optimized to be highly flexible, scalable, and to allow reproducible sample generation from 50 cells to milligrams of protein extracts. This methodology is fast, sensitive, easy-to-use on a wide range of sample types, and accessible to nonspecialists. © 2014 The Authors. PROTEOMICS published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Correlation of acidic and basic carrier ampholyte and immobilized pH gradient two-dimensional gel electrophoresis patterns based on mass spectrometric protein identification

    DEFF Research Database (Denmark)

    Nawrocki, A; Larsen, Martin Røssel; Podtelejnikov, A V

    1998-01-01

    Separation of proteins on either carrier ampholyte-based or immobilized pH gradient-based two-dimensional (2-D) gels gives rise to electrophoretic patterns that are difficult to compare visually. In this paper we have used matrix-assisted laser desorption/ionization mass spectrometry (MALDI......-MS) to determine the identities of 335 protein spots in these two 2-D gel systems, including a substantial number of basic proteins which had never been identified before. Proteins that were identified in both gel systems allowed us to cross-reference the gel patterns. Vector analysis of these cross...

  14. A CCD-based system for the detection of DNA in electrophoresis gels by UV absorption

    International Nuclear Information System (INIS)

    Mahon, A.R.; MacDonald, J.H.; Mainwood, A.; Ott, R.J.

    1999-01-01

    A method and apparatus for the detection and quantification of large fragments of unlabelled nucleic acids in agarose gels is presented. The technique is based on ultraviolet (UV) absorption by nucleotides. A deuterium source illuminates individual sample lanes of an electrophoresis gel via an array of optical fibres. As DNA bands pass through the illuminated region of the gel the amount of UV light transmitted is reduced because of absorption by the DNA. During electrophoresis the regions of DNA are detected on-line using a UV-sensitive charge coupled device (CCD). As the absorption coefficient is proportional to the mass of DNA the technique is inherently quantitative. The mass of DNA in a region of the gel is approximately proportional to the integrated signal in the corresponding section of the CCD image. This system currently has a detection limit of less than 1.25 ng compared with 2-10 ng for the most popular conventional technique, ethidium bromide (EtBr) staining. In addition the DNA sample remains in its native state. The removal of the carcinogenic dye from the detection procedure greatly reduces associated biological hazards. (author)

  15. Study of total seed proteins pattern of sesame (sesamum indicum l.) landraces via sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page)

    International Nuclear Information System (INIS)

    Akbar, F.; Shinwari, Z.K.

    2012-01-01

    The sesame (Sesamum indicum L.) germplasm, comprising of 105 accessions was characterized for total seed storage proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The germplasm was collected from diverse agro-ecological regions of Pakistan. To our information, no studies have yet been carried out in Pakistan on the genetic evaluation of sesame genotypes based on total seed protein. Total seed proteins were electrophoretically separated on 12% polyacrylamide gels by standard protocols. A total of 20 polypeptide bands were observed, of which 14 (70%) were polymorphic and 6 (30%) were monomorphic, with molecular weight ranging from 13.5 to 100 kDa. Six bands i.e., 7, 11, 12, 15, 16 and 18 were common in all genotypes. Similarity coefficients varied fro m 0.50 to 1.00. The dendrogram based on dissimilarity matrix using unweighted pair group method with arithmetic averages (UPGMA) separated all sesame accessions into three main groups i.e., A, B, C, comprising 89, 14 and 2 genotypes, respectively. Overall a low to medium level of genetic variability was observed for SDS-PAGE (single dimension). As SDS-PAGE alone did not reveal high level of genetic variability, hence 2-D gel electrophoresis along with other advanced type DNA markers and more number of sesame accessions from all over the country are recommended for the future genetic evaluation. Our investigation will significantly support the classification, development, genetic evaluation and conservation of sesame germplasm in Pakistan. (author)

  16. Study of total seed proteins pattern of sesame (sesamum indicum l.) landraces via sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page)

    Energy Technology Data Exchange (ETDEWEB)

    Akbar, F; Shinwari, Z K [Quaid-e-Azam University, Islamabad (Pakistan). Dept. of Biotechnology; Yousif, N; Masood, M S [Institute of Agri-Biotechnology and Genetic Resources, Islamabad (Pakistan)

    2012-11-15

    The sesame (Sesamum indicum L.) germplasm, comprising of 105 accessions was characterized for total seed storage proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The germplasm was collected from diverse agro-ecological regions of Pakistan. To our information, no studies have yet been carried out in Pakistan on the genetic evaluation of sesame genotypes based on total seed protein. Total seed proteins were electrophoretically separated on 12% polyacrylamide gels by standard protocols. A total of 20 polypeptide bands were observed, of which 14 (70%) were polymorphic and 6 (30%) were monomorphic, with molecular weight ranging from 13.5 to 100 kDa. Six bands i.e., 7, 11, 12, 15, 16 and 18 were common in all genotypes. Similarity coefficients varied fro m 0.50 to 1.00. The dendrogram based on dissimilarity matrix using unweighted pair group method with arithmetic averages (UPGMA) separated all sesame accessions into three main groups i.e., A, B, C, comprising 89, 14 and 2 genotypes, respectively. Overall a low to medium level of genetic variability was observed for SDS-PAGE (single dimension). As SDS-PAGE alone did not reveal high level of genetic variability, hence 2-D gel electrophoresis along with other advanced type DNA markers and more number of sesame accessions from all over the country are recommended for the future genetic evaluation. Our investigation will significantly support the classification, development, genetic evaluation and conservation of sesame germplasm in Pakistan. (author)

  17. Separation of hydrolytically active components of cellulase from Myrothecium verrucaria by starch gel electrophoresis

    NARCIS (Netherlands)

    Ritter, F.J.; Prins-van der Meulen, P.Y.F.; Marel, T. van der

    1968-01-01

    Using starch gel electrophoresis according to Smithies, desalted crude cellulase from Myrothecium verrucqria was separated into at least 12 protein zones. These were tested on their activity towards p-nitrophenyl-β-D-glucoside, sodium carboxymethylcellulose and α-cellulose. They were all

  18. Application of Fluorescence Two-Dimensional Difference In-Gel Electrophoresis as a Proteomic Biomarker Discovery Tool in Muscular Dystrophy Research

    Science.gov (United States)

    Carberry, Steven; Zweyer, Margit; Swandulla, Dieter; Ohlendieck, Kay

    2013-01-01

    In this article, we illustrate the application of difference in-gel electrophoresis for the proteomic analysis of dystrophic skeletal muscle. The mdx diaphragm was used as a tissue model of dystrophinopathy. Two-dimensional gel electrophoresis is a widely employed protein separation method in proteomic investigations. Although two-dimensional gels usually underestimate the cellular presence of very high molecular mass proteins, integral membrane proteins and low copy number proteins, this method is extremely powerful in the comprehensive analysis of contractile proteins, metabolic enzymes, structural proteins and molecular chaperones. This gives rise to two-dimensional gel electrophoretic separation as the method of choice for studying contractile tissues in health and disease. For comparative studies, fluorescence difference in-gel electrophoresis has been shown to provide an excellent biomarker discovery tool. Since aged diaphragm fibres from the mdx mouse model of Duchenne muscular dystrophy closely resemble the human pathology, we have carried out a mass spectrometry-based comparison of the naturally aged diaphragm versus the senescent dystrophic diaphragm. The proteomic comparison of wild type versus mdx diaphragm resulted in the identification of 84 altered protein species. Novel molecular insights into dystrophic changes suggest increased cellular stress, impaired calcium buffering, cytostructural alterations and disturbances of mitochondrial metabolism in dystrophin-deficient muscle tissue. PMID:24833232

  19. Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin

    Science.gov (United States)

    Gardano, Laura; Holland, Linda; Oulton, Rena; Le Bihan, Thierry; Harrington, Lea

    2012-01-01

    Telomeres, the ends of linear chromosomes, safeguard against genome instability. The enzyme responsible for extension of the telomere 3′ terminus is the ribonucleoprotein telomerase. Whereas telomerase activity can be reconstituted in vitro with only the telomerase RNA (hTR) and telomerase reverse transcriptase (TERT), additional components are required in vivo for enzyme assembly, stability and telomere extension activity. One such associated protein, dyskerin, promotes hTR stability in vivo and is the only component to co-purify with active, endogenous human telomerase. We used oligonucleotide-based affinity purification of hTR followed by native gel electrophoresis and in-gel telomerase activity detection to query the composition of telomerase at different purification stringencies. At low salt concentrations (0.1 M NaCl), affinity-purified telomerase was ‘supershifted’ with an anti-dyskerin antibody, however the association with dyskerin was lost after purification at 0.6 M NaCl, despite the retention of telomerase activity and a comparable yield of hTR. The interaction of purified hTR and dyskerin in vitro displayed a similar salt-sensitive interaction. These results demonstrate that endogenous human telomerase, once assembled and active, does not require dyskerin for catalytic activity. Native gel electrophoresis may prove useful in the characterization of telomerase complexes under various physiological conditions. PMID:22187156

  20. Denaturing gradient gel electrophoresis

    International Nuclear Information System (INIS)

    Kocherginskaya, S.A.; Cann, I.K.O.; Mackie, R.I.

    2005-01-01

    It is worthwhile considering that only some 30 species make up the bulk of the bacterial population in human faeces at any one time based on the classical cultivation-based approach. The situation in the rumen is similar. Thus, it is practical to focus on specific groups of interest within the complex community. These may be the predominant or the most active species, specific physiological groups or readily identifiable (genetic) clusters of phylogenetically related organisms. Several 16S rDNA fingerprinting techniques can be invaluable for selecting and monitoring sequences or phylogenetic groups of interest and are described below. Over the past few decades, considerable attention was focussed on the identification of pure cultures of microbes on the basis of genetic polymorphisms of DNA encoding rRNA such as ribotyping, amplified fragment length polymorphism and randomly amplified polymorphic DNA. However, many of these methods require prior cultivation and are less suitable for use in analysis of complex mixed populations although important in describing cultivated microbial diversity in molecular terms. Much less attention was given to molecular characterization of complex communities. In particular, research into diversity and community structure over time has been revolutionized by the advent of molecular fingerprinting techniques for complex communities. Denaturing or temperature gradient gel electrophoresis (DGGE/TGGE) methods have been successfully applied to the analysis of human, pig, cattle, dog and rodent intestinal populations

  1. Pulsed-field gel electrophoresis (PFGE): application in population structure studies of bovine mastitis-causing streptococci.

    Science.gov (United States)

    Santos-Sanches, Ilda; Chambel, Lélia; Tenreiro, Rogério

    2015-01-01

    Pulsed-field gel electrophoresis (PFGE) separates large DNA molecules by the use of an alternating electrical field, such that greater size resolution can be obtained when compared to normal agarose gel electrophoresis. PFGE is often employed to track pathogens and is a valuable typing scheme to detect and differentiate strains. Particularly, the contour-clamped homogeneous electric field (CHEF) PFGE system is considered to be the gold standard for use in epidemiological studies of many bacterial pathogens. Here we describe a PFGE protocol that was applicable to the study of bovine streptococci, namely, Streptococcus agalactiae (group B Streptococcus, GBS), Streptococcus dysgalactiae subsp. dysgalactiae (group C Streptococcus, GCS), and Streptococcus uberis-which are relevant pathogens causing mastitis, a highly prevalent and costly disease in dairy industry due to antibiotherapy and loss in milk production.

  2. A sol-gel-modified poly(methyl methacrylate) electrophoresis microchip with a hydrophilic channel wall.

    Science.gov (United States)

    Chen, Gang; Xu, Xuejiao; Lin, Yuehe; Wang, Joseph

    2007-01-01

    A sol-gel method was employed to fabricate a poly(methyl methacrylate) (PMMA) electrophoresis microchip that contains a hydrophilic channel wall. To fabricate such a device, tetraethoxysilane (TEOS) was injected into the PMMA channel and was allowed to diffuse into the surface layer for 24 h. After removing the excess TEOS, the channel was filled with an acidic solution for 3 h. Subsequently, the channel was flushed with water and was pretreated in an oven to obtain a sol-gel-modified PMMA microchip. The water contact angle for the sol-gel-modified PMMA was approximately 27.4 degrees compared with approximately 66.3 degrees for the pure PMMA. In addition, the electro-osmotic flow increased from 2.13x10(-4) cm2 V(-1) s(-1) for the native-PMMA channel to 4.86x10(-4) cm2 V(-1) s(-1) for the modified one. The analytical performance of the sol-gel-modified PMMA microchip was demonstrated for the electrophoretic separation of several purines, coupled with amperometric detection. The separation efficiency of uric acid increased to 74,882.3 m(-1) compared with 14,730.5 m(-1) for native-PMMA microchips. The result of this simple modification is a significant improvement in the performance of PMMA for microchip electrophoresis and microfluidic applications.

  3. Radiation-induced DNA damage and repair in radiosensitive and radioresistant human tumour cells measured by field inversion gel electrophoresis

    International Nuclear Information System (INIS)

    Smeets, M.F.M.A.; Mooren, E.H.M.; Begg, A.C.

    1993-01-01

    Radiation-induced DNA damage induction and repair was measured in two human squamous carcinoma cell lines with differing radiosensitivities. Experiments were carried out with field inversion gel electrophoresis (FIGE), adapted to measure DNA double strand break (DSB) induction and repair in unlabelled cells. The sensitivity of the method was increased by introducing a hybridization membrane into the agarose gel. Damaged DNA accumulated on one spot on the membrane resulting in high local concentrations. This DNA was quantified using radioactively-labelled total human DNA as a probe. Radiosensitivity differences at physiological temperatures could not be explained by differences in either induction or repair of DNA damage as measured by pulsed field gel electrophoresis. (author)

  4. Optimization of Large Gel 2D Electrophoresis for Proteomic Studies of Skeletal Muscle

    Science.gov (United States)

    Reed, Patrick W.; Densmore, Allison; Bloch, Robert J.

    2013-01-01

    We describe improved methods for large format, 2-dimensional gel electrophoresis (2-DE) that improve protein solubility and recovery, minimize proteolysis, and reduce the loss of resolution due to contaminants and manipulations of the gels, and thus enhance quantitative analysis of protein spots. Key modifications are: (i) the use of 7M urea + 2 M thiourea, instead of 9M urea, in sample preparation and in the tops of the gel tubes; (ii) standardized deionization of all solutions containing urea with a mixed bed ion exchange resin and removal of urea from the electrode solutions; and (iii) use of a new gel tank and cooling device that eliminate the need to run two separating gels in the SDS dimension. These changes make 2D-GE analysis more reproducible and sensitive, with minimal artifacts. Application of this method to the soluble fraction of muscle tissues reliably resolves ~1800 protein spots in adult human skeletal muscle and over 2800 spots in myotubes. PMID:22589104

  5. Improvement of fragment and primer selection for mutation detection by denaturing gradient gel electrophoresis

    NARCIS (Netherlands)

    Wu, Y; Hayes, VM; Osinga, J; Mulder, IM; Looman, MWG; Buys, CHCM; Hofstra, RMW

    1998-01-01

    Denaturing gradient gel electrophoresis (DGGE) is one of the most powerful methods for mutation detection currently available. For successful application the appropriate selection of PCR fragments and PCR primers is crucial. The sequence of interest should always be within the domain with the lowest

  6. Polymer gel dosimeter with AQUAJOINT® as hydrogel matrix

    Science.gov (United States)

    Maeyama, Takuya; Ishida, Yasuhiro; Kudo, Yoshihiro; Fukasaku, Kazuaki; Ishikawa, Kenichi L.; Fukunishi, Nobuhisa

    2018-05-01

    We report a polymer gel dosimeter based on a new gel matrix (AQUAJOINT®) that is a thermo-irreversible hydrogel formed by mixing two types of water-based liquids at room temperature. Normoxic N-vinylpyrrolidone-based polymer gels were prepared with AQUAJOINT® instead of gelatin. This AQUAJOINT®-based gel dosimeter exhibits a 2.5-fold increase in sensitivity over a gelatin-based gel dosimeter and a linear dose-response in the dose range of 0-8 Gy. This gel has heat resistance in a jar and controlled gel properties such as viscoelastic and mechanical characters, which may be useful for deformable polymer gel dosimetry.

  7. Optimized Protocol for Protein Extraction from the Breast Tissue that is Compatible with Two-Dimensional Gel Electrophoresis

    Directory of Open Access Journals (Sweden)

    Olena Zakharchenko

    2011-01-01

    Full Text Available Proteomics is a highly informative approach to analyze cancer-associated transformation in tissues. The main challenge to use a tissue for proteomics studies is the small sample size and difficulties to extract and preserve proteins. The choice of a buffer compatible with proteomics applications is also a challenge. Here we describe a protocol optimized for the most efficient extraction of proteins from the human breast tissue in a buffer compatible with two-dimensional gel electrophoresis (2D-GE. This protocol is based on mechanically assisted disintegration of tissues directly in the 2D-GE buffer. Our method is simple, robust and easy to apply in clinical practice. We demonstrate high quality of separation of proteins prepared according to the reported here protocol.

  8. Optimizing human synovial fluid preparation for two-dimensional gel electrophoresis.

    Science.gov (United States)

    Chen, Carl Pc; Hsu, Chih-Chin; Yeh, Wen-Lin; Lin, Hsiu-Chu; Hsieh, Sen-Yung; Lin, Shih-Cherng; Chen, Tai-Tzung; Chen, Max Jl; Tang, Simon Ft

    2011-10-11

    Proteome analysis is frequently applied in identifying the proteins or biomarkers in knee synovial fluids (SF) that are associated with osteoarthritis and other arthritic disorders. The 2-dimensional gel electrophoresis (2-DE) is the technique of choice in these studies. Disease biomarkers usually appear in low concentrations and may be masked by high abundant proteins. Therefore, the main aim of this study was to find the most suitable sample preparation method that can optimize the expression of proteins on 2-DE gels that can be used to develop a reference proteome picture for non-osteoarthritic knee synovial fluid samples. Proteome pictures obtained from osteoarthritic knee synovial fluids can then be compared with the reference proteome pictures obtained in this study to assist us in identifying the disease biomarkers more correctly. The proteomic tool of 2-DE with immobilized pH gradients was applied in this study. A total of 12 2-DE gel images were constructed from SF samples that were free of osteoarthritis. In these samples, 3 were not treated with any sample preparation methods, 3 were treated with acetone, 3 were treated with 2-DE Clean-Up Kit, and 3 were treated with the combination of acetone and 2-D Clean-Up Kit prior to 2-DE analysis. Gel images were analyzed using the PDQuest Basic 8.0.1 Analytical software. Protein spots that were of interest were excised from the gels and sent for identification by mass spectrometry. Total SF total protein concentration was calculated to be 21.98 ± 0.86 mg/mL. The untreated SF samples were detected to have 456 ± 33 protein spots on 2-DE gel images. Acetone treated SF samples were detected to have 320 ± 28 protein spots, 2-D Clean-Up Kit treated SF samples were detected to have 413 ± 31 protein spots, and the combined treatment method of acetone and 2-D Clean-Up Kit was detected to have 278 ± 26 protein spots 2-DE gel images. SF samples treated with 2-D Clean-Up Kit revealed clearer presentation of the isoforms

  9. Optimizing Human Synovial Fluid Preparation for Two-Dimensional Gel Electrophoresis

    Directory of Open Access Journals (Sweden)

    Chen Max JL

    2011-10-01

    Full Text Available Abstract Background Proteome analysis is frequently applied in identifying the proteins or biomarkers in knee synovial fluids (SF that are associated with osteoarthritis and other arthritic disorders. The 2-dimensional gel electrophoresis (2-DE is the technique of choice in these studies. Disease biomarkers usually appear in low concentrations and may be masked by high abundant proteins. Therefore, the main aim of this study was to find the most suitable sample preparation method that can optimize the expression of proteins on 2-DE gels that can be used to develop a reference proteome picture for non-osteoarthritic knee synovial fluid samples. Proteome pictures obtained from osteoarthritic knee synovial fluids can then be compared with the reference proteome pictures obtained in this study to assist us in identifying the disease biomarkers more correctly. Results The proteomic tool of 2-DE with immobilized pH gradients was applied in this study. A total of 12 2-DE gel images were constructed from SF samples that were free of osteoarthritis. In these samples, 3 were not treated with any sample preparation methods, 3 were treated with acetone, 3 were treated with 2-DE Clean-Up Kit, and 3 were treated with the combination of acetone and 2-D Clean-Up Kit prior to 2-DE analysis. Gel images were analyzed using the PDQuest Basic 8.0.1 Analytical software. Protein spots that were of interest were excised from the gels and sent for identification by mass spectrometry. Total SF total protein concentration was calculated to be 21.98 ± 0.86 mg/mL. The untreated SF samples were detected to have 456 ± 33 protein spots on 2-DE gel images. Acetone treated SF samples were detected to have 320 ± 28 protein spots, 2-D Clean-Up Kit treated SF samples were detected to have 413 ± 31 protein spots, and the combined treatment method of acetone and 2-D Clean-Up Kit was detected to have 278 ± 26 protein spots 2-DE gel images. SF samples treated with 2-D Clean-Up Kit

  10. Investigation on accordance of DNA double-strand break of blood between in vivo and in vitro irradiation using single cell gel electrophoresis

    International Nuclear Information System (INIS)

    Liu Qiang; Jiang Enhai; Li Jin; Tang Weisheng; Wang Zhiquan; Zhao Yongcheng; Fan Feiyue

    2006-01-01

    Objective: To observe the consistency of DNA double-strand break between in vivo and in vitro irradiation, as a prophase study in radiation biodosimetry using single cell gel electrophoresis (SCGE). Methods: Detect DNA double-strand break after whole-body and in vitro radiation in mice lymphocytes using neutral single cell gel electrophoresis. The comet images were processed by CASP software and all the data were analysed by SPSS12.0. Results: There is no difference between in vivo and in vitro irradiation group in HDNA%, TDNA%, CL, TL, TM and OTM. Conclusion: The result of neutral single cell gel electrophoresis shortly after in vitro irradiation can precisely reflect the DNA double-strand break of lymphocytes in whole-body irradiation. (authors)

  11. On gel electrophoresis of dielectric charged particles with hydrophobic surface: A combined theoretical and numerical study.

    Science.gov (United States)

    Majee, Partha Sarathi; Bhattacharyya, Somnath; Gopmandal, Partha Pratim; Ohshima, Hiroyuki

    2018-03-01

    A theoretical study on the gel electrophoresis of a charged particle incorporating the effects of dielectric polarization and surface hydrophobicity at the particle-liquid interface is made. A simplified model based on the weak applied field and low charge density assumption is also presented and compared with the full numerical model for a nonpolarizable particle to elucidate the nonlinear effects such as double layer polarization and relaxation as well as surface conduction. The main motivation of this study is to analyze the electrophoresis of the surface functionalized nanoparticle with tunable hydrophobicity or charged fluid drop in gel medium by considering the electrokinetic effects and hydrodynamic interactions between the particle and the gel medium. An effective medium approach, in which the transport in the electrolyte-saturated hydrogel medium is governed by the Brinkman equation, is adopted in the present analysis. The governing electrokinetic equations based on the conservation principles are solved numerically. The Navier-slip boundary condition along with the continuity condition of dielectric displacement are imposed on the surface of the hydrophobic polarizable particle. The impact of the slip length on the electrophoresis is profound for a thinner Debye layer, however, surface conduction effect also becomes significant for a hydrophobic particle. Impact of hydrophobicity and relaxation effects are higher for a larger particle. Dielectric polarization creates a reduction in its electrophoretic propulsion and has negligible impact at the thinner Debye length as well as lower gel screening length. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Silica gel matrix immobilized Chlorophyta hydrodictyon africanum ...

    African Journals Online (AJOL)

    Chlorophyta hydrodictyon africanum was immobilized on a silica gel matrix to improve its mechanical properties. The algae-silica gel adsorbent was used for batch sorption studies of a cationic dye, methylene blue (MB). Optimum adsorption was obtained with a dosage of 0.8 g bio sorbent. Results from sorption studies ...

  13. Direct detection of rutin-degrading isozymes with polyacrylamide gel electrophoresis.

    Science.gov (United States)

    Li, Yuping; Deng, Dandan; Zhang, Xuebin; Zhang, Haina; Wang, Cong; Chen, Peng

    2013-12-15

    Rutin-degrading enzymes (RDEs) specifically hydrolyze the glycosidic linkages of rutin, producing quercetin and rutinose. Here we report a reliable and sensitive polyacrylamide gel electrophoresis and staining method for the detection of RDE isozymes, which is based on the aqueous solubility difference between rutin and quercetin, as well as the ultraviolet absorbance of quercetin. With this novel method, we achieved a detection limit of 12 ng with 107 U of RDE activity, enabling us to detect at least five RDE isozymes in tartary buckwheat seeds. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Amplified fragment length polymorphism and pulsed field gel electrophoresis for subspecies differentiation of Serpulina pilosicoli

    DEFF Research Database (Denmark)

    Møller, Kristian; Jensen, Tim Kåre; Boye, Mette

    1999-01-01

    Pulsed field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP) were compared for their ability to differentiate between 50 porcine Serpulina pilosicoli isolates. Both techniques were highly sensitive, dividing the isolates into 36 and 38 groups, respectively. Due...

  15. Detection of hydrolysis of lipid post-translational modifications during gel-electrophoresis-based proteomic protocol

    Czech Academy of Sciences Publication Activity Database

    Žídková, Jitka; Řehulka, Pavel; Chmelík, Josef

    2007-01-01

    Roč. 7, č. 15 (2007), s. 2507-2510 ISSN 1615-9853 R&D Projects: GA MŠk 1M0570 Institutional research plan: CEZ:AV0Z40310501 Keywords : ester hydrolysis * gel electrophoresis * MALDI-TOF MS Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 5.479, year: 2007

  16. Alkaline gel electrophoresis assay to detect DNA strand breaks and repair mechanisms in Escherichia coli

    Directory of Open Access Journals (Sweden)

    José Carlos Pelielo de Mattos

    2008-12-01

    Full Text Available Reactive oxygen species (ROS can induce lesions in different cellular targets, including DNA. Stannous chloride (SnCl2 is a ROS generator, leading to lethality in Escherichia coli (E. coli, with the base excision repair (BER mechanism playing a role in this process. Many techniques have been developed to detect genotoxicity, as comet assay, in eukaryotic cells, and plasmid DNA agarose gel electrophoresis. In this study, an adaptation of the alkaline gel electrophoresis method was carried out to ascertain the induction of strand breaks by SnCl2 in bacterial DNA, from E. coli BER mutants, and its repair pathway. Results obtained show that SnCl2 was able to induce DNA strand breaks in all strains tested. Moreover, endonuclease IV and exonuclease III play a role in DNA repair. On the whole, data has shown that the alkaline gel electrophoresis assay could be used both for studying DNA strand breaks induction and for associated repair mechanisms.Espécies reativas de oxigênio (ERO podem induzir lesões em diferentes alvos celulares, incluindo o DNA. O cloreto estanoso (SnCl2 é um gerador de ERO que induz letalidade em E. coli, sendo o reparo por excisão de bases (BER um mecanismo importante neste processo. Técnicas como o ensaio cometa (em eucariotos e a eletroforese de DNA plasmidial em gel de agarose têm sido utilizadas para detectar genotoxicidade. No presente estudo, uma adaptação do método de eletroforese em gel alcalino de agarose foi usada para verificar a indução de quebras, pelo SnCl2, no DNA de E. coli, bem como a participação de enzimas do BER na restauração das lesões. Os resultados mostraram que o SnCl2 induziu quebras no DNA de todas as cepas testadas. Além disso, endonuclease IV e exonuclease III estão envolvidas na reparação dos danos. Em resumo, os dados obtidos indicam que a metodologia de eletroforese em gel alcalino de agarose pode ser empregada tanto para o estudo de quebras no DNA, quanto para avaliação dos

  17. [Study on the method of two dimensional polycrylamide gel electrophoresis on rat condylar chondrocyte].

    Science.gov (United States)

    Wu, Tuo-jiang; Li, Huang; Ma, Qiao-lin; Wang, Wen-mei

    2010-08-01

    To investigate the protein profile by two dimensional polycrylamide gel electrophoresis on the rat condylar chondrocyte in vitro. The third-passage chondrocytes were harvested from the mandibular condyles of 2-day-old rats in this study. The protein profile of the rat mandibular condylar chondrocytes was examined by two dimensional polycrylamide gel electrophoresis (2-DE-PAGE). The 2-DE gel maps on different pH gradients were obtained. The result of modified coomassi blue-sliver staining and sliver staining was compared using Pdquest 7.1 image analysis software. The results showed that the good protein profile of the condylar chondrocytes was obtained by standard Bio-Rad manual. The protein was mainly in the field from pH4 to pH7. The 1203±86 protein points were examined on 2-DE gel map by modified coomassi blue-sliver staining, and 1769±97 protein points was examined by sliver staining. The silver staining map showed more distinctly but higher background than modified coomassi blue-sliver staining. The protein profile of the condylar chondrocytes enriches the proteomic database and gives evidence to further proteomic research. The 2-DE map obtained by modified coomassi blue-sliver staining is more suitable for MALDI-TOF mass identification. Supported by National Natural Science Foundation of China (Grant No. C30700963), China Postdoctoral Science Foundation(Grant No.20090461088), Jiangsu Provincial Postdoctoral Science Foundation (Grant No.0802003C) and Nanjing City's Science and Technology Foundation (Grant No.200905011).

  18. Impact of quenching failure of Cy dyes in differential gel electrophoresis.

    Directory of Open Access Journals (Sweden)

    Weiqun Wang

    Full Text Available BACKGROUND: Differential gel electrophoresis (DIGE is a technology widely used for protein expression analysis. It is based on labelling with fluorescent Cy dyes. In comparative fluorescence gel electrophoresis experiments, however, unspecific labelling using N-hydroxy-succinimide-ester-based labelling protocols was recently detected. Cross-talk was observed due to failure of the quenching process. Here, the impact of this effect for DIGE experiments was investigated. METHODOLOGY/PRINCIPAL FINDINGS: Experiments to test quenching efficiency were performed in replicate using Escherichia coli lysate. Parameters such as the amount of dye and quencher were varied. Labelling and quenching were reversed in one experiment. Differences in protein spot volumes due to limited quenching were determined. For some spots twice the volume was detected underscoring the importance of proper control of silencing of active dye. CONCLUSIONS/SIGNIFICANCE: It could be demonstrated that uncontrolled labelling increased protein spot volume, even doubling it in some cases. Moreover, proteins responded differently to the protocol. Such unpredictable and unspecific processes are not acceptable in protein regulation studies so that it is necessary to validate the correct amount of quencher for individual samples before the DIGE experiment is performed. Increase of the concentration of lysine, which is used as quencher, from 10 mM to 2500 mM, was sufficient to silence the dye. Alternatively, active dye molecules can be removed by filtration.

  19. Two-dimensional gel electrophoresis of selenized yeast and autoradiography of 75Se-containing proteins

    International Nuclear Information System (INIS)

    Chery, C.C.; Dumont, E.; Cornelis, R.; Moens, L.

    2001-01-01

    Two-dimensional high-resolution gel electrophoresis (2DE) has been applied to the fractionation of 75 Se-containing proteins in yeast, grown in 75 Se-containing medium, and autoradiography was used for detection of the 75 Se-containing proteins. Gel filtration and ultrafiltration were used to check whether the selenium side-chains were stable in the presence of the chemicals used for lysis and 2DE. The mass distribution of the selenium-containing proteins was estimated by use of gel filtration and the results were compared with the distribution obtained by 2DE. A 2DE map of selenium-containing proteins in yeast is presented, and compared with a total protein map of yeast. (orig.)

  20. Comparison of Pseudomonas aeruginosa isolates from mink by serotyping and pulsed-field gel electrophoresis

    DEFF Research Database (Denmark)

    Hammer, Anne Sofie; Pedersen, Karl; Andersen, Thomas Holmen

    2003-01-01

    Isolates of Pseudomonas aeruginosa from clinical infections in mink were subjected to serotyping and pulsed-field gel electrophoresis (PFGE) using SpeI. A total of 212 isolates of P aeruginosa from the year 1998 to 2001 were included in this study: 168 isolates from mink obtained from 74 farm out...

  1. Detection of undeclared animal by-products in commercial canine canned foods: Comparative analyses by ELISA and PCR-RFLP coupled with slab gel electrophoresis or capillary gel electrophoresis.

    Science.gov (United States)

    Hsieh, Ming-Kun; Shih, Pei-Yin; Wei, Chia-Fong; Vickroy, Thomas W; Chou, Chi-Chung

    2016-03-30

    The potential presence of undeclared animal by-products in pet foods is not subject to routine examination. Previously published methods for species-based identification of animal by-products have not been used routinely owing to inconsistent results. The present study evaluated the utility of several approaches for accurate identification of animal by-products in 11 commercial brands of canine canned foods. Canine canned foods from several countries were analysed by ELISA, PCR-RFLP coupled with slab-gel electrophoresis (SGE) and capillary gel electrophoresis (CGE) to test for evidence of by-products derived from cattle, chicken, sheep or pig. While CGE-based analysis detected all (24) animal-derived by-products that were reported for the 11 test samples, SGE and ELISA detected only 22/24 (92%) and 14/24 (58%) of labelled by-products, respectively. In addition, undeclared animal by-products were found using all three analytical approaches with CGE detecting more positives (19) than SGE (17) or ELISA (5). Significant disparities were evident between the labelled contents and the detected content of animal by-products. CGE-based testing for PCR products appears to provide greater sensitivity and accuracy than either SGE or ELISA-based methods. As testing of commercial products becomes more reliable and mainstream, manufacturers will need to develop more thorough and accurate labelling protocols. © 2015 Society of Chemical Industry.

  2. Two-dimensional gel electrophoresis data for proteomic profiling of Sporothrix yeast cells

    Directory of Open Access Journals (Sweden)

    Anderson Messias Rodrigues

    2015-03-01

    Full Text Available Sporotrichosis is a chronic infection of the skin and subcutaneous tissues of human and other mammals caused by a complex of cryptic dimorphic fungi in the plant-associated order Ophiostomatales. With major differences between routes of transmission, Sporothrix infections are emerging as new threat in tropical and subtropical areas, particularly in form of outbreaks. The mechanisms underlying the pathogenesis and invasion of Sporothrix spp. are still poorly understood and many virulence factors remain unidentified. In this scenario, a global analysis of proteins expressed by clinical Sporothrix species combined with the identification of seroreactive proteins is overdue. Optimization of sample preparation and electrophoresis conditions are key steps toward reproducibility of gel-based proteomics assays. We provide the data generated using an efficient protocol of protein extraction for rapid and large-scale proteome analysis using two-dimensional gel electrophoresis. The protocol was established and optimized for pathogenic and non-pathogenic Sporothrix spp. including Sporothrix brasiliensis (CBS 132990, Sporothrix schenckii sensu stricto (CBS 132974, Sporothrix globosa (CBS 132922, and Sporothrix mexicana (CBS 120341. The data, supplied in this article, are related to the research article entitled “Immunoproteomic analysis reveals a convergent humoral response signature in the Sporothrix schenckii complex” (Rodrigues et al., 2014 [1].

  3. A rapid and efficient two-step gel electrophoresis method for the purification of major rye grass pollen allergens.

    Science.gov (United States)

    Levy, D; Davies, J; O'Hehir, R; Suphioglu, C

    2001-06-01

    Purified proteins are mandatory for molecular, immunological and cellular studies. However, purification of proteins from complex mixtures requires specialised chromatography methods (i.e., gel filtration, ion exchange, etc.) using fast protein liquid chromatography (FPLC) or high-performance liquid chromatography (HPLC) systems. Such systems are expensive and certain proteins require two or more different steps for sufficient purity and generally result in low recovery. The aim of this study was to develop a rapid, inexpensive and efficient gel-electrophoresis-based protein purification method using basic and readily available laboratory equipment. We have used crude rye grass pollen extract to purify the major allergens Lol p 1 and Lol p 5 as the model protein candidates. Total proteins were resolved on large primary gel and Coomassie Brilliant Blue (CBB)-stained Lol p 1/5 allergens were excised and purified on a secondary "mini"-gel. Purified proteins were extracted from unstained separating gels and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses. Silver-stained SDS-PAGE gels resolved pure proteins (i.e., 875 microg of Lol p 1 recovered from a 8 mg crude starting material) while immunoblot analysis confirmed immunological reactivity of the purified proteins. Such a purification method is rapid, inexpensive, and efficient in generating proteins of sufficient purity for use in monoclonal antibody (mAb) production, protein sequencing and general molecular, immunological, and cellular studies.

  4. Proteomic analysis of halotolerant proteins under high and low salt stress in Dunaliella salina using two-dimensional differential in-gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Yan-Long Jia

    2016-01-01

    Full Text Available Abstract Dunaliella salina, a single-celled marine alga with extreme salt tolerance, is an important model organism for studying fundamental extremophile survival mechanisms and their potential practical applications. In this study, two-dimensional differential in-gel electrophoresis (2D-DIGE was used to investigate the expression of halotolerant proteins under high (3 M NaCl and low (0.75 M NaCl salt concentrations. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS and bioinformatics were used to identify and characterize the differences among proteins. 2D-DIGE analysis revealed 141 protein spots that were significantly differentially expressed between the two salinities. Twenty-four differentially expressed protein spots were successfully identified by MALDI-TOF/TOF MS, including proteins in the following important categories: molecular chaperones, proteins involved in photosynthesis, proteins involved in respiration and proteins involved in amino acid synthesis. Expression levels of these proteins changed in response to the stress conditions, which suggests that they may be involved in the maintenance of intracellular osmotic pressure, cellular stress responses, physiological changes in metabolism, continuation of photosynthetic activity and other aspects of salt stress. The findings of this study enhance our understanding of the function and mechanisms of various proteins in salt stress.

  5. Multivariate data analysis of two-dimensional gel electrophoresis protein patterns from few samples

    DEFF Research Database (Denmark)

    Jensen, Kristina Nedenskov; Jessen, Flemming; Jørgensen, Bo

    2008-01-01

    One application of 2D gel electrophoresis is to reveal differences in protein pattern between two or more groups of individuals, attributable to their group membership. Multivariate data analytical methods are useful in pinpointing the spots relevant for discrimination by focusing not only...... on single spot differences, but on the covariance structure between proteins. However, their outcome is dependent on data scaling, and they may fail in producing valid multivariate models due to the much higher number of "irrelevant" spots present in the gels. The case where only few gels are available...... and where the aim is to find as many as possible of the group-dependent proteins seems particularly difficult to handle. The present paper investigates such a case regarding the effect of scaling and of prefiltering by univariate nonparametric statistics on the selection of spots. Besides, a modified...

  6. Adapting capillary gel electrophoresis as a sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes.

    Science.gov (United States)

    Greenough, Lucia; Schermerhorn, Kelly M; Mazzola, Laurie; Bybee, Joanna; Rivizzigno, Danielle; Cantin, Elizabeth; Slatko, Barton E; Gardner, Andrew F

    2016-01-29

    Detailed biochemical characterization of nucleic acid enzymes is fundamental to understanding nucleic acid metabolism, genome replication and repair. We report the development of a rapid, high-throughput fluorescence capillary gel electrophoresis method as an alternative to traditional polyacrylamide gel electrophoresis to characterize nucleic acid metabolic enzymes. The principles of assay design described here can be applied to nearly any enzyme system that acts on a fluorescently labeled oligonucleotide substrate. Herein, we describe several assays using this core capillary gel electrophoresis methodology to accelerate study of nucleic acid enzymes. First, assays were designed to examine DNA polymerase activities including nucleotide incorporation kinetics, strand displacement synthesis and 3'-5' exonuclease activity. Next, DNA repair activities of DNA ligase, flap endonuclease and RNase H2 were monitored. In addition, a multicolor assay that uses four different fluorescently labeled substrates in a single reaction was implemented to characterize GAN nuclease specificity. Finally, a dual-color fluorescence assay to monitor coupled enzyme reactions during Okazaki fragment maturation is described. These assays serve as a template to guide further technical development for enzyme characterization or nucleoside and non-nucleoside inhibitor screening in a high-throughput manner. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Quantification and characterization of enzymatically produced hyaluronan with fluorophore-assisted carbohydrate electrophoresis.

    Science.gov (United States)

    Kooy, Floor K; Ma, Muyuan; Beeftink, Hendrik H; Eggink, Gerrit; Tramper, Johannes; Boeriu, Carmen G

    2009-01-15

    Hyaluronan (HA) is a polysaccharide with high-potential medical applications, depending on the chain length and the chain length distribution. Special interest goes to homogeneous HA oligosaccharides, which can be enzymatically produced using Pasteurella multocida hyaluronan synthase (PmHAS). We have developed a sensitive, simple, and fast method, based on fluorophore-assisted carbohydrate electrophoresis (FACE), for characterization and quantification of polymerization products. A chromatographic pure fluorescent template was synthesized from HA tetrasaccharide (HA4) and 2-aminobenzoic acid. HA4-fluor and HA4 were used as template for PmHAS-mediated polymerization of nucleotide sugars. All products, fluorescent and nonfluorescent, were analyzed with gel electrophoresis and quantified using lane densitometry. Comparison of HA4- and HA4-fluor-derived polymers showed that the fluorophore did not negatively influence the PmHAS-mediated polymerization. Only even-numbered oligosaccharide products were observed using HA4-fluor or HA4 as template. The fluorophore intensity was linearly related to its concentration, and the limit of detection was determined to be 7.4pmol per product band. With this assay, we can now differentiate oligosaccharides of size range DP2 (degree of polymerization 2) to approximately DP400, monitor the progress of polymerization reactions, and measure subtle differences in polymerization rate. Quantifying polymerization products enables us to study the influence of experimental conditions on HA synthesis.

  8. Microwave-Assisted Synthesis of Chitosan/Polyvinyl Alcohol Silver Nanoparticles Gel for Wound Dressing Applications

    Directory of Open Access Journals (Sweden)

    Nguyen Thi Hiep

    2016-01-01

    Full Text Available The purpose of this study was to fabricate chitosan/poly(vinyl alcohol/Ag nanoparticles (CPA gels with microwave-assistance for skin applications. Microwave irradiation was employed to reduce silver ions to silver nanoparticles and to crosslink chitosan (CS with polyvinyl alcohol (PVA. The presence of silver nanoparticles in CPA gels matrix was examined using UV-Vis spectroscopy, transmission electron microscopy, and X-ray diffraction. The interaction of CS and PVA was analysed by Fourier transform infrared spectroscopy. The release of silver ions was determined by atomic absorption spectrometry. The antimicrobial properties of CPA gels against P. aeruginosa and S. aureus were investigated using agar diffusion method. Finally, the biocompatibility and wound-healing ability of the gels were studied using fibroblast cells (in vitro and mice models (in vivo. In conclusion, the results showed that CPA gels were successfully fabricated using microwave irradiation method. These gels can be applied to heal an open wound thanks to their antibacterial activity and biocompatibility.

  9. Protein Separation by Capillary Gel Electrophoresis: A Review

    Science.gov (United States)

    Zhu, Zaifang; Lu, Joann J.; Liu, Shaorong

    2011-01-01

    Capillary gel electrophoresis (CGE) has been used for protein separation for more than two decades. Due to the technology advancement, current CGE methods are becoming more and more robust and reliable for protein analysis, and some of the methods have been routinely used for the analysis of protein-based pharmaceuticals and quality controls. In light of this progress, we survey 147 papers related to CGE separations of proteins and present an overview of this technology. We first introduce briefly the early development of CGE. We then review the methodology, in which we specifically describe the matrices, coatings, and detection strategies used in CGE. CGE using microfabricated channels and incorporation of CGE with two-dimensional protein separations are also discussed in this section. We finally present a few representative applications of CGE for separating proteins in real-world samples. PMID:22122927

  10. Matrix-assisted laser desorption-ionization mass spectrometry peptide mass fingerprinting for proteome analysis: identification efficiency after on-blot or in-gel digestion with and without desalting procedures.

    Science.gov (United States)

    Lamer, S; Jungblut, P R

    2001-03-10

    In theory, peptide mass fingerprinting by matrix assisted laser desorption-ionization mass spectrometry (MALDI-MS) has the potential to identify all of the proteins detected by silver staining on gels. In practice, if the genome of the organism investigated is completely sequenced, using current techniques, all proteins stained by Coomassie Brilliant Blue can be identified. This loss of identification sensitivity of ten to hundred-fold is caused by loss of peptides by surface contacts. Therefore, we performed digestion and transfer of peptides in the lower microl range and reduced the number of steps. The peptide mix obtained from in-gel or on-blot digestion was analyzed directly after digestion or after concentration on POROS R2 beads. Eight protein spots of a 2-DE gel from Mycobacterium bovis BCG were identified using these four preparation procedures for MALDI-MS. Overall, on-blot digestion was as effective as in-gel digestion. Whereas higher signal intensities resulted after concentration, hydrophilic peptides are better detected by direct measurement of the peptide mix without POROS R2 concentration.

  11. Agarose-gel electrophoresis for the quality assurance and purity of heparin formulations.

    Science.gov (United States)

    Volpi, Nicola; Buzzega, Dania

    2012-01-01

    The adulteration of raw heparin (Hep) with a synthetic oversulfated chondroitin sulfate (OSCS) not found in nature produced in 2007-2008 a global crisis giving rise to the development of additional, new and specific methods for its quality assurance and purity. In this study, a simple and sensitive agarose-gel electrophoresis method has been developed for the visualization of OSCS in Hep samples along with other natural glycosaminoglycans possibly present as "process-related impurities", in particular dermatan sulfate (DS) and chondroitin sulfate (CS). Agarose-gel electrophoresis under non-conventional conditions is able to separate OSCS from Hep with its two components, the slow-moving and fast-moving species, DS and CS by performing separation for 15 h (overnight) and under high voltage (100 mA, ∼200 V). Densitometric scanning enabled us to calculate a limit of detection of ∼0.5 μg OSCS with a linear behaviour from 0.1 to 5 μg, comparable to CS/DS. Contaminated samples from Hep manufacturers were analyzed and quantitative data were found comparable to previous studies. Due to its capacity to process many samples in a single run and to the equipment commonly available in laboratories, this analytical method would be suitable for the identification and quantification of contamination by other polysaccharides, in particular OSCS and DS, within Hep preparations and formulations. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. DNA unwinding induced by photoaddition of psoralen derivatives and determination of dark-binding equilibrium constants by gel electrophoresis

    International Nuclear Information System (INIS)

    Wiesehahn, G.; Hearst, J.E.

    1978-01-01

    Derivatives of furo[3,2-g]coumarin (psoralen) can bind to the DNA double helix and, in the presence of long-wavelength uv light, the bound psoralen may react covalently with pyrimidine residues on one or both strands of the helix. By using agarose gel electrophoresis, we have determined the unwinding angle associated with each of four different psoralen derivatives to be 28 0 +- 4 0 . For 4,5',8-trimethylpsoralen (trioxsalen) the unwinding angle was found to be independent of the initial DNA superhelix density in the range that is accessible to agarose gel electrophoresis. Also by using agarose gel electrophoresis, we have determined the unwinding angle for ethidium intercalation. This was done by the total relaxation of supercoiled DNA in the presence of a series of ethidium concentrations. By using published values for the association constant for ethidium binding to DNA and evaluating the final superhelix density (after removal of ethidium) of the DNA on gels, we calculated an unwinding angle of 29 0 +- 3 0 . Assuming an unwinding angle of 28 0 for the noncovalent intercalation of psoralen derivatives, we used the same procedure to determine intercalation binding constants. The association constants for 4'-aminomethyltrioxsalen were 300 to 1400 M -1 in NaCl at 0.2 to 0.05 M and 300 to 2500 M -1 in Mg 2+ at 4 to 0.5 mM. The association constant for 4'-hydroxymethyltrioxsalen in 0.5 mM Mg 2+ was determined to be 70 M -1

  13. Electrophoretic analysis of proteins from Mycoplasma hominis strains detected by SDS-PAGE, two-dimensional gel electrophoresis and immunoblotting

    DEFF Research Database (Denmark)

    Andersen, H; Birkelund, Svend; Christiansen, Gunna

    1987-01-01

    The proteins of 14 strains of Mycoplasma hominis were compared by SDS-PAGE in gradient gels, by two-dimensional (2D) gel electrophoresis of extracts of 35S-labelled cells and by immunoblot analysis of cell proteins. The strains examined included the M. hominis type strain PG21 and 13 others...... isolated variously from genital tract, mouth, blood, upper urinary tract and a wound. These 14 strains shared 76-99% of proteins in SDS-gradient gel analysis and 41-72% in the 2D gels. As expected, the immunoblot analysis likewise revealed the existence of an extensive common protein pattern in M. hominis...

  14. Species identification of cooked fish by urea isoelectric focusing and sodium dodecylsulfate polyacrylamide gel electrophoresis : a collaborative study

    DEFF Research Database (Denmark)

    Rehbein, H.; Kundiger, R.; Yman, I.M.

    1999-01-01

    The suitability and reliability of urea IEF and SDS-PAGE for the identification of cooked fish flesh was tested by a collaborative study among nine laboratories. Urea IEF was performed with CleanGels as well as with ImmobilineGels, and ExcelGels were used for SDS-PAGE, enabling all three types...... of gels to be run in the same flat bed electrophoresis chamber. By strictly following optimised standard operation procedures (SOPs), five unknown cooked samples had to be identified with each technique using a set of 10 raw reference samples. With urea IEF, only one out of 35 identifications...

  15. New comprehensive denaturing-gradient-gel-electrophoresis assay for KRAS mutation detection applied to paraffin-embedded tumours

    NARCIS (Netherlands)

    Hayes, VM; Westra, JL; Verlind, E; Bleeker, W; Plukker, JT; Hofstra, RMW; Buys, CHCM

    2000-01-01

    A comprehensive mutation detection assay is presented for the entire coding region and all splice site junctions of the KRAS oncogene. The assay is based on denaturing gradient gel electrophoresis and applicable to archival paraffin-embedded tumour material. All KRAS amplicons are analysed within

  16. Lectin affinity electrophoresis.

    Science.gov (United States)

    Kobayashi, Yuka

    2014-01-01

    An interaction or a binding event typically changes the electrophoretic properties of a molecule. Affinity electrophoresis methods detect changes in the electrophoretic pattern of molecules (mainly macromolecules) that occur as a result of biospecific interactions or complex formation. Lectin affinity electrophoresis is a very effective method for the detection and analysis of trace amounts of glycobiological substances. It is particularly useful for isolating and separating the glycoisomers of target molecules. Here, we describe a sensitive technique for the detection of glycoproteins separated by agarose gel-lectin affinity electrophoresis that uses antibody-affinity blotting. The technique is tested using α-fetoprotein with lectin (Lens culinaris agglutinin and Phaseolus vulgaris agglutinin)-agarose gels.

  17. Evaluation of iron and selenium losses in metalloproteins separated by gel electrophoresis by ICPMS

    International Nuclear Information System (INIS)

    Gherghel, I.; Fernandez, M.L.; Fernandez, B.; Pereiro, R.; Sanz-Medel, A.

    2009-01-01

    Full text: Metallomics addresses the study of metabolism, transport, and metal-protein interactions aiming to obtain relevant information of physiological and pathological alterations in living organisms. Gel electrophoresis is widely employed in proteomics and its use is actually extending to metallomics. Unfortunately, analysis of proteins by molecular techniques does not offer quantitative information. So, a good alternative could be their determination via the quantification of (semi)metal bound to the protein by ICPMS. In this work, we will show a detailed study of possible losses of protein and/or metal in Fe-bound and selenium proteins (transferrin and glutathione peroxidase, respectively) to evaluate the behaviour of the protein-metal interactions during the electrophoresis process. (author)

  18. Sources of variability among replicate samples separated by two-dimensional gel electrophoresis.

    Science.gov (United States)

    Bland, Alison M; Janech, Michael G; Almeida, Jonas S; Arthur, John M

    2010-04-01

    Two-dimensional gel electrophoresis (2DE) offers high-resolution separation for intact proteins. However, variability in the appearance of spots can limit the ability to identify true differences between conditions. Variability can occur at a number of levels. Individual samples can differ because of biological variability. Technical variability can occur during protein extraction, processing, or storage. Another potential source of variability occurs during analysis of the gels and is not a result of any of the causes of variability named above. We performed a study designed to focus only on the variability caused by analysis. We separated three aliquots of rat left ventricle and analyzed differences in protein abundance on the replicate 2D gels. As the samples loaded on each gel were identical, differences in protein abundance are caused by variability in separation or interpretation of the gels. Protein spots were compared across gels by quantile values to determine differences. Fourteen percent of spots had a maximum difference in intensity of 0.4 quantile values or more between replicates. We then looked individually at the spots to determine the cause of differences between the measured intensities. Reasons for differences were: failure to identify a spot (59%), differences in spot boundaries (13%), difference in the peak height (6%), and a combination of these factors (21). This study demonstrates that spot identification and characterization make major contributions to variability seen with 2DE. Methods to highlight why measured protein spot abundance is different could reduce these errors.

  19. Enhanced removal of detergent and recovery of enzymatic activity following sodium dodecyl sulfate-polyacrylamide gel electrophoresis: UUse of casein in gel wash buffer

    International Nuclear Information System (INIS)

    McGrew, B.R.; Green, D.M.

    1990-01-01

    The inclusion of 1% casein or bovine serum albumin in buffer used to reactivate enzymes subjected to sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis resulted in accelerated removal of SDS and restoration of nuclease and beta-galactosidase enzyme activities. Nuclease and beta-galactosidase activities which are absent from gels after longer wash procedures are detectable with this technique. Enzyme activity in gels prepared with SDS which contained inhibitory contaminants was partially restored by the casein wash procedure. The threshold of detection of two-dimensionally separated deoxyribonuclease I using the casein wash procedure was 1 picogram

  20. Effect of gamma-irradiation on cereal DNA investigated by pulsed-field gel electrophoresis

    International Nuclear Information System (INIS)

    Kawamura, Yoko; Miura, Aya; Imura, Hiromi; Yamada, Takashi; Saito, Yukio

    1996-01-01

    The effects of gamma-irradiation on the DNA of corn, soybean and wheat were investigated using a pulsed-field gel electrophoresis technique. In order to avoid strand breaks during the DNA extracting steps, protoplasts prepared from seeds were embedded in agarose plugs and the DNA was purified by the digesting membranes and proteins. Pulsed-field gel electrophoresis can separate large DNA strands of about a few Mb in length. The DNA from unirradiated corn, soybean and wheat had mainly 3 fragments, about 6Mb(Fr.1), 5Mb(Fr.2), a few hundred kb(Fr.3) and so on. After gamma-irradiation, Fr.1 and Fr.2 had decreased depend on irradiation dose. The Fr.4(about 200 kb) of corn and Fr.3 of soybean DNA increased while Fr.3 of wheat did not increase under 10 kGy irradiation, however, the Fr.3 of all samples and the Fr.4 of corn decreased by over 10 kGy irradiation. It can be assumed that the large DNA strands were broken into smaller strands which increased at low irradiation doses, whereas both large and small DNA strands were broken down at higher irradiation doses. The Fr.6(2.5Mb) and Fr.7(1.5Mb) appeared in irradiated wheat DNA. (author)

  1. Improved methods for the fluorographic detection of weak β-emitting radioisotopes in agarose and acrylamide gel electrophoresis media

    International Nuclear Information System (INIS)

    Pulleyblank, D.E.; Booth, G.M.

    1981-01-01

    The use of acetic acid as a solvent for diphenyloxazole (PPO) in fluorographic procedures has been investigated. It is demonstrated to be superior to both dimethyl sulfoxide and methanol with respect to its suitability in both agarose and acrylamide gel electrophoresis systems. In addition, a method has been developed for impregnating fragile gels such as those used for immunodiffusion with PPO in preparation for fluorography. (Auth.)

  2. Radioiodination of surface proteins of bull spermatozoa and their characterization by sodium dodecyl sulphate-polyacrylamide gel electrophoresis

    International Nuclear Information System (INIS)

    Vierula, M.

    1980-01-01

    Surface proteins of ejaculated bull spermatozoa were radioiodinated using Ma 125 I, solubilized and characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The electron microscopic autoradiographs showed that the labelling was equally distributed to all parts of the spermatozoon and restricted to the sperm surface. The electrophoresis of solubilized radioactivity revealed 6 radioactive fractions with approximate molecular weights of 67 000-69 000, 47 000-50 000, 34 000-37 000, 25 000-28 000 and 14 000-16 000. The 6th fraction probably represented labelled lipids. The electrophoresis of radioiodinated seminal plasma proteins revealed only 2 radioactive protein peaks which coincided with the sperm surface protein fractions IV and V. (author)

  3. Influence of ionic constituents and electrical conductivity on the propagation of charged nanoscale objects in passivated gel electrophoresis.

    Science.gov (United States)

    Bikos, Dimitri A; Mason, Thomas G

    2018-01-01

    When determining the electric field E acting on charged objects in gel electrophoresis, the electrical conductivity of the buffer solution is often overlooked; E is typically calculated by dividing the applied voltage by a separation distance between electrodes. However, as a consequence of electrolytic reactions, which occur at the electrodes, gradients in the ionic content of the buffer solution and its conductivity can potentially develop over time, thereby impacting E and affecting propagation velocities of charged objects, v, directly. Here, we explore how the types and concentrations of ionic constituents of the buffer solution, which largely control its conductivity, when used in passivated gel electrophoresis (P-gelEP), can influence E, thereby altering v of charged nanospheres propagating through large-pore gels. We measure the conductivity of the buffer solution in the center of the gel region near propagating bands of nanospheres, and we show that predictions of E based on conductivity closely correlate with v. We also explore P-gelEP involving two different types of passivation agents: nonionic polyethylene glycol (PEG) and anionic sodium dodecyl sulfate (SDS). Our observations indicate that using a conductivity model to determine E from the local current density and the conductivity where spheres are propagating can lead to a better estimate than the standard approach of a voltage divided by a separation. Moreover, this conductivity model also provides a starting point for interpreting the complex behavior created by amphiphilic ionic passivation agents, such as SDS, on propagating nanospheres used in some P-gelEP experiments. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Protein identification from two-dimensional gel electrophoresis analysis of Klebsiella pneumoniae by combined use of mass spectrometry data and raw genome sequences

    Directory of Open Access Journals (Sweden)

    Zeng An-Ping

    2003-12-01

    Full Text Available Abstract Separation of proteins by two-dimensional gel electrophoresis (2-DE coupled with identification of proteins through peptide mass fingerprinting (PMF by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS is the widely used technique for proteomic analysis. This approach relies, however, on the presence of the proteins studied in public-accessible protein databases or the availability of annotated genome sequences of an organism. In this work, we investigated the reliability of using raw genome sequences for identifying proteins by PMF without the need of additional information such as amino acid sequences. The method is demonstrated for proteomic analysis of Klebsiella pneumoniae grown anaerobically on glycerol. For 197 spots excised from 2-DE gels and submitted for mass spectrometric analysis 164 spots were clearly identified as 122 individual proteins. 95% of the 164 spots can be successfully identified merely by using peptide mass fingerprints and a strain-specific protein database (ProtKpn constructed from the raw genome sequences of K. pneumoniae. Cross-species protein searching in the public databases mainly resulted in the identification of 57% of the 66 high expressed protein spots in comparison to 97% by using the ProtKpn database. 10 dha regulon related proteins that are essential for the initial enzymatic steps of anaerobic glycerol metabolism were successfully identified using the ProtKpn database, whereas none of them could be identified by cross-species searching. In conclusion, the use of strain-specific protein database constructed from raw genome sequences makes it possible to reliably identify most of the proteins from 2-DE analysis simply through peptide mass fingerprinting.

  5. Comparison between pulsed-field and constant-field gel electrophoresis for measurement of DNA double-strand breaks in irradiated Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Wlodek, D.; Banath, J.; Olive, P.L.

    1991-01-01

    Pulsed-field gel electrophoresis (PFGE) is one of the most sensitive methods for detecting DNA double-strand breaks in mammalian cells. However, it has been observed that constant-field gel electrophoresis (CFGE), when optimized, can detect breaks with equal efficiency. The migration of DNA from the well and the separation of DNA molecules according to size appear to be different processes; only the latter requires the application of PFGE. CFGE is very sensitive and can detect DNA damage produced by less than 5Gy of radiation. Low voltage (ca.0.6V/cm) during electrophoresis appears to be essential for the migration of the largest fraction of DNA from the agarose plug containing the cells; the electrophoresis run time, cell density in the plug, agarose concentration, nature of detergent and extent of radiolabelling are less important. It is concluded that CFGE is equally sensitive but more rapid and economical than PFGE for the measurement of DNA damage. (author)

  6. Genotypic characterization of Salmonella by multilocus sequence typing, pulsed-field gel electrophoresis and amplified fragment length polymorphism

    DEFF Research Database (Denmark)

    Torpdahl, Mia; Skov, Marianne N.; Sandvang, Dorthe

    2005-01-01

    subspecies enterica isolates. A total of 25 serotypes were investigated that had been isolated from humans or veterinary sources in Denmark between 1995 and 2001. All isolates were genotyped by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and amplified fragment length...

  7. Chromatin structure influence of DNA damage measurements by four assays: pulsed- and constant-field gel electrophoresis, DNA precipitation and non-denaturing filter elution

    International Nuclear Information System (INIS)

    Wlodek, D.; Olive, P.L.

    1996-01-01

    The of elution of DNA during non-denaturing filter elution (NFE) often correlates with cell sensitivity to radiation. The elution rate is influenced by two cellular factors: chromatin structure and the number of DNA-strand breaks (DSBs) produced in an intact cell by ionizing radiation. To determine which of the above factors is relevant to cell radiosensitivity, four assays were used to measure induction of DNA damage in three cell lines varying in radiosensitivity (V79, CHO, and L5178Y-R). Each of the assays, neutral filter elution (NFE), DNA precipitation, constant (CFGE) and pulsed field gel electrophoresis (PFGE) have different physical basis for DNA damage measurement and might be differently affected by chromatin structure. Three of the methods used to measure DNA double-strand breaks gave different results: NFE was dependent on cell type and location of DNA relative to the replication fork, gel electrophoresis was independent of cell type but was affected by proximity to the replication fork, and the precipitation assay was independent of both cell type and replication status. Pulsed field gel electrophoresis produced the same results and constant field gel electrophoresis for 3 cell lines examined. Only NFE showed differences in sensitivity which correlated with cell survival following irradiation. The results suggest that three is the same initial amount of DSBs in cells from all three lines and that the sensitivity to radiation is determined by some additional factors, probably chromatin structure. (author). 18 refs, 5 figs

  8. Development of methods to measure hemoglobin adducts by gel electrophoresis - Preliminary results

    International Nuclear Information System (INIS)

    Sun, J.D.; McBride, S.M.

    1988-01-01

    Chemical adducts formed on blood hemoglobin may be a useful biomarker for assessing human exposures to these compounds. This paper reports preliminary results in the development of methods to measure such adducts that may be generally applicable for a wide variety of chemicals. Male F344/N rats were intraperitoneally injected with 14 C-BaP dissolved in corn oil. Twenty-four hours later, the rats were sacrificed. Blood samples were collected and globin was isolated. Globin protein was then cleaved into peptide fragments using cyanogen bromide and the fragments separated using 2-dimensional gel electrophoresis. The results showed that the adducted 14 C-globin fragments migrated to different areas of the gel than did unadducted fragments. Further research is being conducted to develop methods that will allow quantitation of separated adducted globin fragments from human blood samples without the use of a radiolabel. (author)

  9. Detection of metalloproteins in human liver cytosol by synchrotron radiation X-ray fluorescence after sodium dodecyl sulphate polyacrylamide gel electrophoresis

    International Nuclear Information System (INIS)

    Gao Yuxi; Chen Chunying; Zhang Peiqun; Chai Zhifang; He Wei; Huang Yuying

    2003-01-01

    An improved method of analysis of metals in protein bands with synchrotron radiation X-ray fluorescence (SRXRF) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation is introduced and applied to human liver cytosol. Through a step of drying the gel before SRXRF determination, the continuous background resulting mainly from the Compton-scattering of X-rays by the gel matrix was substantially reduced, and the detection of biological trace elements, such as Cu, Fe, and Zn in protein bands was thereby made possible. With the new procedure, six Zn-containing proteins with molecular weights (MWs) of 17.5, 20.5, 27, 35, 55, and 63 kDa, respectively were found in human liver cytosol, among which the 63 kDa Zn-containing band was shown to be the dominant form of zinc. In addition, at least four Fe containing proteins with MWs of 20, 23, 43, and 83.5 kDa, respectively, were present in the samples. The metal contents in some metalloproteins, such as the 63 kDa Zn-containing protein, the 23 and 83.5 kDa Fe-containing proteins, and a 22 kDa Cu-containing protein were more closely related to the metal level in the sample. It is demonstrated that the procedure could be widely used to further investigate metal-binding proteins in biological samples

  10. Application of the Single Cell Gel Electrophoresis (SCGE) Assay to Genotoxicity Evaluation in Plants and Animals

    International Nuclear Information System (INIS)

    Kim, Jin Kyu

    2007-10-01

    Application of the Single Cell Gel Electrophoresis (SCGE) Assay to Genotoxicity Evaluation in Plants and Animals. Recently, the importance of ionizing radiation and chemicals has been recognized since radio- and chemical therapy is directly related to the control of various diseases such as cancer. Radiation and the chemicals can cause biological damages while they have great applicability. It is of necessity to analyze rapidly, easily and accurately the biological effects, especially DNA damage due to those factors. Recently SCGE (single cell gel electrophoresis assay, alias comet assay) has been developed for the efficient evaluation of DNA damage. In this report, the comprehensive review will be given on the rationale, the technical applications and the advantages and shortcomings of SCGE assay. This method can be directly applied to study on toxicity, cancer, and aging in terms of the evaluation of DNA damages due to radiation and chemicals on human cellular level. It is also suggested that comet assay be used for testing genotoxicity of suspected substances, detecting irradiated foods, screening radioprotective candidates, and studying DNA repair process in various biological systems

  11. Application of the Single Cell Gel Electrophoresis (SCGE) Assay to Genotoxicity Evaluation in Plants and Animals

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jin Kyu

    2007-10-15

    Application of the Single Cell Gel Electrophoresis (SCGE) Assay to Genotoxicity Evaluation in Plants and Animals. Recently, the importance of ionizing radiation and chemicals has been recognized since radio- and chemical therapy is directly related to the control of various diseases such as cancer. Radiation and the chemicals can cause biological damages while they have great applicability. It is of necessity to analyze rapidly, easily and accurately the biological effects, especially DNA damage due to those factors. Recently SCGE (single cell gel electrophoresis assay, alias comet assay) has been developed for the efficient evaluation of DNA damage. In this report, the comprehensive review will be given on the rationale, the technical applications and the advantages and shortcomings of SCGE assay. This method can be directly applied to study on toxicity, cancer, and aging in terms of the evaluation of DNA damages due to radiation and chemicals on human cellular level. It is also suggested that comet assay be used for testing genotoxicity of suspected substances, detecting irradiated foods, screening radioprotective candidates, and studying DNA repair process in various biological systems.

  12. Characterization and identification of early proteins in Chlamydia trachomatis serovar L2 by two-dimensional gel electrophoresis

    DEFF Research Database (Denmark)

    Lundemose, AG; Birkelund, Svend; Larsen, PM

    1990-01-01

    The synthesis of early proteins from Chlamydia trachomatis serovar L2 was analyzed by two-dimensional gel electrophoresis. By pulse-label experiments, the synthesis of seven proteins was observed at 2 to 8 h postinfection before the major outer membrane protein was detected at 8 to 10 h after...

  13. Electrophoresis forum '80

    International Nuclear Information System (INIS)

    Radola, B.J.

    1980-01-01

    In this volume the contributions of the electrophoresis meeting are presented in a short term form. The main topics are gel-electrophoresis, ultra thin film isoelectric focusing, one- and two-dimensional electrophoresis, electrophoretical separation techniques, electric focusing (for phorensic studies), substrate free and substrate electrophoresis. In the poster session of this meeting subjects such as (ultra) thin film isoelectric focusing, identification of radioactive proteins, labelling of cell surfaces, autoradiography and 3 H-labelled proteins. Separate abstracts were prepared for 4 papers in this report. (HK) [de

  14. Optimization of a pulsed-field gel electrophoresis for molecular typing of Proteus mirabilis

    Directory of Open Access Journals (Sweden)

    Alper Karagöz

    2013-09-01

    Full Text Available Objective: For the detection of outbreaks caused byProteus mirabilis, strains clonal relations are determinedmethods as “pulsed-field gel electrophoresis (PFGE”.The aim of this study was optimization of a pulsed-fieldgel electrophoresis for molecular typing of P. mirabilis.Methods: In this study, PFGE’ protocol is optimized foruse in molecular typing of P. mirabilis. Phylogenetic analyzesof strains were evaluated with Bionumerics softwaresystem (version 6.01; Applied Maths, Sint-Martens-Latem, Belgium.Results: This protocol compared with Gram-negativebacteria PFGE protocols, NotI enzyme is suitable for thisbacterium. Electrophoresis conditions should be revealedas; - block 1: initial pulse duration 1 sec, ending pulseduration 30 sec, striking angle 120°, the current 6 V/cm2,temperature 14°C, time 8 hours; - block 2: initial pulseduration 30 sec, ending pulse duration 70 sec, strikingangle 120°, the current 6 V/cm2, temperature 14°C, time16 hours; - TBE, pH=8.4.Conclusion: P. mirabilis strains were typed by PFGE andBionumerics analysis program were determined clonal relationships.The procedure was simple, reproducible andsuitable for these bacteria. Also it was evaluated, becauseof reducing time, the solution volumes and enzymes canbe economically. Outbreaks of nosocomial infections dueto bacteria studied assessment and the potential to provideuseful information about the degree of prevalence.This optimized protocol is allowed different centers’ PFGEresults to compare with other laboratories results. J ClinExp Invest 2013; 4 (3: 306-312Key words: Proteus mirabilis, molecular typing, pulsedfieldgel electrophoresis.

  15. Analysis of aqueous humour in uveitis by high performance liquid chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis

    NARCIS (Netherlands)

    Murray, P. I.; Hoekzema, R.; Luyendijk, L.; Kijlstra, A.

    1992-01-01

    Aqueous humour from patients with Fuchs' heterochromic cyclitis (FHC) and other types of uveitis was analysed by high performance liquid chromatography (HPLC) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Using HPLC, the number of peaks and their respective elution times

  16. Introducing Proteomics in the Undergraduate Curriculum: A Simple 2D Gel Electrophoresis Exercise with Serum Proteins

    Science.gov (United States)

    Kim, Thomas D.; Craig, Paul A.

    2010-01-01

    Two-dimensional gel electrophoresis (2DGE) remains an important tool in the study of biological systems by proteomics. While the use of 2DGE is commonplace in research publications, there are few instructional laboratories that address the use of 2DGE for analyzing complex protein samples. One reason for this lack is the fact that the preparation…

  17. A neutral glyoxal gel electrophoresis method for the detection and semi-quantitation of DNA single-strand breaks.

    Science.gov (United States)

    Pachkowski, Brian; Nakamura, Jun

    2013-01-01

    Single-strand breaks are among the most prevalent lesions found in DNA. Traditional electrophoretic methods (e.g., the Comet assay) used for investigating these lesions rely on alkaline conditions to denature DNA prior to electrophoresis. However, the presence of alkali-labile sites in DNA can result in the introduction of additional single-strand breaks upon alkali treatment during DNA sample processing. Herein, we describe a neutral glyoxal gel electrophoresis assay which is based on alkali-free DNA denaturation and is suitable for qualitative and semi-quantitative analyses of single-strand breaks in DNA isolated from different organisms.

  18. Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS- PAGE) of Irradiated Wheat Flour Proteins

    International Nuclear Information System (INIS)

    Souzan, R.M.

    1999-01-01

    Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) of wheat (Triticum aestivum L) flour have revealed 23 polypeptides of molecular weights between 170 and 11.57 KDa, High molecular weight glutenin subunits (LMW-GS) were distinguished. Densitometric analysis of the gel showed the effect of radiation on polypeptide constitution at radiation energy up to 7.5 kGy. Irradiation of wheat flour with 2.5 kGy have resulted in a slight increase in the molecular weight of wheat flour protein subunits. The increase of irradiation dose to 5.0 kGy has also induced an additional increase of molecular weight of protein subunits. The continuity in application of more radiation energy to a level of 7.5 kGy have resulted in the prevalence of degradation processes of all protein subunits more than the aggregation

  19. High-resolution gel electrophoresis and sodium dodecyl sulphate-agarose gel electrophoresis on urine samples for qualitative analysis of proteinuria in dogs.

    Science.gov (United States)

    Giori, Luca; Tricomi, Flavia Marcella; Zatelli, Andrea; Roura, Xavier; Paltrinieri, Saverio

    2011-07-01

    The aims of the current study were to assess whether sodium dodecyl sulphate-agarose gel electrophoresis (SDS-AGE) and high-resolution electrophoresis (HRE) can identify dogs with a urinary protein-to-creatinine ratio (UPC ratio) >0.2 and whether HRE can provide preliminary information about the type of proteinuria, using SDS-AGE as a reference method. HRE and SDS-AGE were conducted on 87 urine samples classified according to the International Renal Interest Society as non-proteinuric (NP; UPC ratio: 0.51; 40/87). SDS-AGE and HRE were positive in 14 out of 32 and 3 out of 32 NP samples and in 52 out of 55 and 40 out of 55 samples with a UPC ratio >0.20, respectively. The concordance between HRE or SDS and UPC ratio was comparable (κ = 0.59; κ = 0.55). However, specificity (90%) and positive likelihood ratio (7.76) were higher for HRE than for SDS-AGE (56% and 2.16) while sensitivity was lower (73% vs. 94%). The analysis of HRE results revealed that a percentage of albumin >41.4% and an albumin/α(1)-globulin ratio (alb/α(1) ratio) >1.46 can identify samples classified by SDS-AGE as affected by glomerular proteinuria while a percentage of α(1)-globulin >40.8% and an alb/α(1) ratio HRE could misclassify samples with a UPC ratio higher or lower than 0.20. Therefore, UPC ratio must always be determined before conducting these tests. The percentage of albumin and α(1)-globulin or the alb/α(1) ratio determined by HRE can provide preliminary information about the origin of proteinuria.

  20. Procedures for two-dimensional electrophoresis of proteins

    Energy Technology Data Exchange (ETDEWEB)

    Tollaksen, S.L.; Giometti, C.S.

    1996-10-01

    High-resolution two-dimensional gel electrophoresis (2DE) of proteins, using isoelectric focusing in the first dimension and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) in the second, was first described in 1975. In the 20 years since those publications, numerous modifications of the original method have evolved. The ISO-DALT system of 2DE is a high-throughput approach that has stood the test of time. The problem of casting many isoelectric focusing gels and SDS-PAGE slab gels (up to 20) in a reproducible manner has been solved by the use of the techniques and equipment described in this manual. The ISO-DALT system of two-dimensional gel electrophoresis originated in the late 1970s and has been modified many times to improve its high-resolution, high-throughput capabilities. This report provides the detailed procedures used with the current ISO-DALT system to prepare, run, stain, and photograph two-dimensional gels for protein analysis.

  1. pI-Control in comparative fluorescence gel electrophoresis (CoFGE) using amphoteric azo dyes

    Czech Academy of Sciences Publication Activity Database

    Hanneken, M.; Šlais, Karel; König, S.

    2015-01-01

    Roč. 8, SEP (2015), s. 36-39 ISSN 2212-9685 Institutional support: RVO:68081715 Keywords : comparative fluorescence gel * electrophoresis * protein grid * azo dyes Subject RIV: CB - Analytical Chemistry, Separation http://ac.els-cdn.com/S2212968515000094/1-s2.0-S2212968515000094-main.pdf?_tid=7c92fa40-56e6-11e5-b36a-00000aab0f01&acdnat=1441798543_19612c0d7466780944bc4ae22173da92

  2. A metrological study of autoradiographs from two-dimensional gel electrophoresis.

    Science.gov (United States)

    Valiron, O; Lefkovits, I; Garderet, P; Steinberg, C

    1984-12-01

    Samples prepared from a single batch of labeled cells were subjected to two-dimensional gel electrophoresis and autoradiography. Three factors were varied: total quantity of protein, quantity of labeled protein, and exposure time. The mean background absorbance of the film remained identical (about 0.5 A) for all the treated series, whatever the exposure time and whether or not there were unlabeled proteins in the sample. Hence any spot with a peak A of the same order of magnitude can be seen. The standard deviation was about 0.05 A. Thus, the measurement precision is 2.5% of full scale for digitalization over 0 to 2 A. We derived experimental calibration curves, which are neither linear nor logarithmic because of the film response and which can be used on randomly chosen spots.

  3. Proteomic Analysis of Bovine Pregnancy-specific Serum Proteins by 2D Fluorescence Difference Gel Electrophoresis

    OpenAIRE

    Lee, Jae Eun; Lee, Jae Young; Kim, Hong Rye; Shin, Hyun Young; Lin, Tao; Jin, Dong Il

    2015-01-01

    Two dimensional-fluorescence difference gel electrophoresis (2D DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The purpose of this study was to investigate bovine pregnancy-specific proteins in the proteome between bovine pregnant and non-pregnant serum using DIGE technique. Serums of 2 pregnant Holstein dairy cattle at day 21 after artificial insemination and those of 2...

  4. Screening for mutations in the uroporphyrinogen decarboxylase gene using denaturing gradient gel electrophoresis

    DEFF Research Database (Denmark)

    Christiansen, L; Ged, C; Hombrados, I

    1999-01-01

    to exon skipping, and a 2-bp deletion (415-416delTA) resulting in a frameshift and the introduction of a premature stop codon. Heterologous expression and enzymatic studies of the mutant proteins demonstrate that the three mutations leading to shortening or truncation of the UROD protein have no residual......, confirming the heterogeneity of the underlying genetic defects of these diseases. We have established a denaturing gradient gel electrophoresis (DGGE) assay for mutation detection in the UROD gene, enabling the simultaneous screening for known and unknown mutations. The established assay has proved able...

  5. Characterization of royal jelly proteins in both Africanized and European honeybees (Apis mellifera) by two-dimensional gel electrophoresis.

    Science.gov (United States)

    Sano, Osamu; Kunikata, Toshio; Kohno, Keizo; Iwaki, Kanso; Ikeda, Masao; Kurimoto, Masashi

    2004-01-14

    In this study, the proteins contained in royal jelly (RJ) produced by Africanized honeybees and European honeybees (Apis mellifera) haven been analyzed in detail and compared using two-dimensional gel electrophoresis, and the N-terminal amino acid sequence of each spot has been determined. Most spots were assigned to major royal jelly proteins (MRJPs). Remarkable differences were found in the heterogeneity of the MRJPs, in particular MRJP3, in terms of molecular weights and isoelectric points between the two species of RJ. Furthermore, during the determination of the N-terminal amino acid sequence of each spot, for the first time, MRJP4 protein has been identified, the existence of which had been only implied by cloning of its cDNA sequence. The presence of heterogeneous bands of glucose oxidase was also identified. Thus, the results suggest that two-dimensional gel electrophoresis provides a suitable method for the qualitative analysis of the proteins contained in RJ derived from different honeybee species.

  6. Application of pulsed field gel electrophoresis to determine γ-ray-induced double-strand breaks in yeast chromosomal molecules

    International Nuclear Information System (INIS)

    Friedl, A.A.; Hahn, K.; Eckardt-Schupp, F.; Kellerer, A.M.; Beisker, W.

    1993-01-01

    The frequency of DNA double-strand breaks (dsb) was determined in yeast cells exposed to γ-rays under anoxic conditions. Genomic DNA of treated cells was separated by pulsed field gel electrophoresis, and two different approaches for the evaluation of the gels were employed: (1) The DNA mass distribution profile obtained by electrophoresis was compared to computed profiles, and the number of DSB per unit length was then derived in terms of a fitting procedure; (2) hybridization of selected chromosomes was performed, and a comparison of the hybridization signals in treated and untreated samples was then used to derive the frequency of dsb. The two assays gave similar results for the frequency of dsb ((1.07 ± 0.06) x 10 -9 Gy -1 bp -1 and (0.93 ± 0.09) x 10 -9 Gy -1 bp -1 , respectively). The dsb frequency was found to be linearly dependent on dose. (author)

  7. Physical and genetic mapping of the genomes of five Mycoplasma hominis strains by pulsed-field gel electrophoresis

    DEFF Research Database (Denmark)

    Ladefoged, Søren; Christiansen, Gunna

    1992-01-01

    -field gel electrophoresis. All the ApaI, SmaI, BamHI, XhoI, and SalI restriction sites (total of 21 to 33 sites in each strain) were placed on the physical map, yielding an average resolution of 26 kb. The maps were constructed using three different approaches: (i) size determination of DNA fragments...

  8. Speciation of iodine-containing proteins in Nori seaweed by gel electrophoresis laser ablation ICP-MS.

    Science.gov (United States)

    Romarís-Hortas, V; Bianga, J; Moreda-Piñeiro, A; Bermejo-Barrera, P; Szpunar, J

    2014-09-01

    An analytical approach providing an insight into speciation of iodine in water insoluble fraction of edible seaweed (Nori) was developed. The seaweed, harvested in the Galician coast (Northwestern Spain), contained 67.7±1.3 μg g(-1) iodine of which 25% was water soluble and could be identifies as iodide. Extraction conditions of water insoluble residue using urea, NaOH, SDS and Triton X-100 were investigated. The protein pellets obtained in optimized conditions (after precipitation of urea extracts with acetone), were digested with trypsin and protease XIV. Size exclusion chromatography-ICP-MS of both enzymatic digests demonstrated the occurrence of iodoaminoacids putatively present in proteins. Intact proteins could be separated by gel electrophoresis after an additional extraction of the protein extract with phenol. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) with laser ablation ICP-MS detection of (127)I indicated the presence of iodine in protein bands corresponding to molecular masses of 110 kDa, 40 kDa, 27 kDa, 20 kDa and 10 kDa. 2D IEF-SDS PAGE with laser ablation ICP-MS (127)I imaging allowed the detection of 5 iodine containing protein spots in the alkaline pI range. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Analysis of Endonuclease R·EcoRI Fragments of DNA from Lambdoid Bacteriophages and Other Viruses by Agarose-Gel Electrophoresis

    Science.gov (United States)

    Helling, Robert B.; Goodman, Howard M.; Boyer, Herbert W.

    1974-01-01

    By means of agarose-gel electrophoresis, endonuclease R·EcoRI-generated fragments of DNA from various viruses were separated, their molecular weights were determined, and complete or partial fragment maps for lambda, φ80, and hybrid phages were constructed. Images PMID:4372397

  10. Automated DNA electrophoresis, hybridization and detection

    International Nuclear Information System (INIS)

    Zapolski, E.J.; Gersten, D.M.; Golab, T.J.; Ledley, R.S.

    1986-01-01

    A fully automated, computer controlled system for nucleic acid hybridization analysis has been devised and constructed. In practice, DNA is digested with restriction endonuclease enzyme(s) and loaded into the system by pipette; 32 P-labelled nucleic acid probe(s) is loaded into the nine hybridization chambers. Instructions for all the steps in the automated process are specified by answering questions that appear on the computer screen at the start of the experiment. Subsequent steps are performed automatically. The system performs horizontal electrophoresis in agarose gel, fixed the fragments to a solid phase matrix, denatures, neutralizes, prehybridizes, hybridizes, washes, dries and detects the radioactivity according to the specifications given by the operator. The results, printed out at the end, give the positions on the matrix to which radioactivity remains hybridized following stringent washing

  11. Compatibility between weak gel and microorganisms in weak gel-assisted microbial enhanced oil recovery.

    Science.gov (United States)

    Qi, Yi-Bin; Zheng, Cheng-Gang; Lv, Cheng-Yuan; Lun, Zeng-Min; Ma, Tao

    2018-03-20

    To investigate weak gel-assisted microbial flooding in Block Wang Long Zhuang in the Jiangsu Oilfield, the compatibility of weak gel and microbe was evaluated using laboratory experiments. Bacillus sp. W5 was isolated from the formation water in Block Wang Long Zhuang. The rate of oil degradation reached 178 mg/day, and the rate of viscosity reduction reached 75.3%. Strain W5 could produce lipopeptide with a yield of 1254 mg/L. Emulsified crude oil was dispersed in the microbial degradation system, and the average diameter of the emulsified oil particles was 18.54 μm. Bacillus sp. W5 did not affect the rheological properties of the weak gel, and the presence of the weak gel did not significantly affect bacterial reproduction (as indicated by an unchanged microbial biomass), emulsification (surface tension is 35.56 mN/m and average oil particles size is 21.38 μm), oil degradation (162 mg/day) and oil viscosity reduction (72.7%). Core-flooding experiments indicated oil recovery of 23.6% when both weak gel and Bacillus sp. W5 were injected into the system, 14.76% when only the weak gel was injected, and 9.78% with strain W5 was injected without the weak gel. The results demonstrate good compatibility between strains W5 and the weak gel and highlight the application potential of weak gel-assisted microbial flooding. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  12. Affinity in electrophoresis.

    Science.gov (United States)

    Heegaard, Niels H H

    2009-06-01

    The journal Electrophoresis has greatly influenced my approaches to biomolecular affinity studies. The methods that I have chosen as my main tools to study interacting biomolecules--native gel and later capillary zone electrophoresis--have been the topic of numerous articles in Electrophoresis. Below, the role of the journal in the development and dissemination of these techniques and applications reviewed. Many exhaustive reviews on affinity electrophoresis and affinity CE have been published in the last few years and are not in any way replaced by the present deliberations that are focused on papers published by the journal.

  13. The use of the 2-aminobenzoic acid tag for oligosaccharide gel electrophoresis.

    Science.gov (United States)

    Huang, Z; Prickett, T; Potts, M; Helm, R F

    2000-08-18

    Gel electrophoresis of fluorophore labeled saccharides provides a rapid and reliable method to screen enzymatic and/or chemical treatments of polysaccharides and glycoconjugates, as well as a sensitive and efficient microscale method to separate and purify oligosaccharides for further analysis. A simple and inexpensive method of derivatization and analysis using 2-aminobenzoic acid (anthranilic acid, AA) is described and applied to the extracellular polysaccharide released by the desiccation tolerant cyanobacterium Nostoc commune DRH-1. The results of these analyses suggest a possible protective functionality of two pendent groups, as well as a potential relationship between these groups and the desiccation tolerance of the organism.

  14. Accommodating brightness and exposure levels in densitometry of stained polyacrylamide electrophoresis gels

    International Nuclear Information System (INIS)

    Tan, Han Yen; Ng, Tuck Wah; Liew, Oi Wah

    2010-01-01

    Flatbed scanner densitometers can be operated under various illumination and recording exposure levels. In this work, we show that optical density measurement accuracy, sensitivity, and stability of stained polyacrylamide electrophoresis gel densitometry are crucially dependent on these two factors (brightness and exposure level), notwithstanding that the source is monochromatic, spatially uniform, and the measurements are made using an accurately calibrated step wedge in tandem. We further outline a method to accommodate the intensity deviations over a range of illumination and exposure levels in order to maintain sensitivity and repeatability in the computed optical densities. Comparisons were also made with results from a commercial densitometer.

  15. In vitro evaluation of calcium alginate gels as matrix for iontophoresis electrodes.

    Science.gov (United States)

    Haida, Haruka; Ando, Shizuka; Ogami, Saori; Wakita, Ryo; Kohase, Hikaru; Saito, Norio; Yoshioka, Tomohiko; Ikoma, Toshiyuki; Tanaka, Junzo; Umino, Masahiro; Fukayama, Haruhisa

    2012-03-13

    Calcium alginate gel has some unique properties, such as the capability to keep the drugs, bioadhesiveness, safety, and low cost. The purpose of this study is to determine whether calcium alginate gel can be used as a matrix of electrodes for iontophoresis (IOP). We measured the concentration of lidocaine transported from calcium alginate gels with various concentrations of alginic acid using an in vitro experimental cell with square-wave alternating current (AC) application. Temperature and pH changes were also determined during AC-IOP. The results revealed that lidocaine was released from calcium alginate gels at concentrations nearly 1.71-fold larger at 5 V, 60 min after AC application than in the case of passive diffusion. Lidocaine transport depended on the alginic acid concentration in the gels. Although there were slight increases in temperature and pH, chemical and thermal burns were not severe enough to be a concern. In conclusion, the calcium alginate gel can be used as a possible matrix for IOP electrodes.

  16. Radiobiological study on DNA strand breaks and repair using single cell gel electrophoresis

    International Nuclear Information System (INIS)

    Ikushima, Takaji

    1994-01-01

    Single cell gel electrophoresis (SCGE) provides a novel method to measure DNA damage in individual cells and more importantly, to assess heterogeneity in response within a mixed population of cells. Cells embedded in agarose are lysed, subjected to electrophoresis, stained with a fluorescent DNA-specific dye, and viewed under a fluorescence microscope. Damaged cells display 'comets', broken DNA migrating farther to the anode in the electric field. We have previously used this technique to quantify DNA damage induced by moderate doses of low and high LET radiations in cultured Chinese hamster cells. The assay has been optimized in terms of lysing and electrophoresis conditions, and applied to analyse the DNA strand breaks, their repair kinetics and heterogeneity in response in individual Chinese hamster cells exposed to gamma-rays, and to KUR thermal neutrons with and without 10 B or to KEK PF monochromatic soft X-rays as well as to a radio-mimetic agent, neocarzinostatin. The DNA double-strand breaks induced by boron-neutron captured reactions were repaired at a slower rate, but a heterogeneity in response might not contribute to the difference. The neocarzinostatin-induced DNA damage were efficiently repaired in a dose-dependent fashion. The initial amount of gamma-ray induced DNA double-strand breaks was not significantly altered in cells pre-exposed to very low adapting dose. (author)

  17. Polyacrylamide gel electrophoresis-SDS as a tool to study myofibrillar proteins. A review.

    Directory of Open Access Journals (Sweden)

    Perez-Chabela, M. Lourdes

    2015-12-01

    Full Text Available Miofibrillar proteins are part of land and sea animals’ muscle. Nonetheless, even when muscle proteins are the same type of proteins, their structure, rigor mortis time, and biochemical process associated to muscle to meat conversion, are different among animal species. This review has the aim to describe the advantages of SDS-polyacrylamide gel electrophoresis (SDS-PAGE in the study of myofibrillar proteins structure, besides the influence of many parameters on this technique to obtain an electrophoretic profile. Applications of this technique as a diagnostic tool in the food science, ecology and health are described as well.

  18. The limitations of pulsed-field gel electrophoresis for analysis of Yersinia enterocolitica isolates.

    Science.gov (United States)

    Gilpin, B J; Robson, B; Lin, S; Hudson, J A; Weaver, L; Dufour, M; Strydom, H

    2014-09-01

    This study describes the analysis of 432 isolates of Yersinia enterocolitica by pulsed-field gel electrophoresis (PFGE). PFGE had a high level of discrimination with biotype 1A isolates (Simpson's Diversity Index 0.997), but with the clinically important biotypes 2, 3 and 4, the discriminatory ability of PFGE was so low as to severely limit its usefulness (DI enterocolitica biotypes 2, 3 and 4, and inferences based on finding indistinguishable PFGE profiles among cases or between cases and sources need to be substantiated using alternative typing tools, or strong epidemiological evidence. © 2013 Blackwell Verlag GmbH.

  19. In-gel and OFFGEL-based proteomic approach for authentication of meat species from minced meat and meat products.

    Science.gov (United States)

    Naveena, Basappa M; Jagadeesh, Deepak S; Kamuni, Veeranna; Muthukumar, Muthupalani; Kulkarni, Vinayak V; Kiran, Mohan; Rapole, Srikanth

    2018-02-01

    Fraudulent mislabelling of processed meat products on a global scale that cannot be detected using conventional techniques necessitates sensitive, robust and accurate methods of meat authentication to ensure food safety and public health. In the present study, we developed an in-gel (two-dimensional gel electrophoresis, 2DE) and OFFGEL-based proteomic method for authenticating raw and cooked water buffalo (Bubalus bubalis), sheep (Ovis aries) and goat (Caprus hircus) meat and their mixes. The matrix-assisted liquid desorption/ionization time-of-flight mass spectrometric analysis of proteins separated using 2DE or OFFGEL electrophoresis delineated species-specific peptide biomarkers derived from myosin light chain 1 and 2 (MLC1 and MLC2) of buffalo-sheep-goat meat mix in definite proportions at 98:1:1, 99:0.5:0.5 and 99.8:0.1:0.1 that were found stable to resist thermal processing. In-gel and OFFGEL-based proteomic approaches are efficient in authenticating meat mixes spiked at minimum 1.0% and 0.1% levels, respectively, in triple meat mix for both raw and cooked samples. The study demonstrated that authentication of meat from a complex mix of three closely related species requires identification of more than one species-specific peptide due to close similarity between their amino acid sequences. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  20. Enhanced detergent extraction for analysis of membrane proteomes by two-dimensional gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Hsu Kimberly K

    2005-06-01

    Full Text Available Abstract Background The analysis of hydrophobic membrane proteins by two-dimensional gel electrophoresis has long been hampered by the concept of inherent difficulty due to solubility issues. We have optimized extraction protocols by varying the detergent composition of the solubilization buffer with a variety of commercially available non-ionic and zwitterionic detergents and detergent-like phospholipids. Results After initial analyses by one-dimensional SDS-PAGE, quantitative two-dimensional analyses of human erythrocyte membranes, mouse liver membranes, and mouse brain membranes, extracted with buffers that included the zwitterionic detergent MEGA 10 (decanoyl-N-methylglucamide and the zwitterionic lipid LPC (1-lauroyl lysophosphatidylcholine, showed selective improvement over extraction with the common 2-DE detergent CHAPS (3 [(3-cholamidopropyldimethylammonio]-1-propanesulfonate. Mixtures of the three detergents showed additive improvements in spot number, density, and resolution. Substantial improvements in the analysis of a brain membrane proteome were observed. Conclusion This study demonstrates that an optimized detergent mix, coupled with rigorous sample handling and electrophoretic protocols, enables simple and effective analysis of membrane proteomes using two-dimensional electrophoresis.

  1. Modeling the Dynamics of Gel Electrophorresis in the High School Classroom

    Science.gov (United States)

    Saucedo, Skyler R.

    2013-01-01

    Gel electrophoresis, used by geneticists and forensic experts alike, is an immensely popular technique that utilizes an electric field to separate molecules and proteins by size and charge. At the microscopic level, a dye or complex protein like DNA is passed through agarose, a gelatinous three-dimensional matrix of pores and nano-sized tunnels.…

  2. Analysis of the surface membrane of iodinated leukemic cells by SDS-polyacrylamide gel electrophoresis

    International Nuclear Information System (INIS)

    Ishitani, Kunihiko; Ikeda, Akira; Tamura, Minoru; Takeuchi, Hidekazu; Ihara, Koji

    1980-01-01

    Surface proteins of human leukemic cells were labeled selectively by lactoperoxydase catalysed-iodination and examined by SDS-polyacrylamide gel electrophoresis. The electrophoretic pattern of the surface membranes of cells from a patients with chronic mylogeneous leukemia in blast crisis was of B cell type and showed Ia like antigen. Leukemic cells from a patient with hairly cell leukemia also expressed the pattern of B cell type when tested by this method the technique of iodinating cell surface with lactoperoxidase is useful in characterization of leukemia cells for diagnosis and monitoring of clinical course. (author)

  3. Microchip capillary gel electrophoresis using programmed field strength gradients for the ultra-fast analysis of genetically modified organisms in soybeans.

    Science.gov (United States)

    Kim, Yun-Jeong; Chae, Joon-Seok; Chang, Jun Keun; Kang, Seong Ho

    2005-08-12

    We have developed a novel method for the ultra-fast analysis of genetically modified organisms (GMOs) in soybeans by microchip capillary gel electrophoresis (MCGE) using programmed field strength gradients (PFSG) in a conventional glass double-T microchip. Under the programmed electric field strength and 0.3% poly(ethylene oxide) sieving matrix, the GMO in soybeans was analyzed within only 11 s of the microchip. The MCGE-PFSG method was a program that changes the electric field strength during GMO analysis, and was also applied to the ultra-fast analysis of PCR products. Compared to MCGE using a conventional and constantly applied electric field, the MCGE-PFSG analysis generated faster results without the loss of resolving power and reproducibility for specific DNA fragments (100- and 250-bp DNA) of GM-soybeans. The MCGE-PFSG technique may prove to be a new tool in the GMO analysis due to its speed, simplicity, and high efficiency.

  4. Proteome profile of functional mitochondria from human skeletal muscle using one-dimensional gel electrophoresis and HPLC-ESI-MS/MS

    DEFF Research Database (Denmark)

    Lefort, Natalie; Yi, Zhengping; Bowen, Benjamin

    2009-01-01

    were functional as evidenced by their response to carbohydrate and fat-derived fuels. Using one-dimensional gel electrophoresis and HPLC-ESI-MS/MS, 823 unique proteins were detected, and 487 of these were assigned to the mitochondrion, including the newly characterized SIRT5, MitoNEET and RDH13...

  5. Modeling the Dynamics of Gel Electrophorresis in the High School Classroom

    Science.gov (United States)

    Saucedo, Skyler R.

    2013-01-01

    Gel electrophoresis, used by geneticists and forensic experts alike, is an immensely popular technique that utilizes an electric field to separate molecules and proteins by size and charge. At the microscopic level, a dye or complex protein like DNA is passed through agarose, a gelatinous three-dimensional matrix of pores and nano-sized tunnels. When forced through a maze of holes, the molecule unravels, forming a long chain, slithering through the field of pores in a process colloquially coined "reputation." As a result, the smaller molecules travel farther through the gel when compared to molecules of larger molecular weight. This highly effective "molecular sieve" provides consistent data and allows scientists to compare similar sequences of DNA base pairs in a routine fashion.2 When performed at the high school level, gel electrophoresis provides students the opportunity to learn about a contemporary lab technique of great scientific relevance. Doing real science certainly excites students and motivates them to learn more.

  6. Structural analysis of protein complexes with sodium alkyl sulfates by small-angle scattering and polyacrylamide gel electrophoresis.

    Science.gov (United States)

    Ospinal-Jiménez, Mónica; Pozzo, Danilo C

    2011-02-01

    Small-angle X-ray (SAXS) and neutron (SANS) scattering is used to probe the structure of protein-surfactant complexes in solution and to correlate this information with their performance in gel electrophoresis. Proteins with sizes between 6.5 to 116 kDa are denatured with sodium alkyl sulfates (SC(x)S) of variable tail lengths. Several combinations of proteins and surfactants are analyzed to measure micelle radii, the distance between micelles, the extension of the complex, the radius of gyration, and the electrophoretic mobility. The structural characterization shows that most protein-surfactant complexes can be accurately described as pearl-necklace structures with spherical micelles. However, protein complexes with short surfactants (SC(8)S) bind with micelles that deviate significantly from spherical shape. Sodium decyl (SC(10)S) and dodecyl (SC(12)S, more commonly abbreviated as SDS) sulfates result in the best protein separations in standard gel electrophoresis. Particularly, SC(10)S shows higher resolutions for complexes of low molecular weight. The systematic characterization of alkyl sulfate surfactants demonstrates that changes in the chain architecture can significantly affect electrophoretic migration so that protein-surfactant structures could be optimized for high resolution protein separations.

  7. 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.

  8. Analysis of glycoprotein-derived oligosaccharides in glycoproteins detected on two-dimensional gel by capillary electrophoresis using on-line concentration method.

    Science.gov (United States)

    Kamoda, Satoru; Nakanishi, Yasuharu; Kinoshita, Mitsuhiro; Ishikawa, Rika; Kakehi, Kazuaki

    2006-02-17

    Capillary electrophoresis (CE) is an effective tool to analyze carbohydrate mixture derived from glycoproteins with high resolution. However, CE has a disadvantage that a few nanoliters of a sample solution are injected to a narrow capillary. Therefore, we have to prepare a sample solution of high concentration for CE analysis. In the present study, we applied head column field-amplified sample stacking method to the analysis of N-linked oligosaccharides derived from glycoprotein separated by two-dimensional gel electrophoresis. Model studies demonstrated that we achieved 60-360 times concentration effect on the analysis of carbohydrate chains labeled with 3-aminobenzoic acid (3-AA). The method was applied to the analysis of N-linked oligosaccharides from glycoproteins separated and detected on PAGE gel. Heterogeneity of alpha1-acid glycoprotein (AGP), i.e. glycoforms, was examined by 2D-PAGE and N-linked oligosaccharides were released by in-gel digestion with PNGase F. The released oligosaccharides were derivatized with 3-AA and analyzed by CE. The results showed that glycoforms having lower pI values contained a larger amount of tetra- and tri-antennary oligosaccharides. In contrast, glycoforms having higher pI values contained bi-antennary oligosaccharides abundantly. The result clearly indicated that the spot of a glycoprotein glycoform detected by Coomassie brilliant blue staining on 2D-PAGE gel is sufficient for quantitative profiling of oligosaccharides.

  9. Pulsed Field Gel Electrophoresis (PFGE: a DNA finger printing technique to study the genetic diversity of blood disease bacterium of banana

    Directory of Open Access Journals (Sweden)

    HADIWIYONO

    2011-01-01

    Full Text Available Hadiwiyono, Widada J, Subandiyah S, Fegan F (2011 Pulsed Field Gel Electrophoresis (PFGE: a DNA finger printing technique to study the genetic diversity of blood disease bacterium of banana. Biodiversitas 12: 12-16. Blood disease bacterium (BDB is the most important pathogen of bananas in Indonesia. In some field, the disease incidence reaches over 80%. Epidemiologically, the disease is similar to moko disease in South America and bugtok disease in the Philippines caused by Ralstonia solanacearum race 2. However, BDB is different in phenotype and genotype from the two diseases. Previously BDB was limited in South Sulawesi since 1920s – 1980s and recently was reported in 27 of 30 provinces in Indonesia. Pulsed-Field Gel Electrophoresis (PFGE is a genomic DNA fingerprinting method, which employs rare cutting restriction endonucleases to digest genome prior to electrophoresis using specialized condition to separate of large DNA fragments. The results showed that PFGE analysis was a discriminative tool to study the genetic diversity of BDB. Based on the PFGE analysis, BDB isolates obtained from different localities in Yogyakarta and Central Java were quit diverse.

  10. Proteomic Analysis of Bovine Pregnancy-specific Serum Proteins by 2D Fluorescence Difference Gel Electrophoresis

    Science.gov (United States)

    Lee, Jae Eun; Lee, Jae Young; Kim, Hong Rye; Shin, Hyun Young; Lin, Tao; Jin, Dong Il

    2015-01-01

    Two dimensional-fluorescence difference gel electrophoresis (2D DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The purpose of this study was to investigate bovine pregnancy-specific proteins in the proteome between bovine pregnant and non-pregnant serum using DIGE technique. Serums of 2 pregnant Holstein dairy cattle at day 21 after artificial insemination and those of 2 non-pregnant were used in this study. The pre-electrophoretic labeling of pregnant and non-pregnant serum proteins were mixed with Cy3 and Cy5 fluorescent dyes, respectively, and an internal standard was labeled with Cy2. Labeled proteins with Cy2, Cy3, and Cy5 were separated together in a single gel, and then were detected by fluorescence image analyzer. The 2D DIGE method using fluorescence CyDye DIGE flour had higher sensitivity than conventional 2D gel electrophoresis, and showed reproducible results. Approximately 1,500 protein spots were detected by 2D DIGE. Several proteins showed a more than 1.5-fold up and down regulation between non-pregnant and pregnant serum proteins. The differentially expressed proteins were identified by MALDI-TOF mass spectrometer. A total 16 protein spots were detected to regulate differentially in the pregnant serum, among which 7 spots were up-regulated proteins such as conglutinin precursor, modified bovine fibrinogen and IgG1, and 6 spots were down-regulated proteins such as hemoglobin, complement component 3, bovine fibrinogen and IgG2a three spots were not identified. The identified proteins demonstrate that early pregnant bovine serum may have several pregnancy-specific proteins, and these could be a valuable information for the development of pregnancy-diagnostic markers in early pregnancy bovine serum. PMID:25925056

  11. Proteomic Analysis of Bovine Pregnancy-specific Serum Proteins by 2D Fluorescence Difference Gel Electrophoresis

    Directory of Open Access Journals (Sweden)

    Jae Eun Lee

    2015-06-01

    Full Text Available Two dimensional-fluorescence difference gel electrophoresis (2D DIGE is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The purpose of this study was to investigate bovine pregnancy-specific proteins in the proteome between bovine pregnant and non-pregnant serum using DIGE technique. Serums of 2 pregnant Holstein dairy cattle at day 21 after artificial insemination and those of 2 non-pregnant were used in this study. The pre-electrophoretic labeling of pregnant and non-pregnant serum proteins were mixed with Cy3 and Cy5 fluorescent dyes, respectively, and an internal standard was labeled with Cy2. Labeled proteins with Cy2, Cy3, and Cy5 were separated together in a single gel, and then were detected by fluorescence image analyzer. The 2D DIGE method using fluorescence CyDye DIGE flour had higher sensitivity than conventional 2D gel electrophoresis, and showed reproducible results. Approximately 1,500 protein spots were detected by 2D DIGE. Several proteins showed a more than 1.5-fold up and down regulation between non-pregnant and pregnant serum proteins. The differentially expressed proteins were identified by MALDI-TOF mass spectrometer. A total 16 protein spots were detected to regulate differentially in the pregnant serum, among which 7 spots were up-regulated proteins such as conglutinin precursor, modified bovine fibrinogen and IgG1, and 6 spots were down-regulated proteins such as hemoglobin, complement component 3, bovine fibrinogen and IgG2a three spots were not identified. The identified proteins demonstrate that early pregnant bovine serum may have several pregnancy-specific proteins, and these could be a valuable information for the development of pregnancy-diagnostic markers in early pregnancy bovine serum.

  12. Separation of oligopeptides, nucleobases, nucleosides and nucleotides using capillary electrophoresis/electrochromatography with sol-gel modified inner capillary wall.

    Science.gov (United States)

    Svobodová, Jana; Kofroňová, Olga; Benada, Oldřich; Král, Vladimír; Mikšík, Ivan

    2017-09-29

    The aim of this article is to study the modification of an inner capillary wall with sol-gel coating (pure silica sol-gel or silica sol-gel containing porphyrin-brucine conjugate) and determine its influence on the separation process using capillary electrophoresis/electrochromatography method. After modification of the inner capillary surface the separation of analytes was performed using two different phosphate buffers (pH 2.5 and 9.0) and finally the changes in electrophoretic mobilities of various samples were calculated. To confirm that the modification of the inner capillary surface was successful, the parts of the inner surfaces of capillaries were observed using scanning electron microscopy. The analytes used as testing samples were oligopeptides, nucleosides, nucleobases and finally nucleotides. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Identification of proteins of human colorectal carcinoma cell line SW480 by two-dimensional electrophoresis and MALDI-TOF mass spectrometry

    Institute of Scientific and Technical Information of China (English)

    Ying-Tao Zhang; Yi-Ping Geng; Le Zhou; Bao-Chang Lai; Lv-Sheng Si; Yi-Li Wang

    2005-01-01

    AIM: To conduct the proteomic analysis of human colorectal carcinoma cell line, SW480 by using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption /ionization-time of flight mass spectrometry (MALDITOFMS).METHODS: The total proteins of human colorectal carcinoma cell line, SW480 were separated with 2-DE by using immobilized pH gradient strips and visualized by staining with silver nitrate. The gel images were acquired by scanner and 2-DE analysis software, Image Master 2D Elite. Nineteen distinct protein spots were excised from gel randomly and digested in gel by TPCK-trypsin. Mass analysis ofthe tryptic digest peptides mixture was performed by using MALDI-TOF MS. Peptide mass fingerprints (PMFs) obtained by the MALDI-TOF analysis were used to search NCBI,SWISS-PROT and MSDB databases by using Mascot software.RESULTS: PMF maps of all spots were obtained by MALDI-TOF MS and thirteen proteins were preliminarily identified.CONCLUSION: The methods of analysis and identification of protein spots of tumor cells in 2-DE gel with silver staining by MALDI-TOF MS derived PMF have been established.Protein expression profile of SW480 has been obtained.It is demonstrated that a combination of proteomics and cell culture is a useful approach to comprehend the process of colon carcinogenesis.

  14. Calibration of pulsed field gel electrophoresis for measurement of DNA double-strand breaks

    International Nuclear Information System (INIS)

    Ager, D.D.; Dewey, W.C.

    1990-01-01

    Pulsed field gel electrophoresis (PFGE) assay was calibrated for the measurement of X-ray induced DNA double-strand breaks in Chinese hamster ovary (CHO) cells. Calibration was conducted by incorporating [ 125 I] deoxyuridine into DNA, which induces one double-strand break for every disintegration that occurs in frozen cells. Based on the percentage of DNA migrating into the gel, the number of breaks/dalton/Gy was estimated to be (9.3±1.0) x 10 -12 . This value is close to (10 to 12) x 10 -12 determined by neutral filter elution using similar cell lysis procedures at 24 o C and at pH8.0. The estimate is in good agreement with the value of (11.7±2) x 10 -12 breaks/dalton/Gy as measured in Ehrlich ascites tumour cells using the neutral sucrose gradient method (Bloecher 1988), and (6 to 9) x 10 -12 breaks/dalton/Gy as measured in mouse L and Chinese hamster V79 cells using neutral filter elution (Radford and Hodgson 1985). (author)

  15. Quantitative gel electrophoresis: new records in precision by elaborated staining and detection protocols.

    Science.gov (United States)

    Deng, Xi; Schröder, Simone; Redweik, Sabine; Wätzig, Hermann

    2011-06-01

    Gel electrophoresis (GE) is a very common analytical technique for proteome research and protein analysis. Despite being developed decades ago, there is still a considerable need to improve its precision. Using the fluorescence of Colloidal Coomassie Blue -stained proteins in near-infrared (NIR), the major error source caused by the unpredictable background staining is strongly reduced. This result was generalized for various types of detectors. Since GE is a multi-step procedure, standardization of every single step is required. After detailed analysis of all steps, the staining and destaining were identified as the major source of the remaining variation. By employing standardized protocols, pooled percent relative standard deviations of 1.2-3.1% for band intensities were achieved for one-dimensional separations in repetitive experiments. The analysis of variance suggests that the same batch of staining solution should be used for gels of one experimental series to minimize day-to-day variation and to obtain high precision. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Study on the non-target effect of ionizing radiation using single cell gel electrophoresis

    International Nuclear Information System (INIS)

    Wang Yan; Li Deguan; Liu Jianfeng; Chu Liping; Liu Qiang

    2008-01-01

    Objective: To assess the non-target effect of ionizing radiation by single cell gel electrophoresis (SCGE). Methods: Cross incubated the irradiated( 137 Cs; 2Gy) or non-irradiated lymphocytes of human peripheral blood in the irradiated or non-irradiated plasma respectively, then, assess the DNA damage of lymphocytes using SCGE analysis. Results: The lymphocytes incubated in the irradiated plasma presented more obvious DNA damage than the incubated in the non-irradiated plasma dose (P<0.05). Conclusion: The non-target effect of ionizing radiation can be assessed by SCGE, and the results confirm that cytokines may play a great role in it. (authors)

  17. Radiation-induced DNA breaks detected by immuno labelling of poly(ADP-ribose) in CHO cells. Standardization by pulsed-field gel electrophoresis

    International Nuclear Information System (INIS)

    Varlet, P.; Bidon, N.; Noel, G.; Averbeck, D.; Salamero, J.; DeMurcia, G.

    1998-01-01

    The poly (ADP-ribose) polymerase is an ubiquitous nuclear protein capable of binding specifically to DNA strand breaks. It synthesizes ADP-ribose polymers proportionally to DNA breaks. The actual method of reference to determine DNA double strand breaks is pulsed-field gel electrophoresis, but this requires many cells. It thus appeared of interest to use poly (ADP-ribos)ylation to follow and estimate γ-ray-induced DNA fragmentation at the level of isolated cells after γ-irradiation in chinese hamster ovary cells (CHO-K1). The results obtained by the immuno-labelling technique of ADP-ribose polymers were compared to those obtained by pulsed-field gel electrophoresis. They show that poly (ADP-ribos)ylation reflects the occurrence of radiation-induced DNA strand breaks. A clear relationship exists between the amount of ADP-ribose polymers detected and DNA double strand breaks after γ-irradiation. (authors)

  18. Classification of wheat varieties: Use of two-dimensional gel electrophoresis for varieties that can not be classified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry and an artificial neural network

    DEFF Research Database (Denmark)

    Jacobsen, Susanne; Nesic, Ljiljana; Petersen, Marianne Kjerstine

    2001-01-01

    Analyzing a gliadin extract by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI- TOF-MS) combined with an artificial neural network (ANN) is a suitable method for identification of wheat varieties. However, the ANN can not distinguish between all different wheat...

  19. Characterization of Mycoplasma hyosynoviae strains by amplified fragment length polymorphism analysis, pulsed-field gel electrophoresis and 16S ribosomal DNA sequencing

    DEFF Research Database (Denmark)

    Kokotovic, Branko; Friis, N.F.; Ahrens, Peter

    2002-01-01

    , were investigated by analysis of amplified fragment length polymorphisms of the Bgl II and Mfe I restriction sites and by pulsed-field gel electrophoresis of a Bss HII digest of chromosomal DNA. Both methods allowed unambiguous differentiation of the analysed strains and showed similar discriminatory...

  20. Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes.

    Science.gov (United States)

    García-Cañas, Virginia; Mondello, Monica; Cifuentes, Alejandro

    2011-05-01

    New DNA amplification methods are continuously developed for sensitive detection and quantification of specific DNA target sequences for, e.g. clinical, environmental or food applications. These new applications often require the use of long DNA oligonucleotides as probes for target sequences hybridization. Depending on the molecular technique, the length of DNA probes ranges from 40 to 450 nucleotides, solid-phase chemical synthesis being the strategy generally used for their production. However, the fidelity of chemical synthesis of DNA decreases for larger DNA probes. Defects in the oligonucleotide sequence result in the loss of hybridization efficiency, affecting the sensitivity and selectivity of the amplification method. In this work, an enzymatic procedure has been developed as an alternative to solid-phase chemical synthesis for the production of long oligonucleotides. The enzymatic procedure for probe production was based on ligation of short DNA sequences. Long DNA probes were obtained from smaller oligonucleotides together with a short sequence that acts as bridge stabilizing the molecular complex for DNA ligation. The ligation reactions were monitored by capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) using a bare fused-silica capillary. The capillary gel electrophoresis-LIF method demonstrated to be very useful and informative for the characterization of the ligation reaction, providing important information about the nature of some impurities, as well as for the fine optimization of the ligation conditions (i.e. ligation cycles, oligonucleotide and enzyme concentration). As a result, the yield and quality of the ligation product were highly improved. The in-lab prepared DNA probes were used in a novel multiplex ligation-dependent genome amplification (MLGA) method for the detection of genetically modified maize in samples. The great possibilities of the whole approach were demonstrated by the specific and sensitive

  1. Comparison of DNA double-strand break rejoining as measured by pulsed field gel electrophoresis, neutral sucrose gradient centrifugation and non-unwinding filter elution in irradiated plateau-phase CHO cells

    International Nuclear Information System (INIS)

    Iliakis, G.; Metzger, L.; Pantelias, G.

    1991-01-01

    The initial (up to 30 min) rate of DNA double-strand break (dsb) rejoining was measured in irradiated plateau-phase CHO cells, in a set of parallel experiments using the same cell suspension, by means of non-unwinding filter elution, neutral sucrose gradient centrifugation, and two pulsed-field gel electrophoresis assays: asymmetric field inversion gel electrophoresis (AFIGE) and clamped homogeneous electric field (CHEF) gel electrophoresis. The rate of DNA dsb rejoining was compared to the rate of rejoining of chromatin breaks measured, also in the same cell population, using the technique of premature chromosome condensation (PCC). Two radiation exposures, 25 Gy and/or 50 Gy, were used and applied to the individual parts of the experiments according to the sensitivity of the assay under investigation. The results suggest all major techniques currently used for assaying rejoining of DNA dsb give similar results, and indicate that more information is required before a direct correlation between rejoining of DNA dsb and rejoining of chromatin breaks can be established. (author)

  2. Application of single cell gel electrophoresis in post-evaluation of organism radiation damage

    International Nuclear Information System (INIS)

    Jiang Lin; Mu Wanjun; Liu Guoping; Xu Yunshu; Luo Shunzhong; Gao Qingxiang

    2009-01-01

    The transient irradiation-caused DNA damage in the human peripheral blood lymphocytes,mouse peripheral blood lymphocytes and alive mouse irradiated by α-ray and γ-ray was investigated, and the single cell gel electrophoresis(SCGE, Comet Assay) was used to detect the extent of DNA damage. On this basis, the dose-effect curve and the evaluating method for radiant after-effect were established, the absorbed dose of alive mouse A irradiated by γ-rays was computed. The results indicate that not only the dose-effect can be described by using SCGE, but also the dose-computed after organism irradiated by radiant rays is achieved with it, and SCGE may be used as a new biological dosimeter. (authors)

  3. Application of single cell gel electrophoresis in post-evaluation of organism radiation damage

    International Nuclear Information System (INIS)

    Jiang Lin; Mu Wanjun; Liu Guoping; Xu Yunshu; Gao Qingxiang

    2007-01-01

    The immediate irradiation-caused DNA damage in the human Peripheral Blood Lymphocytes, mouse Peripheral Blood Lymphocytes and alive mouse irradiated by α-Rays and γ-rays was investigated, and the single cell gel electrophoresis(SCGE, Comet Assay) was used to detect the extent of DNA damage. On this base, the dose-effect curve and the evaluating method for radiant aftereffect were established, the absorbed does of alive mouse A irradiated by γ-rays was computed. The results indicated that not only the does-effect could be described by using SCGE, but also the does-computed after organism irradiated by radiant rays was achieved with it, and SCGE might be used as a new biological dosimeter. (authors)

  4. Calibration of low molecular weight polypeptides by sodium dodecylsulphate polyacrylamide gel electrophoresis

    International Nuclear Information System (INIS)

    Glyn, M.C.P.; Bull, J.; Wright, R.

    1982-01-01

    Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is a technique commonly used in determining molecular weights of large proteins and peptides. This technique is used to analyse viral peptides, available in amounts too small to be monitored by an ultraviolet spectrophotometer. An experiment is described (with the limiting factor to use the SDS-PAGE technique), to determine the molecular weight peptides and the results are given to fit the linear relationship log M=4.286 - 0.42 V(e)/V(o). The results given by the SDS-PAGE system, described in the article, show that the experimental values describe a linear relationship with good resolution of low molecular weight peptides in the range 3 000 to 14 000 and that a partial cyanogen bromide digest of cytochrome c is suitable for calibration standards

  5. Comparative analyses of amplicon migration behavior in differing denaturing gradient gel electrophoresis (DGGE) systems

    Science.gov (United States)

    Thornhill, D. J.; Kemp, D. W.; Sampayo, E. M.; Schmidt, G. W.

    2010-03-01

    Denaturing gradient gel electrophoresis (DGGE) is commonly utilized to identify and quantify microbial diversity, but the conditions required for different electrophoretic systems to yield equivalent results and optimal resolution have not been assessed. Herein, the influence of different DGGE system configuration parameters on microbial diversity estimates was tested using Symbiodinium, a group of marine eukaryotic microbes that are important constituents of coral reef ecosystems. To accomplish this, bacterial clone libraries were constructed and sequenced from cultured isolates of Symbiodinium for the ribosomal DNA internal transcribed spacer 2 (ITS2) region. From these, 15 clones were subjected to PCR with a GC clamped primer set for DGGE analyses. Migration behaviors of the resulting amplicons were analyzed using a range of conditions, including variation in the composition of the denaturing gradient, electrophoresis time, and applied voltage. All tests were conducted in parallel on two commercial DGGE systems, a C.B.S. Scientific DGGE-2001, and the Bio-Rad DCode system. In this context, identical nucleotide fragments exhibited differing migration behaviors depending on the model of apparatus utilized, with fragments denaturing at a lower gradient concentration and applied voltage on the Bio-Rad DCode system than on the C.B.S. Scientific DGGE-2001 system. Although equivalent PCR-DGGE profiles could be achieved with both brands of DGGE system, the composition of the denaturing gradient and application of electrophoresis time × voltage must be appropriately optimized to achieve congruent results across platforms.

  6. Development and application of a selective pcr-denaturing gradient gel electrophoresis approach to detect a recently cultivated Bacillus group predominant in soil

    NARCIS (Netherlands)

    Tzeneva, V.A.; Li, Y.; Felske, A.; Vos, de W.M.; Akkermans, A.D.L.; Vaughan, E.E.; Smidt, H.

    2004-01-01

    The worldwide presence of a hitherto-nondescribed group of predominant soil microorganisms related to Bacillus benzoevorans was analyzed after development of two sets of selective primers targeting 16S rRNA genes in combination with denaturing gradient gel electrophoresis (DGGE). The high abundance

  7. Titanium Dioxide Photocatalytic Polymerization of Acrylamide for Gel Electrophoresis (TIPPAGE) of Proteins and Structural Identification by Mass Spectrometry

    Science.gov (United States)

    Zhang, Wenyang; Yuan, Zhiwei; Huang, Lulu; Kang, Jie; Jiang, Ruowei; Zhong, Hongying

    2016-01-01

    Polyacrylamide gel electrophoresis (PAGE) coupled with mass spectrometry has been well established for separating, identifying and quantifying protein mixtures from cell lines, tissues or other biological samples. The copolymerization process of acrylamide and bis-acrylamide is the key to mastering this powerful technique. In general, this is a vinyl addition reaction initiated by free radical-generating reagents such as ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) under basic pH and degassing experimental condition. We report herein a photocatalytic polymerization approach that is based on photo-generated hydroxyl radicals with nanoparticles of titanium dioxide. It was shown that the polymerization process is greatly accelerated in acidic condition when ultraviolet light shots on the gel solution containing TiO2 nanoparticles without degassing. This feature makes it very useful in preparing Triton X-100 acid urea (TAU) gel that has been developed for separating basic proteins such as histones and variants in acidic experimental condition. Additionally, the presence of titanium dioxide in the gel not only improves mechanistic property of gels but also changes the migration pattern of different proteins that have different affinities to titanium dioxide. PMID:26865351

  8. Application of native agarose gel electrophoresis of serum proteins in veterinary diagnostics

    Directory of Open Access Journals (Sweden)

    Jania Bartosz

    2016-12-01

    Full Text Available Electrophoretic techniques, used to separate mixtures of electrically charged particles, are widely used in science. One of these techniques, native protein electrophoresis in an agarose gel, is applied in human and veterinary medicine. Changes in the proportions of individual protein fractions correspond to significant changes in the physiology of the body. Although the pattern obtained by electrophoretic separation rarely indicates a specific disease, it provides valuable information for the differential diagnosis. Decades of research on the types of patterns obtained in the case of particular diseases have led to the accumulation of substantial knowledge. The paper presents the available information on this topic. Serum protein electrophoresis is recommended in cases of increased levels of total protein in order to reveal the nature of the process. The basic information which can be obtained from electrophoretic separation includes the immune status of the organism. Both increased antigenic stimulation and immunodeficiency are clearly visible in electropherograms. Moreover, the level of heterogeneity of the corresponding protein fractions can help to distinguish between infectious diseases and cancer - multiple myeloma - the latter producing a homogeneous immunoglobulin fraction. Analysis of other protein fractions helps to detect or confirm an ongoing inflammatory process and provides information regarding liver function. Even when the concentration of total protein is within the reference range, this analysis can be recommended as a basic laboratory test.

  9. Protein profile analysis of Malaysian snake venoms by two-dimensional gel electrophoresis

    Directory of Open Access Journals (Sweden)

    J Vejayan

    2010-01-01

    Full Text Available Snake venoms comprise a highly complex mixture of proteins, which requires for their characterization the use of versatile two-dimensional electrophoresis techniques. In the present study, venoms obtained from eight snakes (Ophiophagus hannah, Naja kaouthia, Naja sumatrana, Bungarus fasciatus, Trimeresurus sumatranus, Tropidolaemus wagleri, Enhydrina schistosa and Calloselasma rhodostoma commonly found in Malaysia were separated based on two independent properties, isoelectric point (pI and molecular weight (MW. Many differences in snake venoms at the inter-family, inter-subfamily, inter-genus and inter-species levels were revealed. Notably, proteins from individuals of the Viperidae family - Trimeresurus sumatranus, Tropidolaemus wagleri and Calloselasma rhodostoma - were found to be numerous and scattered by the two-dimensional gel electrophoresis (2DE specifically in regions between 37 and 100 kDa compared to the Elapidae venom proteins. The latter were clustered at the basic and lower molecular mass region (less than 20 kDa. Trains of spots were commonly observed, indicating that these proteins may be derived from post-translational modifications. Ophiophagus hannah (Elapidae revealed a great amount of protein spots in the higher molecular mass range when compared to Enhydrina schistosa, Naja kaouthia, Naja sumatrana and Bungarus fasciatus. Overall 2DE showed large differences in the venom profile of each species, which might be employed as an ancillary tool to the identification of venomous snake species.

  10. Evaluation of a capillary zone electrophoresis system versus a conventional agarose gel system for routine serum protein separation and monoclonal component typing.

    Science.gov (United States)

    Roudiere, L; Boularan, A M; Bonardet, A; Vallat, C; Cristol, J P; Dupuy, A M

    2006-01-01

    Capillary zone electrophoresis of serum proteins is increasingly gaining impact in clinical laboratories. During 2003, we compared the fully automated capillary electrophoresis (CE) system from Beckman (Paragon CZE 2000) with the method agarose gel electrophoresis Sebia (Hydrasis-Hyris, AGE). This new study focused on the evaluation of analytical performance and a comparison including 115 fresh routine samples (group A) and a series of 97 frozen pathologic sera with suspicion of monoclonal protein (group B). Coefficients of variation (CVs %) for the five classical protein fractions have been reported to be consistenly serum samples (group B), there were 90 in which we detected a monoclonal protein by immunofixation (IF) (immunosubtraction (IS) was not used). AGE and Paragon 2000 failed to detect 7 and 12 monoclonal proteins, respectively, leading to a concordance to 92% for AGE and 87% for Paragon 2000 for identifying electrophoretic abnormalities in this group. Beta-globulin abnormalities and M paraprotein were well detected with Paragon 2000. Only 81% (21 vs 26) of the gammopathies were immunotyped with IS by two readers blinded to the IF immunotype. The Paragon 2000 is a reliable alternative to conventional agarose gel electrophoresis combining the advantages of full automation (rapidity, ease of use and cost) with high analytical performance. Qualified interpretation of results requires an adaptation period which could further improve concordance between the methods. Recently, this CE system has been improved by the manufacturer (Beckman) concerning the migration buffer and detection of beta-globulin abnormalities.

  11. Effects of interferon gamma on Chlamydia trachomatis serovar A and L2 protein expression investigated by two-dimensional gel electrophoresis

    DEFF Research Database (Denmark)

    Shaw, A; Christiansen, Gunna; Birkelund, Svend

    1999-01-01

    ]methionine and two-dimensional gel electrophoresis with immobilized pH gradients in order to investigate changes in the protein expression of C. trachomatis serovar A and L2 caused by treatment with IFN-gamma. In contrast to what was observed in C. trachomatis L2, our results showed that, in C. trachomatis A, down...

  12. Combining two-dimensional gel electrophoresis and metabolomic data in support of dry-season survival in the two main species of the malarial mosquito Anopheles gambiae

    Directory of Open Access Journals (Sweden)

    Hidalgo K.

    2015-12-01

    Full Text Available In dry savannahs of West-Africa, the malarial mosquitoes of the Anopheles gambiae sensu stricto complex annually survive the harsh desiccating conditions of the dry season. However, the physiological and biochemical mechanisms underlying how these mosquitoes survive such desiccating conditions are still undefined, and controversial. In this context, we provide the first work examining both proteomic and metabolomic changes in the two molecular forms of A. gambiae s.s (M and S forms experimentally exposed to the rainy and dry season conditions as they experience in the field. Protein abundances of the mosquitoes were measured using a two-dimensional fluorescence difference gel electrophoresis (2D DIGE coupled with a matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF and tandem mass spectrometry (MS for protein identification. These assays were conducted by Applied Biomics (http://www.appliedbiomics.com, Applied Biomics, Inc. Hayward, CA, USA, and the mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org via the PRIDE partner repository with the dataset identifier PXD000294. The metabolomic analysis was conducted using both Acquity UPLC® system (for amino acid identification, and a gas-chromatography-mass spectrometry platform (for sugars identification. Metabolomic fingerprintings were assessed in the University of Rennes 1, UMR CNRS 6553 EcoBio (France. A detailed interpretation of the obtained data can be found in Hidalgo et al. (2014 [1] (Journal of Insect Physiology (2014.

  13. Ultrasound-assisted sol-gel synthesis of ZrO2.

    Science.gov (United States)

    Guel, Marlene Lariza Andrade; Jiménez, Lourdes Díaz; Hernández, Dora Alicia Cortés

    2017-03-01

    Synthesis of tetragonal ZrO 2 by both conventional sol-gel and ultrasound-assisted sol-gel methods and using a non-ionic surfactant Tween-20, was performed. A porous microstructure composed of nanometric particles was observed. Tetragonal ZrO 2 was obtained using a low heat treatment temperature of powders, 500°C by both methods. A higher crystallinity and a shorter reaction time were observed when ultrasound was used in the sol-gel method due to the cavitation phenomenon. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. The laboratory technology of discrete molecular separation: the historical development of gel electrophoresis and the material epistemology of biomolecular science, 1945-1970.

    Science.gov (United States)

    Chiang, Howard Hsueh-hao

    2009-01-01

    Preparative and analytical methods developed by separation scientists have played an important role in the history of molecular biology. One such early method is gel electrophoresis, a technique that uses various types of gel as its supporting medium to separate charged molecules based on size and other properties. Historians of science, however, have only recently begun to pay closer attention to this material epistemological dimension of biomolecular science. This paper substantiates the historiographical thread that explores the relationship between modern laboratory practice and the production of scientific knowledge. It traces the historical development of gel electrophoresis from the mid-1940s to the mid-1960s, with careful attention to the interplay between technical developments and disciplinary shifts, especially the rise of molecular biology in this time-frame. Claiming that the early 1950s marked a decisive shift in the evolution of electrophoretic methods from moving boundary to zone electrophoresis, I reconstruct various trajectories in which scientists such as Oliver Smithies sought out the most desirable solid supporting medium for electrophoretic instrumentation. Biomolecular knowledge, I argue, emerged in part from this process of seeking the most appropriate supporting medium that allowed for discrete molecular separation and visualization. The early 1950s, therefore, marked not only an important turning point in the history of separation science, but also a transformative moment in the history of the life sciences as the growth of molecular biology depended in part on the epistemological access to the molecular realm available through these evolving technologies.

  15. Rapid identification of Pseudomonas aeruginosa by pulsed-field gel electrophoresis.

    Science.gov (United States)

    Selim, Samy; El Kholy, Iman; Hagagy, Nashwa; El Alfay, Sahar; Aziz, Mohamed Abdel

    2015-01-02

    Twenty clinical Pseudomonas aeruginosa isolates recovered from patients admitted to The General Hospital in Ismailia Governorate (Egypt) were examined in this study. We analysed P. aeruginosa ATCC 9027 (as a control strain) and 19 of the isolates after digestion with SpeI restriction endonuclease. After this we conducted a pulsed-field gel electrophoresis (PFGE) and typed the obtained 10 unique patterns, designated as A, A1, B, B1, C, C1, D, D1, E and F. We evaluated the genetic relatedness between all strains, based on ≥87% band identity. As a result, the isolates were grouped in the 10 clusters as follows: patterns A, A1, B, B1, C contained two strains each and patterns C1, D, D1, E contained a single strain each; the five remaining strains were closely related (genomic pattern F). One isolate belonged to antibiotype 'b'. The genotype patterns of the P. aeruginosa ATCC 9027 control strain and isolate no. 11 were closely related and had two different antibiotypes 'd' and 'c', respectively.

  16. Antigenic profile of heat-killed versus thimerosal-treated Leishmania major using sodium dodecyl sulfate-polyacrylamide gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Reza Arjmand

    2015-01-01

    Full Text Available Background: Leishmania is a parasitic protozoan of trypanosomatidae family which causes a wide spectrum of diseases ranging from self-healing cutaneous lesions to deadly visceral forms. In endemic areas, field trials of different preparations of Leishmania total antigen were tested as leishmaniasis vaccine. Two preparations of killed Leishmania major were produced In Iran, which were heat-killed vaccine called autoclaved L. major (ALM and thimerosal-treated freeze-thawed vaccine called killed L. major (KLM. In this study, the protein content of both ALM and KLM were compared with that of freshly harvested intact L. major promastigotes using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE. Materials and Methods: L. major (MRHO/IR/75/ER from pre-infected Balb/c mice was isolated with modified Novy-MacNeal-Nicolle (NNN medium and then subcultured in liquid RPMI 1640 medium supplemented with fetal calf serum (FCS 20% for mass production. Two preparations of KLM and ALM were produced by Razi Vaccine and Serum Research Institute, Iran, under WHO/TDR supervision. Electrophoresis was performed by SDS-PAGE method and the gel was stained by Coomassie brilliant blue dye. The resultant unit bands were compared using standard molecular proteins. Results: Electrophoresis of the two preparations produced many bands from 10 kDa to 100 kDa. KLM bands were much like those of freshly harvested intact L. major. Conclusion: It is concluded that although there are similar bands in the three forms of Leishmania antigens, there are some variations which might be considered for identification and purification of protective immunogens in a total crude antigen, and detection of their stability is essential for the production and marketing of a putative vaccine.

  17. Salt-assisted and salt-suppressed sol-gel transitions of methylcellulose in water.

    Science.gov (United States)

    Xu, Y; Wang, C; Tam, K C; Li, L

    2004-02-03

    The effects of various salts on the sol-gel transition of aqueous methylcellulose (MC) solutions have been studied systematically by means of a micro differential scanning calorimeter. It was found that the heating process was endothermic while the cooling process was exothermic for both MC solutions with and without salts. The addition of salts did not change the patterns of gelation and degelation of MC. However, the salts could shift the sol-gel transition and the gel-sol transition to lower or higher temperatures from a pure MC solution, depending on the salt type. These opposite effects were termed the salt-assisted and salt-suppressed sol-gel transitions. Either the salt-assisted transition or the salt-suppressed sol-gel transition was a function of salt concentration. In addition, each salt was found to have its own concentration limit for producing a stable aqueous solution of MC at a given concentration of MC, which was related to the anionic charge density of the salt. Cations were proved to have weaker effects than anions. The "salt-out strength", defined as the salt effect per mole of anion, was obtained for each anion studied. The thermodynamic mechanisms involved in the salt-assisted and salt-suppressed sol-gel transitions are discussed.

  18. One-day pulsed-field gel electrophoresis protocol for rapid determination of emetic Bacillus cereus isolates.

    Science.gov (United States)

    Kaminska, Paulina S; Fiedoruk, Krzysztof; Jankowska, Dominika; Mahillon, Jacques; Nowosad, Karol; Drewicka, Ewa; Zambrzycka, Monika; Swiecicka, Izabela

    2015-04-01

    Bacillus cereus, the Gram-positive and spore-forming ubiquitous bacterium, may cause emesis as the result of food intoxication with cereulide, a heat-stable emetic toxin. Rapid determination of cereulide-positive B. cereus isolates is of highest importance due to consequences of this intoxication for human health and life. Here we present a 1-day pulsed-field gel electrophoresis for emetic B. cereus isolates, which allows rapid and efficient determination of their genomic relatedness and helps determining the source of intoxication in case of outbreaks caused by these bacilli. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Electrophoresis Gel Quantification with a Flatbed Scanner and Versatile Lighting from a Screen Scavenged from a Liquid Crystal Display (LCD) Monitor

    Science.gov (United States)

    Yeung, Brendan; Ng, Tuck Wah; Tan, Han Yen; Liew, Oi Wah

    2012-01-01

    The use of different types of stains in the quantification of proteins separated on gels using electrophoresis offers the capability of deriving good outcomes in terms of linear dynamic range, sensitivity, and compatibility with specific proteins. An inexpensive, simple, and versatile lighting system based on liquid crystal display backlighting is…

  20. Pseudomonas aeruginosa endophthalmitis after penetrating keratoplasty transmitted from the same donor to two recipients confirmed by pulsed-field gel electrophoresis.

    Science.gov (United States)

    Oguido, Ana Paula Miyagusko Taba; Casella, Antonio Marcelo Barbante; Hofling-Lima, Ana Luisa; Pacheco, Sergio Arruda; Bispo, Paulo José Martins; Marques, Fernanda

    2011-09-01

    Two devastating cases of multidrug-resistant Pseudomonas aeruginosa endophthalmitis after keratoplasty as the result of transmission from the same donor were confirmed by pulsed-field gel electrophoresis. Strategies for preventing donor-to-host transmission, such as the use of antimicrobial agents of greater efficacy and better methods for detecting microorganisms in preservation medium, could minimize this type of transmission.

  1. Detection of Aspartic Proteinase Activities Using Gel Zymography.

    Science.gov (United States)

    Perera, Handunge Kumudu Irani

    2017-01-01

    Gel zymography is a two-stage process where the proteins from the test sample are first separated by electrophoresis followed by the detection of the activity of hydrolytic enzymes. Many zymography procedures use sodium dodecyl sulfate (SDS) polyacrylamide gels copolymerized with an appropriate substrate. The procedure described here uses native polyacrylamide gel electrophoresis (PAGE) in the absence of both SDS and substrate. In order to visualize aspartic proteinase activity, the gel is impregnated in bovine hemoglobin at pH 3.0 for 15 min after the electrophoresis procedure. Subsequently, the gel is incubated in a humid container in the absence of hemoglobin for 1 h at 37 °C. At the end, the gel is stained with amido black and destained. Clear areas against a dark background corresponding to aspartic proteinase activities can be detected.

  2. Comparative proteomic analysis of the ribosomes in 5-fluorouracil resistance of a human colon cancer cell line using the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis.

    Science.gov (United States)

    Kimura, Kosei; Wada, Akira; Ueta, Masami; Ogata, Akihiko; Tanaka, Satoru; Sakai, Akiko; Yoshida, Hideji; Fushitani, Hideo; Miyamoto, Akiko; Fukushima, Masakazu; Uchiumi, Toshio; Tanigawa, Nobuhiko

    2010-11-01

    Many auxiliary functions of ribosomal proteins (r-proteins) have received considerable attention in recent years. However, human r-proteins have hardly been examined by proteomic analysis. In this study, we isolated ribosomal particles and subsequently compared the proteome of r-proteins between the DLD-1 human colon cancer cell line and its 5-fluorouracil (5-FU)-resistant sub-line, DLD-1/5-FU, using the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis, which has a superior ability to separate basic proteins, and we discuss the role of r-proteins in 5-FU resistance. Densitometric analysis was performed to quantify modulated proteins, and protein spots showing significant changes were identified by employing matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Three basic proteins (L15, L37 and prohibitin) which were significantly modulated between DLD-1 and DLD-1/5-FU were identified. Two proteins, L15 and L37, showed down-regulated expression in DLD-1/5-FU in comparison to DLD-1. Prohibitin, which is not an r-protein and is known to be localized in the mitochondria, showed up-regulated expression in DLD-1/5-FU. These 3 proteins may be related to 5-FU resistance.

  3. Two-dimensional gel electrophoresis and FTIR spectroscopy reveal both forms of yeast plasma membrane H(+)-ATPase in activated and basal-level enzyme preparations

    Czech Academy of Sciences Publication Activity Database

    Lapathitis, Georgios; Tanfani, F.; Kotyk, Arnošt; Bertoli, E.

    2001-01-01

    Roč. 505, č. 1 (2001), s. 155-158 ISSN 0014-5793 R&D Projects: GA ČR GA204/98/0474 Keywords : H+-ATPase * plasma membrane * two-dimensional gel electrophoresis Subject RIV: CE - Biochemistry Impact factor: 3.644, year: 2001

  4. Synthesis of hydrogel via click chemistry for DNA electrophoresis.

    Science.gov (United States)

    Finetti, Chiara; Sola, Laura; Elliott, Jim; Chiari, Marcella

    2017-09-01

    This work introduces a novel sieving gel for DNA electrophoresis using a classical click chemistry reaction, the copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC), to cross-link functional polymer chains. The efficiency of this reaction provides, under mild conditions, hydrogels with near-ideal network connectivity and improved physical properties. Hydrogel formation via click chemistry condensation of functional polymers does not involve the use of toxic monomers and UV initiation. The performance of the new hydrogel in the separation of double stranded DNA fragments was evaluated in the 2200 TapeStation system, an analytical platform, recently introduced by Agilent that combines the advantages of CE in terms of miniaturization and automation with the simplicity of use of slab gel electrophoresis. The click gel enables addition of florescent dyes prior to electrophoresis with considerable improvement of resolution and separation efficiency over conventional cross-linked polyacrylamide gels. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Determination of saccharides in fruit juices by capillary electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry

    Czech Academy of Sciences Publication Activity Database

    Žídková, Jitka; Chmelík, Josef

    2001-01-01

    Roč. 36, č. 4 (2001), s. 417-421 ISSN 1076-5174 R&D Projects: GA MZe EP9410 Institutional research plan: CEZ:AV0Z4031919 Keywords : fruit juices * adulteration * matrix-assisted laser deasorption Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 2.685, year: 2001

  6. Fast screening of glycosaminoglycan disaccharides by fluorophore-assisted carbohydrate electrophoresis (FACE): applications to biologic samples and pharmaceutical formulations.

    Science.gov (United States)

    Karousou, Evgenia; Asimakopoulou, Athanasia P; Zafeiropoulou, Vassiliki; Viola, Manuela; Monti, Luca; Rossi, Antonio; Passi, Alberto; Karamanos, Nikos

    2015-01-01

    Hyaluronan (HA), chondroitin sulfate (CS), and heparan sulfate (HS) are glycosaminoglycans (GAGs) with a great importance in biological processes as they participate in functional cell properties, such as migration, adhesion, and proliferation. A perturbation of the quantity and/or the sulfation of GAGs is often associated with pathological conditions. In this chapter, we present valuable and validated protocols for the analysis of HA-, CS-, and HS-derived disaccharides after derivatization with 2-aminoacridone and by using the fluorophore-assisted carbohydrate electrophoresis (FACE). FACE is a well-known technique and a reliable tool for a fast screening of GAGs, as it is possible to analyze 16 samples at the same time with one electrophoretic apparatus. The protocols for the gel preparation are based on the variations of the acrylamide/bisacrylamide and buffer concentrations. Different approaches for the extraction and purification of the disaccharides of various biologic samples and pharmaceutical preparations are also stressed.

  7. Meta-analysis of pulsed-field gel electrophoresis fingerprints based on a constructed Salmonella database.

    Directory of Open Access Journals (Sweden)

    Wen Zou

    Full Text Available A database was constructed consisting of 45,923 Salmonella pulsed-field gel electrophoresis (PFGE patterns. The patterns, randomly selected from all submissions to CDC PulseNet during 2005 to 2010, included the 20 most frequent serotypes and 12 less frequent serotypes. Meta-analysis was applied to all of the PFGE patterns in the database. In the range of 20 to 1100 kb, serotype Enteritidis averaged the fewest bands at 12 bands and Paratyphi A the most with 19, with most serotypes in the 13-15 range among the 32 serptypes. The 10 most frequent bands for each of the 32 serotypes were sorted and distinguished, and the results were in concordance with those from distance matrix and two-way hierarchical cluster analyses of the patterns in the database. The hierarchical cluster analysis divided the 32 serotypes into three major groups according to dissimilarity measures, and revealed for the first time the similarities among the PFGE patterns of serotype Saintpaul to serotypes Typhimurium, Typhimurium var. 5-, and I 4,[5],12:i:-; of serotype Hadar to serotype Infantis; and of serotype Muenchen to serotype Newport. The results of the meta-analysis indicated that the pattern similarities/dissimilarities determined the serotype discrimination of PFGE method, and that the possible PFGE markers may have utility for serotype identification. The presence of distinct, serotype specific patterns may provide useful information to aid in the distribution of serotypes in the population and potentially reduce the need for laborious analyses, such as traditional serotyping.

  8. Differences in serum protein 2D gel electrophoresis patterns of Przewalski's (Mongolian wild horse) and thoroughbred horses.

    Science.gov (United States)

    Barsuren, Enkhbolor; Namkhai, Bandi; Kong, Hong Sik

    2015-04-01

    The objective of this study was to assess differences in serum protein expression profiles of Przewalski's (Mongolian wild horse) and thoroughbred horses using proteome analysis. The serum proteins were separated by two-dimensional electrophoresis (2-DE) and five different gene products were identified. Proteins represented by the five spots were identified by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS)/MS technology. The identities of all proteins were deduced based on their similarity to proteins in the human plasma protein database. Three proteins (a haptoglobin-2 alpha glycoprotein and two haptoglobin-2beta glycoproteins with different accession numbers) were downregulated in Przewalski's horse sera compared to thoroughbred horse sera. Moreover, two proteins (tetraspanin-18 and pM5) were upregulated in Przewalski's horses compared to thoroughbred horses. Haptoglobin-2 alpha and haptoglobin-2beta may serve as candidate molecules in future studies of inflammation, coagulation, immune modulation and pro-oxidant and antioxidant activity with consequential effects on the entire metabolism of the horse. © 2014 Japanese Society of Animal Science.

  9. Assessment of Matrix Metalloproteinases by Gelatin Zymography.

    Science.gov (United States)

    Cathcart, Jillian

    2016-01-01

    Matrix metalloproteinases are endopeptidases responsible for remodeling of the extracellular matrix and have been identified as critical contributors to breast cancer progression. Gelatin zymography is a valuable tool which allows the analysis of MMP expression. In this approach, enzymes are resolved electrophoretically on a sodium dodecyl sulfate-polyacrylamide gel copolymerized with the substrate for the MMP of interest. Post electrophoresis, the enzymes are refolded in order for proteolysis of the incorporated substrate to occur. This assay yields valuable information about MMP isoforms or changes in activation and can be used to analyze the role of MMPs in normal versus pathological conditions.

  10. Characterization of the Human Skeletal Muscle Proteome by One-dimensional Gel Electrophoresis and HPLC-ESI-MS/MS

    DEFF Research Database (Denmark)

    Højlund, Kurt; Yi, Zhengping; Hwang, Hyonson

    2008-01-01

    and pathophysiological conditions. However, to date, the number of proteins identified by this approach has been limited, with 107 different proteins being the maximum reported so far. Using a combination of one-dimensional gel electrophoresis and high performance liquid chromatography electrospray ionization tandem...... muscle. Mitochondrial proteins accounted for 22% of all proteins identified, including 55 subunits of the respiratory complexes I-V. Moreover, a number of enzymes involved in endocrine and metabolic signaling pathways as well as calcium homeostasis were identified. These results provide the most...

  11. Method for the typing of Clostridium difficile based on polyacrylamide gel electrophoresis of [35S]methionine-labeled proteins

    International Nuclear Information System (INIS)

    Tabaqchali, S.; O'Farrell, S.; Holland, D.; Silman, R.

    1986-01-01

    A typing method for Clostridium difficile based on the incorporation of [ 35 S]methionine into cellular proteins, their separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their visualization by autoradiography is described. On analysis of the radiolabeled-protein profiles, nine distinct groups were observed (A to E and W to Z). The method, which is simple, reproducible, and readily expandable, has been applied in epidemiological studies to demonstrate cross-infection and hospital acquisition of C. difficile

  12. Development of an open source laboratory information management system for 2-D gel electrophoresis-based proteomics workflow

    Directory of Open Access Journals (Sweden)

    Toda Tosifusa

    2006-10-01

    Full Text Available Abstract Background In the post-genome era, most research scientists working in the field of proteomics are confronted with difficulties in management of large volumes of data, which they are required to keep in formats suitable for subsequent data mining. Therefore, a well-developed open source laboratory information management system (LIMS should be available for their proteomics research studies. Results We developed an open source LIMS appropriately customized for 2-D gel electrophoresis-based proteomics workflow. The main features of its design are compactness, flexibility and connectivity to public databases. It supports the handling of data imported from mass spectrometry software and 2-D gel image analysis software. The LIMS is equipped with the same input interface for 2-D gel information as a clickable map on public 2DPAGE databases. The LIMS allows researchers to follow their own experimental procedures by reviewing the illustrations of 2-D gel maps and well layouts on the digestion plates and MS sample plates. Conclusion Our new open source LIMS is now available as a basic model for proteome informatics, and is accessible for further improvement. We hope that many research scientists working in the field of proteomics will evaluate our LIMS and suggest ways in which it can be improved.

  13. Study on dose-effect relationship of radiation-induced DNA damage using single cell gel electrophoresis

    International Nuclear Information System (INIS)

    Liu Qiang; Jiang Enhai; Li Jin; Tang Weisheng; Wang Zhiquan; Zhao Yongcheng; Fan Feiyue

    2006-01-01

    Objective: To explore a new, simple and quick radiation biodosimeter, which can be applied to estimate biological dose in case of radiation accident. Methods: DNA double-strand break were detected using neutral single cell gel electrophoresis (SCGE), and all the indexes of comet assay including HDNA%, TDNA%, CL, TL, TM, and OTM were analyzed by CASP (Comet Assay Software Project). The curve of dose-effect was fitted using SPSS 12.0 software. Results: Statistically significant dose-effect relationships were observed in all the indexes of comet assay, OTM was superior to other indexes. Conclusions: Application of neutral comet assay combined with CASP analysis could serve as a new radiation biodosimeter. (authors)

  14. Studies on irradiated lactate dehydrogenase using gel electrophoresis

    International Nuclear Information System (INIS)

    Huth, O.

    1981-01-01

    LDH in aqueous phosphate buffer solution was x-irradiated under nitrogen and dinitrogen oxide. The pre-treated samples were separated by SDS-phosphate gel electrophoresis. Colour yields of the protein bands were determined by planimetry following staining and photometric densitometry. It could be shown that aggregates formed up to the tetramer size were mainly covalently bonded and that negligent amounts of fragments are formed. No exact statements could be made concerning products formed after longer irradiation treatment. Measurements under UV-light also revealed an elevated base from the point of application to the band at the native LDH level; this supports the theory of a reduced migration path through intra-molecular bonding. No firm conclusions can be drawn from the process of staining because CBB itself leads to an increased background colouring between closely-lying protein bands. It could be shown that OH-radicals cause greater changes than H-radicals or solvated electrons since the native LDH peak diminished fastest when irradiated under dinitrogen oxide. With regard to radiation effect it could be demonstrated that a higher radiation dose was required for degradation of the LDH molecule than for its inactivation. These values approach each other with increasing concentration. This indicates that at low concentrations inactivation of the enzyme is mainly caused by conformational changes and at high concentrations by aggregation. Disulphide bridges were found to contribute to aggregate formation in 5-15% of the aggregates formed, this increasing linearly with concentration. (orig./MG) [de

  15. Characterization of vancomycin-resistant and vancomycin-susceptible Enterococcus faecium isolates from humans, chickens and pigs by RiboPrinting and pulsed-field gel electrophoresis

    DEFF Research Database (Denmark)

    Hammerum, Anette Marie; Fussing, Vivian; Aarestrup, Frank Møller

    2000-01-01

    Forty-eight vancomycin-resistant and 35 vancomycin-sensitive Danish Enterococcus faecium isolates obtained from pigs, chickens and humans, as well as the human vanA reference isolate BM4147, were characterized by EcoRI RiboPrinting and Smal pulsed-field gel electrophoresis. RiboPrinting of the 84...

  16. Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis

    Directory of Open Access Journals (Sweden)

    Hoseok Choi

    2016-04-01

    Full Text Available Assaying the glycogen synthase kinase-3 (GSK3 activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed peptide substrate for GSK3 was employed, a distinct mobility shift in the fluorescent bands on the agarose was observed by GSK3-induced phosphorylation of the primed peptides. The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts.

  17. Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis

    Science.gov (United States)

    Choi, Hoseok; Choi, Bomi; Seo, Ju Tae; Lee, Kyung Jin; Gye, Myung Chan; Kim, Young-Pil

    2016-01-01

    Assaying the glycogen synthase kinase-3 (GSK3) activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed) peptide substrate for GSK3 was employed, a distinct mobility shift in the fluorescent bands on the agarose was observed by GSK3-induced phosphorylation of the primed peptides. The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts. PMID:27092510

  18. Detection by denaturing gradient gel electrophoresis of ammonia-oxidizing bacteria in microcosms of crude oil-contaminated mangrove sediments.

    Science.gov (United States)

    dos Santos, A C F; Marques, E L S; Gross, E; Souza, S S; Dias, J C T; Brendel, M; Rezende, R P

    2012-01-27

    Currently, the effect of crude oil on ammonia-oxidizing bacterium communities from mangrove sediments is little understood. We studied the diversity of ammonia-oxidizing bacteria in mangrove microcosm experiments using mangrove sediments contaminated with 0.1, 0.5, 1, 2, and 5% crude oil as well as non-contaminated control and landfarm soil from near an oil refinery in Camamu Bay in Bahia, Brazil. The evolution of CO(2) production in all crude oil-contaminated microcosms showed potential for mineralization. Cluster analysis of denaturing gradient gel electrophoresis-derived samples generated with primers for gene amoA, which encodes the functional enzyme ammonia monooxygenase, showed differences in the sample contaminated with 5% compared to the other samples. Principal component analysis showed divergence of the non-contaminated samples from the 5% crude oil-contaminated sediment. A Venn diagram generated from the banding pattern of PCR-denaturing gradient gel electrophoresis was used to look for operational taxonomic units (OTUs) in common. Eight OTUs were found in non-contaminated sediments and in samples contaminated with 0.5, 1, or 2% crude oil. A Jaccard similarity index of 50% was found for samples contaminated with 0.1, 0.5, 1, and 2% crude oil. This is the first study that focuses on the impact of crude oil on the ammonia-oxidizing bacterium community in mangrove sediments from Camamu Bay.

  19. Suitability of PCR fingerprinting, infrequent-restriction-site PCR, and pulsed-field gel electrophoresis, combined with computerized gel analysis, in library typing of Salmonella enterica serovar enteritidis

    DEFF Research Database (Denmark)

    Garaizar, J.; Lopez-Molina, N.; Laconcha, I.

    2000-01-01

    Strains of Salmonella enterica (n = 212) of different serovars and phage types were used to establish a library typing computerized system for serovar Enteritidis on the basis of PCR fingerprinting, infrequent-restriction-site PCR (IRS-PCR), or pulsed-field gel electrophoresis (PFGE). The rate...... showed an intercenter reproducibility value of 93.3%. The high reproducibility of PFGE combined with the previously determined high discrimination directed its use for library typing. The use of PFGE with enzymes XbaI, BlnI, and SpeI for library typing of serovar Enteritidis was assessed with GelCompar 4.......0 software, Three computer libraries of PFGE DNA profiles were constructed, and their ability to recognize new DNA profiles was analyzed. The results obtained pointed out that the combination of PFGE with computerized analysis could be suitable in long-term epidemiological comparison and surveillance...

  20. Construction of physical and genetic maps of Chlamydia trachomatis serovar L2 by pulsed-field gel electrophoresis

    DEFF Research Database (Denmark)

    Birkelund, Svend; Stephens, RS

    1992-01-01

    We constructed the physical map of Chlamydia trachomatis serovar L2 by using three restriction endonucleases, NotI (GC[GGCCGC), SgrAI (C(A/G)[CCGG(T/G)G), and Sse8387I (CCTGCA[GG), and we analyzed the fragments by pulsed-field gel electrophoresis. A total of 25 restriction endonuclease sites and 13...... genes and/or operons were located on the map. The genome size was determined to be 1,045 kb. Neither highly transcribed chlamydia genes nor developmental cycle-specific genes were clustered on the genome....

  1. Induction of DNA double-strand breaks by restriction enzymes in X-ray-sensitive mutant Chinese hamster ovary cells measured by pulsed-field gel electrophoresis

    International Nuclear Information System (INIS)

    Kinashi, Yuko; Nagasawa, Hatsumi; Little, J.B.; Okayasu, Ryuichi; Iliakis, G.E.

    1995-01-01

    This investigation was designed to determine whether the cytotoxic effects of different restriction endonucleases are related to the number and type of DNA double-strand breaks (DSBs) they produce. Chinese hamster ovary (CHO) K1 and xrs-5 cells, a radiosensitive mutant of CHO K1, were exposed to restriction endonucleases HaeIII, HinfI, PvuII and BamHI by electroporation. These enzymes represent both blunt and sticky end cutters with differing recognition sequence lengths. The number of DSBs was measured by pulsed-field gel electrophoresis (PFGE). Two forms of PFGE were employed: asymmetric field-inversion gel electrophoresis (AFIGE) for measuring the kinetics of DNA breaks by enzyme digestion and clamped homogeneous gel electrophoresis (CHEF) for examining the size distributions of damaged DNA. The amount of DNA damage induced by exposure to all four restriction enzymes was significantly greater in xrs-5 compared to CHO K1 cells, consistent with the reported DSB repair deficiency in these cells. Since restriction endonucleases produce DSBs alone as opposed to the various types of DNA damage induced by X rays, these results confirm that the repair defect in this mutant involves the rejoining of DSBs. Although the cutting frequency was directly related to the length of the recognition sequence for four restriction enzymes, there was no simple correlation between the cytotoxic effect and the amount of DNA damage produced by each enzyme in either cell line. This finding suggests that the type or nature of the cutting sequence itself may play a role in restriction enzyme-induced cell killing. 32 refs., 6 figs., 3 tabs

  2. Specific proteins synthesized during the viral lytic cycle in vaccinia virus-infected HeLa cells: analysis by high-resolution, two-dimensional gel electrophoresis

    International Nuclear Information System (INIS)

    Carrasco, L.; Bravo, R.

    1986-01-01

    The proteins synthesized in vaccinia-infected HeLa cells have been analyzed at different times after infection by using two-dimensional gel electrophoresis. Vaccinia-infected cells present up to 198 polypeptides (138 acidic, isoelectric focusing; 60 basic, nonequilibrium pH gradient electrophoresis) not detected in control cells. Cells infected in the presence of cycloheximide show 81 additional polypeptides after cycloheximide removal, resulting in a total estimate of 279 proteins induced after vaccinia infection. The glycoproteins made at various time postinfection were also analyzed. At least 13 proteins labeled with [ 3 H]glucosamine were detected in vaccinia-infected HeLa cells

  3. Purification of Escherichia coli L-asparaginase mutants by a native polyacrylamide gel electrophoresis.

    Science.gov (United States)

    Wei, Yujun; Chen, Jianhua; Jia, Ruibo; Wang, Min; Wu, Wutong

    2008-07-01

    The antigenicity of L-asaparaginase (L-ASP) has been problematic for the treatment of leukemia for many years. In order to establish a relationship between the antigenic epitope of L-asparaginase and its antigenicity, several L-asparaginase mutants (mL-ASPs) are constructed and expressed. To effectively purify these enzyme mutants for further investigation, a native preparative polyacrylamide gel electrophoresis is developed. The simplicity and reproducibility of this approach permits the purification of different mutants from the crude enzyme extracts, with a sufficient activity to perform immunological and biological studies. Furthermore, the newly developed method is efficient and cost-effective compared with other methods, such as column chromatography and affinity chromatography. As a result, the enzyme mutants with specific activity of 300 approximately 400 U/mg are obtained by the single-step purification with a high degree of purity.

  4. A novel mechanism of “metal gel-shift” by histidine-rich Ni2+-binding Hpn protein from Helicobacter pylori strain SS1

    Science.gov (United States)

    Ito, Yuki; Masumoto, Junya; Morita, Eugene Hayato; Hayashi, Hidenori

    2017-01-01

    Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) is a universally used method for determining approximate molecular weight (MW) in protein research. Migration of protein that does not correlate with formula MW, termed “gel shifting” appears to be common for histidine-rich proteins but not yet studied in detail. We investigated “gel shifting” in Ni2+-binding histidine-rich Hpn protein cloned from Helicobacter pylori strain SS1. Our data demonstrate two important factors determining “gel shifting” of Hpn, polyacrylamide-gel concentration and metal binding. Higher polyacrylamide-gel concentrations resulted in faster Hpn migration. Irrespective of polyacrylamide-gel concentration, preserved Hpn-Ni2+ complex migrated faster (3–4 kDa) than apo-Hpn, phenomenon termed “metal gel-shift” demonstrating an intimate link between Ni2+ binding and “gel shifting”. To examine this discrepancy, eluted samples from corresponding spots on SDS-gel were analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). The MW of all samples was the same (6945.66±0.34 Da) and identical to formula MW with or without added mass of Ni2+. MALDI-TOF-MS of Ni2+-treated Hpn revealed that monomer bound up to six Ni2+ ions non-cooperatively, and equilibrium between protein-metal species was reliant on Ni2+ availability. This corroborates with gradually increased heterogeneity of apo-Hpn band followed by compact "metal-gel shift" band on SDS-PAGE. In view of presented data metal-binding and “metal-gel shift” models are discussed. PMID:28207866

  5. Study of Streptavidin-Modified Quantum Dots by Capillary Electrophoresis

    Czech Academy of Sciences Publication Activity Database

    Stanisavljevic, M.; Janů, L.; Šmerková, K.; Křížková, S.; Pizúrová, Naděžda; Ryvolová, M.; Adam, V.; Hubálek, J.; Kizek, R.

    2013-01-01

    Roč. 76, 7-8 (2013), s. 335-343 ISSN 0009-5893 Institutional support: RVO:68081723 Keywords : Capillary electrophoresis * Gel electrophoresis * Avidin-biotin technology * Oligonucleotide * Nanoparticle * quantum dots Subject RIV: CE - Biochemistry Impact factor: 1.370, year: 2013

  6. Radiosensitivity evaluation of Human tumor cell lines by single cell gel electrophoresis

    International Nuclear Information System (INIS)

    Zhang Yipei; Cao Jia; Wang Yan; Du Liqing; Li Jin; Wang Qin; Fan Feiyue; Liu Qiang

    2011-01-01

    Objective: To explore the feasibility of determining radiosensitivity of human tumor cell lines in vitro using single cell gel electrophoresis (SCGE). Methods: Three human tumor cell lines were selected in this study, HepG 2 , EC-9706 and MCF-7. The surviving fraction (SF) and DNA damage were detected by MTT assay, nested PCR technique and comet assay respectively. Results: MTT assay: The SF of HepG 2 and EC-9706 after irradiated by 2, 4 and 8 Gy was lower significantly than that of MCF-7, which showed that the radiosensitivity of HepG 2 and EC-9706 was higher than that of MCF-7. But there was no statistical difference of SF between HepG 2 and EC-9706. SCGE: The difference of radiosensitivity among these three tumor cell lines was significant after 8 Gy γ-ray irradiation. Conclusion: The multi-utilization of many biological parameter is hopeful to evaluate the radiosensitivity of tumor cells more objectively and exactly. (authors)

  7. Separation of intron 22 inversion type 1 and 2 of hemophilia A by modified inverse-shifting polymerase chain reaction and capillary gel electrophoresis.

    Science.gov (United States)

    Pan, Tzu-Yu; Chiou, Shyh-Shin; Wang, Chun-Chi; Wu, Shou-Mei

    2014-12-01

    An inverse-shifting polymerase chain reaction (IS-PCR) combined with short-end capillary gel electrophoresis (CGE) was developed for genotyping of intron 22 inversion Type 1 (Inv22-1) and Type 2 (Inv22-2) of hemophilia A (HA). Severe HA cases are affected by intron 22 inversion around 45-50%. Inv22-1 has higher frequency than Inv22-2. The aim of this study is to distinguish them by genotyping. In order to improve Inv22 genotyping efficiency, five primers were designed and applied to differentiate the wild type, Inv22-1, Inv22-2 and carrier. Three amplicons of 405, 457 and 512 bp were recognized for wild type; 333, 457 and 584 bp for Inv22-1; 385, 405 and 584 bp for Inv22-2. The Inv22-1 carrier has 5 amplicons including 333, 405, 457, 512, 584 bp and Inv22-2 carrier is differentiated by 385, 405, 457, 512 and 584 bp. The amplicons between Inv22-1 and Inv22-2 carriers are only different in 333 bp for Inv22-1 carrier and 385 bp for Inv22-2 carrier. Capillary gel electrophoresis (CGE) was used for separation within 5 min. The separation voltage was set at 8 kV (cathode at detector), and the temperature was kept at 25°C. The sieving matrix was 89 mM Tris, 89 mM boric acid, 2mM EDTA containing 0.4% (w/v) HPMC and 1 μM of YO-PRO(®)-1 Iodide. Total of 50 HA patients (including 35 non-Inv22, 14 Inv22-1, and one Inv22-2 patients) and 7 HA carriers were diagnosed in the application. Seven random samples (5 patients and 2 carriers) were subjected to comparison and gave identical results of DNA sequencing and this modified IS-PCR. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. ISOLATION OF EGG DROP SYNDROME VIRUS AND ITS MOLECULAR CHARACTERIZATION USING SODIUM DODECYL SULPHATE POLYACRYLAMIDE GEL ELECTROPHORESIS

    Directory of Open Access Journals (Sweden)

    M. H. Rasool, S. U. Rahman and M. K. Mansoor

    2005-10-01

    Full Text Available Six isolates of egg drop syndrome (EDS virus were recovered from five different outbreaks of EDS in commercial laying hens in and around Faisalabad. The aberrant eggs were fed to the susceptible laying hens for experimental induction of infection. The samples from infected birds (egg washing, cloacal swabs, oviducts and spleens were collected, processed and inoculated into 11-day old duck embryos. The presence of virus in harvested allanto-amniotic fluid was monitored by spot and microhaemagglutination tests and confirmed by haemagglutination inhibition and agar gel precipitation tests. The EDS virus grew well in duck embryos and agglutinated only avian but not mammalian red blood cells. These isolates were purified through velocity density gradient centrifugation. Protein concentration was determined through Lowry method and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE was conducted by loading 300 µg protein concentration on 12.5% gel using discontinuous buffer system. All the six isolates showed 13 polypeptides, which were identical to those described in the referral EDS-76 virus (strain-127. The molecular weights of the polypeptides ranged from 6.5 KDa to 126 KDa.

  9. Correlation between the viscoelastic properties of the gel layer of swollen HPMC matrix tablets and their in vitro drug release.

    Science.gov (United States)

    Hamed, Rania; Al Baraghthi, Tamadur; Sunoqrot, Suhair

    2016-11-21

    Drug release from hydroxypropyl methylcellulose (HPMC) hydrophilic matrix tablets is controlled by drug diffusion through the gel layer of the matrix-forming polymer upon hydration, matrix erosion or combination of diffusion and erosion mechanisms. In this study, the relationship between viscoelastic properties of the gel layer of swollen intact matrix tablets and drug release was investigated. Two sets of quetiapine fumarate (QF) matrix tablets were prepared using the high viscosity grade HPMC K4M at low (70 mg/tablet) and high (170 mg/tablet) polymer concentrations. Viscoelastic studies using a controlled stress rheometer were performed on swollen matrices following hydration in the dissolution medium for predetermined time intervals. The gel layer of swollen tablets exhibited predominantly elastic behavior. Results from the in vitro release study showed that drug release was strongly influenced by the viscoelastic properties of the gel layer of K4M tablets, which was further corroborated by results from water uptake studies conducted on intact tablets. The results provide evidence that the viscoelastic properties of the gel layer can be exploited to guide the selection of an appropriate matrix-forming polymer, to better understand the rate of drug release from matrix tablets in vitro and to develop hydrophilic controlled-release formulations.

  10. Matrix-assisted peptide synthesis on nanoparticles.

    Science.gov (United States)

    Khandadash, Raz; Machtey, Victoria; Weiss, Aryeh; Byk, Gerardo

    2014-09-01

    We report a new method for multistep peptide synthesis on polymeric nanoparticles of differing sizes. Polymeric nanoparticles were functionalized via their temporary embedment into a magnetic inorganic matrix that allows multistep peptide synthesis. The matrix is removed at the end of the process for obtaining nanoparticles functionalized with peptides. The matrix-assisted synthesis on nanoparticles was proved by generating various biologically relevant peptides. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

  11. Method for the typing of Clostridium difficile based on polyacrylamide gel electrophoresis of (/sup 35/S)methionine-labeled proteins

    Energy Technology Data Exchange (ETDEWEB)

    Tabaqchali, S.; O' Farrell, S.; Holland, D.; Silman, R.

    1986-01-01

    A typing method for Clostridium difficile based on the incorporation of (/sup 35/S)methionine into cellular proteins, their separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their visualization by autoradiography is described. On analysis of the radiolabeled-protein profiles, nine distinct groups were observed (A to E and W to Z). The method, which is simple, reproducible, and readily expandable, has been applied in epidemiological studies to demonstrate cross-infection and hospital acquisition of C. difficile.

  12. An Improved 2-Dimensional Gel Electrophoresis Method for Resolving Human Erythrocyte Membrane Proteins.

    Science.gov (United States)

    Kumar, Manoj; Singh, Rajendra; Meena, Anil; Patidar, Bhagwan S; Prasad, Rajendra; Chhabra, Sunil K; Bansal, Surendra K

    2017-01-01

    The 2-dimensional gel electrophoresis (2-DE) technique is widely used for the analysis of complex protein mixtures extracted from biological samples. It is one of the most commonly used analytical techniques in proteomics to study qualitative and quantitative protein changes between different states of a cell or an organism (eg, healthy and diseased), conditionally expressed proteins, posttranslational modifications, and so on. The 2-DE technique is used for its unparalleled ability to separate thousands of proteins simultaneously. The resolution of the proteins by 2-DE largely depends on the quality of sample prepared during protein extraction which increases results in terms of reproducibility and minimizes protein modifications that may result in artifactual spots on 2-DE gels. The buffer used for the extraction and solubilization of proteins influences the quality and reproducibility of the resolution of proteins on 2-DE gel. The purification by cleanup kit is another powerful process to prevent horizontal streaking which occurs during isoelectric focusing due to the presence of contaminants such as salts, lipids, nucleic acids, and detergents. Erythrocyte membrane proteins serve as prototypes for multifunctional proteins in various erythroid and nonerythroid cells. In this study, we therefore optimized the selected major conditions of 2-DE for resolving various proteins of human erythrocyte membrane. The modification included the optimization of conditions for sample preparation, cleanup of protein sample, isoelectric focusing, equilibration, and storage of immobilized pH gradient strips, which were further carefully examined to achieve optimum conditions for improving the quality of protein spots on 2-DE gels. The present improved 2-DE analysis method enabled better detection of protein spots with higher quality and reproducibility. Therefore, the conditions established in this study may be used for the 2-DE analysis of erythrocyte membrane proteins for

  13. Identification of column edges of DNA fragments by using K-means clustering and mean algorithm on lane histograms of DNA agarose gel electrophoresis images

    Science.gov (United States)

    Turan, Muhammed K.; Sehirli, Eftal; Elen, Abdullah; Karas, Ismail R.

    2015-07-01

    Gel electrophoresis (GE) is one of the most used method to separate DNA, RNA, protein molecules according to size, weight and quantity parameters in many areas such as genetics, molecular biology, biochemistry, microbiology. The main way to separate each molecule is to find borders of each molecule fragment. This paper presents a software application that show columns edges of DNA fragments in 3 steps. In the first step the application obtains lane histograms of agarose gel electrophoresis images by doing projection based on x-axis. In the second step, it utilizes k-means clustering algorithm to classify point values of lane histogram such as left side values, right side values and undesired values. In the third step, column edges of DNA fragments is shown by using mean algorithm and mathematical processes to separate DNA fragments from the background in a fully automated way. In addition to this, the application presents locations of DNA fragments and how many DNA fragments exist on images captured by a scientific camera.

  14. Avoiding acidic region streaking in two-dimensional gel electrophoresis: case study with two bacterial whole cell protein extracts.

    Science.gov (United States)

    Roy, Arnab; Varshney, Umesh; Pal, Debnath

    2014-09-01

    Acidic region streaking (ARS) is one of the lacunae in two-dimensional gel electrophoresis (2DE) of bacterial proteome. This streaking is primarily caused by nucleic acid (NuA) contamination and poses major problem in the downstream processes like image analysis and protein identification. Although cleanup and nuclease digestion are practiced as remedial options, these strategies may incur loss in protein recovery and perform incomplete removal of NuA. As a result, ARS has remained a common observation across publications, including the recent ones. In this work, we demonstrate how ultrasound wave can be used to shear NuA in plain ice-cooled water, facilitating the elimination of ARS in the 2DE gels without the need for any additional sample cleanup tasks. In combination with a suitable buffer recipe, IEF program and frequent paper-wick changing approach, we are able to reproducibly demonstrate the production of clean 2DE gels with improved protein recovery and negligible or no ARS. We illustrate our procedure using whole cell protein extracts from two diverse organisms, Escherichia coli and Mycobacterium smegmatis. Our designed protocols are straightforward and expected to provide good 2DE gels without ARS, with comparable times and significantly lower cost.

  15. An accurate determination of human grawth hormone content in different pituitary extracts, using a radioimmunoassay with polyacrylamide gel electrophoresis as a bound-free separation system

    International Nuclear Information System (INIS)

    Bartoline, P.; Assis, L.M. de; Scwarz, I.; Pieroni, R.R.

    1977-01-01

    Human growth hormone was extracted and purified according to the method of Roos et al. A first control of its purification and integrity was performed through molecular weight determination by gel filtration on Sephadex G-100 and on plyacrylamide gel electrophoresis (PAGE). Its biological activity was confirmed by the growth promoted in non-hypophysectomized rats at plateau. The main object, however, was the setting up of an accurate, reproducible method that could furnish the more absolute and comparable value of rafioimmunoassayable HGH content in perfect agreement with the results obtained by other laboratories. This was accomplished through a radioimmunoassay system that uses HGH labelled with 125 I, where the separation of the bound from the free antigen is achieved on polyacrylamide gel electrophoresis, by a modification introduced in the original method of Davis. The resulting values, extremely close to that stated by the KABI-Laboratories (Stockolm), through obtained in quite different conditions of incubation, antibody concentration and with no use of second antibody, represent a confident approach to a comparable measure of this hormone in extract, which can also be applied to plasma determinations [pt

  16. Measurement of DNA double-strand breaks in CHO cells at various stages of the cell cycle using pulsed field gel electrophoresis: calibration by means of 125I decay

    International Nuclear Information System (INIS)

    Iliakis, G.E.; Cicilioni, O.; Metzger, L.

    1991-01-01

    Experiments were performed to calibrate a recently developed pulsed field gel electrophoresis assay, the asymmetric field inversion gel electrophoresis (AFIGE), for the measurement of double-strand breaks (dsb) in the DNA of mammalian cells. Calibration was carried out by means of 125 I decay accumulation, under conditions preventing repair, based on the observation that each 125 I decay in the DNA produces approximately one dsb. Results suggest that that observed fluctuations in the fraction of DNA activity released (FAR) per Gy throughout the cycle reflect cell-cycle-associated differences in the physicochemical properties of the DNA molecules that alter their electrophoretic mobility, rather than variations in the induction of dsb per Gy, i.e. the sensitivity of the assay fluctuates throughout the cycle. (author)

  17. Exploration of beer proteome using OFFGEL prefractionation in combination with two-dimensional gel electrophoresis with narrow pH range gradients.

    Science.gov (United States)

    Konečná, Hana; Müller, Lukáš; Dosoudilová, Hana; Potěšil, David; Buršíková, Jana; Sedo, Ondrej; Márová, Ivana; Zdráhal, Zbyněk

    2012-03-14

    Two-dimensional gel electrophoresis in combination with mass spectrometry has already been applied successfully to study beer proteome. Due to the abundance of protein Z in beer samples, prefractionation techniques might help to improve beer proteome coverage. Proteins from four lager beers of different origins were separated by two-dimensional electrophoresis (2-DE) followed by tandem mass spectrometric analysis. Initially 52 proteins mostly from Hordeum vulgare (22 proteins) and Saccharomyces species (25 proteins) were identified. Preparative isoelectric focusing by OFFGEL Fractionator was applied prior to 2-DE to improve its resolution power. As a result of this combined approach, a total of 70 beer proteins from Hordeum vulgare (30 proteins), from Saccharomyces species (31 proteins), and from other sources (9 proteins) were identified. Of these, 37 proteins have not been previously reported in beer samples.

  18. Comparison of antimicrobial peptide purification via free-flow electrophoresis and gel filtration chromatography.

    Science.gov (United States)

    Xia, Zhi-Jun; Liu, Zhen; Kong, Fan-Zhi; Fan, Liu-Yin; Xiao, Hua; Cao, Cheng-Xi

    2017-12-01

    Antimicrobial peptides (AMPs) are usually small and cationic biomolecules with broad-spectrum antimicrobial activities against pathogens. Purifying them from complex samples is essential to study their physiochemical properties. In this work, free-flow zone electrophoresis (FFZE) was utilized to purify AMPs from yeast fermentation broth. Meanwhile, gel filtration chromatography (GFC) was conducted for comparison. The separation efficiency was evaluated by SDS-PAGE analysis of the fractions from both methods. Our results demonstrated as follows: (i) FFZE had more than 30-fold higher processing capacity as compared with GFC; (ii) FFZE could achieve 87% purity and 89% recovery rate while in GFC these parameters were about 93 and 82%, respectively; (iii) the former had ∼2-fold dilution but the latter had ∼13-fold dilution. Furthermore, Tricine-SDS-PAGE, Native-PAGE, and gel IEF were carried out to characterize the purified AMPs. We found that two peptides existed as a pair with the molecular mass of ∼5.5 and 7.0 kDa, while the same pI 7.8. These two peptides were proved to have the antimicrobial activity through the standardized agar diffusion method. Therefore, FFZE could be used to continuously purify AMPs with high bioactivity, which will lead to its wide application in the clinical and pharmaceutical fields. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Agarose gel electrophoresis of cerebrospinal fluid proteins of dogs after sample concentration using a membrane microconcentrator technique.

    Science.gov (United States)

    Gama, Fernanda Gomes Velasque; Santana, Aureo Evangelista; Filho, Eugênio de Campos; Nogueira, Cláudia Aparecida da Silva

    2007-03-01

    Cerebrospinal fluid (CSF) is produced in the cerebral ventricles through ultrafiltration of plasma and active transport mechanisms. Evaluation of proteins in CSF may provide important information about the production of immunoglobulins within the central nervous system as well as possible disturbances in the blood-brain barrier. The objective of this study was to measure the concentration and fractions of protein in CSF samples using a membrane microconcentrator technique followed by electrophoresis, and to compare the protein fractions obtained with those in serum. CSF samples from 3 healthy dogs and 3 dogs with canine distemper virus infection were concentrated using a membrane microconcentrator having a 0.5 to 30,000 d nominal molecular weight limit (Ultrafree, Millipore, Billerica, MA, USA). Protein concentration was determined before and after concentration. Agarose gel electrophoresis was done on concentrated CSF samples, serum, and serial dilutions of one of the CSF samples. Electrophoretic bands were clearly identified in densitometer tracings in CSF samples with protein concentrations as low as 1.3 g/dL. The higher CSF protein concentration in dogs with distemper was mainly the result of increased albumin concentration. The microconcentrating method used in this study enables characterization of the main protein fractions in CSF by routine electrophoresis and may be useful for interpreting the underlying cause of changes in CSF protein concentrations.

  20. Discriminatory usefulness of pulsed-field gel electrophoresis and sequence-based typing in Legionella outbreaks.

    Science.gov (United States)

    Quero, Sara; García-Núñez, Marian; Párraga-Niño, Noemí; Barrabeig, Irene; Pedro-Botet, Maria L; de Simon, Mercè; Sopena, Nieves; Sabrià, Miquel

    2016-06-01

    To compare the discriminatory power of pulsed-field gel electrophoresis (PFGE) and sequence-based typing (SBT) in Legionella outbreaks for determining the infection source. Twenty-five investigations of Legionnaires' disease were analyzed by PFGE, SBT and Dresden monoclonal antibody. The results suggested that monoclonal antibody could reduce the number of Legionella isolates to be characterized by molecular methods. The epidemiological concordance PFGE-SBT was 100%, while the molecular concordance was 64%. Adjusted Wallace index (AW) showed that PFGE has better discriminatory power than SBT (AWSBT→PFGE = 0.767; AWPFGE→SBT = 1). The discrepancies appeared mostly in sequence type (ST) 1, a worldwide distributed ST for which PFGE discriminated different profiles. SBT discriminatory power was not sufficient verifying the infection source, especially in worldwide distributed STs, which were classified into different PFGE patterns.

  1. Changes in the diversity of pig ileal lactobacilli around weaning determined by means of 16S rRNA gene amplification and denaturing gradient gel electrophoresis

    NARCIS (Netherlands)

    Janzcyk, P.; Pieper, R.; Smidt, H.; Souffrant, W.B.

    2007-01-01

    Our study aimed to provide a comprehensive characterization of changes in porcine intestinal Lactobacillus populations around the time of weaning based on 16S rRNA gene amplification and denaturing gradient gel electrophoresis (DGGE). DNA was extracted from the ileal contents of piglets at weaning

  2. Population Genetic Structure of Listeria monocytogenes Strains as Determined by Pulsed-Field Gel Electrophoresis and Multilocus Sequence Typing

    DEFF Research Database (Denmark)

    Henri, Clémentine; Félix, Benjamin; Guillier, Laurent

    2016-01-01

    on the basis of different pulsed-field gel electrophoresis (PFGE) clusters, serotypes, and strain origins and typed by multilocus sequence typing (MLST), and the MLST results were supplemented with MLST data available from Institut Pasteur, representing human and additional food strains from France....... The distribution of sequence types (STs) was compared between food and clinical strains on a panel of 675 strains. High congruence between PFGE and MLST was found. Out of 73 PFGE clusters, the two most prevalent corresponded to ST9 and ST121. Using original statistical analysis, we demonstrated that (i...

  3. Behavior of variable V3 region from 16S rDNA of lactic acid bacteria in denaturing gradient gel electrophoresis.

    Science.gov (United States)

    Ercolini, D; Moschetti, G; Blaiotta, G; Coppola, S

    2001-03-01

    Separation of amplified V3 region from 16S rDNA by denaturing gradient gel electrophoresis (DGGE) was tested as a tool for differentiation of lactic acid bacteria commonly isolated from food. Variable V3 regions of 21 reference strains and 34 wild strains referred to species belonging to the genera Pediococcus, Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, Weissella, and Streptococcus were analyzed. DGGE profiles obtained were species-specific for most of the cultures tested. Moreover, it was possible to group the remaining LAB reference strains according to the migration of their 16S V3 region in the denaturing gel. The results are discussed with reference to their potential in the analysis of LAB communities in food, besides shedding light on taxonomic aspects.

  4. Comparative Studies of Two-Dimensional Electrophoresis on Galactosidase Relating to Bombyx Lectin Activity

    OpenAIRE

    加藤, 靖夫; カトウ, ヤスオ; Yasuo, Kato

    2005-01-01

    "Comparative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis on the haemolymph of the domesticated silkworm, Bombyx mori and Fraction II obtained by gel filtration from the haemolymph of B. mori was performed using the 2-D mini-slab system (Atto Co.) (the first method of 2-D PAGE) and the Mini-PROTEAN mini tube gel 2-D PAGE system (Bio-Rad Laboratories, Inc.) (the second method). Moreover, two-dimensionnal electrophoresis analysis on standard β-galactosidase, grade III ...

  5. Bacterial analysis of combined periodontal-endodontic lesions by polymerase chain reaction-denaturing gradient gel electrophoresis.

    Science.gov (United States)

    Xia, Minghui; Qi, Qingguo

    2013-01-01

    We used denaturing gradient gel electrophoresis (DGGE) to compare bacterial profiles in periodontium and root canals of teeth with combined periodontal-endodontic lesions. Samples of dental plaque and necrotic pulp were collected from thirteen extracted teeth with advanced periodontitis. Genomic DNA was extracted for polymerase chain reaction (PCR) analysis using universal bacterial primers. The PCR products were then loaded onto DGGE gels to obtain fractionated bands. Characteristic DGGE bands were excised and DNA was cloned and sequenced. The number of bands, which indicates the number of bacterial species, was compared between dental plaques and necrotic pulp tissues from the same tooth. Although the difference was statistically significant (P bacteria species were present in both the periodontal pockets and root canals of the same tooth; however, periodontal bacteria did not always invade the root canals, and some bacteria in root canals were not present in periodontal pockets of the same tooth. In some teeth, unique bacteria in root canals had not passed from periodontal pockets. A basic local alignment search tool (BLAST) sequence search in Genbank indicated that new bacteria species were present in periodontal pockets and root canals. Their characteristics must thus be further analyzed.

  6. Dynamic changes in proteins during apple (Malus x domestica) fruit ripening and storage

    OpenAIRE

    Shi, Yun; Jiang, Li; Zhang, Li; Kang, Ruoyi; Yu, Zhifang

    2014-01-01

    A proteomic study, using two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight, was conducted in apple fruit (cv. ‘Golden Delicious’) starting at 10 days prior to harvest through 50 days in storage. Total protein was extracted using a phenol/sodium dodecyl sulfate protocol. More than 400 protein spots were detected in each gel and 55 differentially expressed proteins (p

  7. PVC gel soft actuator-based wearable assist wear for hip joint support during walking

    Science.gov (United States)

    Li, Yi; Hashimoto, Minoru

    2017-12-01

    Plasticized polyvinyl chloride (PVC) gel and mesh electrode-based soft actuators have considerable potential to provide new types of artificial muscle, exhibiting similar responsiveness to biological muscle in air, >10% deformation, >90 kPa output stress, variable stiffness, long cycle life (>5 million cycles), and low power consumption. We have designed and fabricated a prototype of walking assist wear using the PVC gel actuator in previous study. The system has several advantages compared with traditional motor-based exoskeletons, including lower weight and power consumption, and no requirement for rigid external structures that constrain the wearer’s joints. In this study, we designed and established a control and power system to making the whole system portable and wearable outdoors. And we designed two control strategies based on the characteristics of the assist wear and the biological kinematics. In a preliminary experimental evaluation, a hemiparetic stroke patient performed a 10 m to-and-fro straight line walking task with and without assist wear on the affected side. We found that the assist wear enabled natural movement, increasing step length and decreasing muscular activity during straight line walking. We demonstrated that the assistance effect could be adjusted by controlling the on-off time of the PVC gel soft actuators. The results show the effectiveness of the proposed system and suggest the feasibility of PVC gel soft actuators for developing practical soft wearable assistive devices, informing the development of future wearable robots and the other soft actuator technologies for human movement assistance and rehabilitation.

  8. DNA double-strand breaks measured by pulsed-field gel electrophoresis in irradiated lymphocytes from normal humans and those with Alzheimer's disease

    International Nuclear Information System (INIS)

    Tobi, S.E.; Itzhaki, R.F.

    1993-01-01

    The authors previously found that radiation-induced chromosome aberrations (dicentrics) are more numerous in lymphocytes from Alzheimer's disease (AD) patients than in those from age-matched normal individuals (Tobi et al. 1990). They have examined double-strand breaks (dsb) produced by g amma - irradiation in the DNA of AD and normal lymphocytes by using pulsed-field gel electrophoresis. The percentage of DNA migrating into the gels is an indirect measure of the number of dsb; DNA content of sequential slices of the gel was assayed by direct fluorometry and the percentage migrating was dose dependent. Results show that the level of damage is similar in AD and normal lymphocytes and preliminary assays of the rate of repair suggest that the half-time is also similar, the value being > 1 h. The latter is consistent with the known rate of rejoining of chromosome fragments in interphase lymphocytes (Pantelias and Maillie 1985). (Author)

  9. TCA precipitation and ethanol/HCl single-step purification evaluation: One-dimensional gel electrophoresis, bradford assays, spectrofluorometry and Raman spectroscopy data on HSA, Rnase, lysozyme - Mascots and Skyline data

    Directory of Open Access Journals (Sweden)

    Balkis Eddhif

    2018-04-01

    Full Text Available The data presented here are related to the research paper entitled “Study of a Novel Agent for TCA Precipitated Proteins Washing - Comprehensive Insights into the Role of Ethanol/HCl on Molten Globule State by Multi-Spectroscopic Analyses” (Eddhif et al., submitted for publication [1]. The suitability of ethanol/HCl for the washing of TCA-precipitated proteins was first investigated on standard solution of HSA, cellulase, ribonuclease and lysozyme. Recoveries were assessed by one-dimensional gel electrophoresis, Bradford assays and UPLC-HRMS. The mechanistic that triggers protein conformational changes at each purification stage was then investigated by Raman spectroscopy and spectrofluorometry. Finally, the efficiency of the method was evaluated on three different complex samples (mouse liver, river biofilm, loamy soil surface. Proteins profiling was assessed by gel electrophoresis and by UPLC-HRMS.

  10. Two dimensional gel electrophoresis using narrow pH 3-5.6 immobilised pH gradient strips identifies potential novel disease biomarkers in plasma or serum

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Bevin Gangadharan & Nicole Zitzmann ### Abstract Two-dimensional gel electrophoresis (2-DE) is a protein separation technique often used to separate plasma or serum proteins in an attempt to identify novel biomarkers. This protocol describes how to run 2-DE gels using narrow pH 3-5.6 immobilised pH gradient strips to separate 2 mg of serum proteins. pH 3-6 ampholytes are used to enhance the solubility of proteins in this pH range before the serum proteins are separated in the...

  11. Tunable separations based on a molecular size effect for biomolecules by poly(ethylene glycol) gel-based capillary electrophoresis.

    Science.gov (United States)

    Kubo, Takuya; Nishimura, Naoki; Furuta, Hayato; Kubota, Kei; Naito, Toyohiro; Otsuka, Koji

    2017-11-10

    We report novel capillary gel electrophoresis (CGE) with poly(ethylene glycol) (PEG)-based hydrogels for the effective separations of biomolecules containing sugars and DNAs based on a molecular size effect. The gel capillaries were prepared in a fused silica capillary modified with 3-(trimethoxysilyl)propylmethacrylate using a variety of the PEG-based hydrogels. After the fundamental evaluations in CGE regarding the separation based on the molecular size effect depending on the crosslinking density, the optimized capillary provided the efficient separation of glucose ladder (G1 to G20). In addition, another capillary showed the successful separation of DNA ladder in the range of 10-1100 base pair, which is superior to an authentic acrylamide-based gel capillary. For both glucose and DNA ladders, the separation ranges against the molecular size were simply controllable by alteration of the concentration and/or units of ethylene oxide in the PEG-based crosslinker. Finally, we demonstrated the separations of real samples, which included sugars carved out from monoclonal antibodies, mAbs, and then the efficient separations based on the molecular size effect were achieved. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Detection of telomerase activity using microchip electrophoresis.

    Science.gov (United States)

    Karasawa, Koji; Arakawa, Hidetoshi

    2015-07-01

    Telomerase participates in malignant transformation or immortalization of cells and thus has attracted attention as an anticancer drug target and diagnostic tumor marker. The telomeric repeat amplification protocol (TRAP) and improved TRAP methods (TRAP-fluorescence, TRAP-hybridization, etc.) are widely used forms of this telomerase assay. However, these approaches generally employ acrylamide gel electrophoresis after amplification of telomeric repeats by polymerase chain reaction (PCR), making these TRAP methods time consuming and technically demanding. In this study we developed a novel telomerase assay using microchip electrophoresis for rapid and highly sensitive detection of telomerase activity in cancer cells. The mixed gel of 0.8% hydroxypropyl methylcellulose (HPMC) and 0.3% polyethylene oxide (PEO) with SYBR Gold (fluorescent reagent) was used for microchip electrophoresis. As a result, the product amplified by a telomerase-positive cell could be measured in one cell per assay and detected with high reproducibility (CV=0.67%) in the short time of 100s. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Analysis of purified gp96 preparations from rat and mouse livers using 2-D gel electrophoresis and tandem mass spectrometry.

    Science.gov (United States)

    Fairburn, B; Muthana, M; Hopkinson, K; Slack, L K; Mirza, S; Georgiou, A S; Espigares, E; Wong, C; Pockley, A G

    2006-09-01

    The stress protein gp96 exhibits a number of immunological activities, the majority of studies into which have used gp96 purified from a variety of tissues. On the basis of 1-D gel electrophoresis, the purity of these preparations has been reported to range between 70% and 99%. This study analyzed gp96 preparations from rat and mouse livers using 2-D gel electrophoresis and liquid chromatography electrospray ionization tandem mass spectrometry (MS-MS). The procedure for purifying gp96 was reproducible, as similar protein profiles were observed in replicate gels of gp96 preparations. The purity of the preparations was typically around 70%, with minor co-purified proteins of varying molecular weights and mobilities being present. Dominant bands at 95-100 kDa in preparations from Wistar rats and C57BL/6 mice were identified as gp96 by ECL Western blotting. Multiple bands having similar, yet distinct molecular weights and differing pI mobility on ECL Western blots were confirmed as being gp96 in preparations from Wistar rats using MS-MS. The most striking feature of the 2-D gel analysis was the presence of additional dominant bands at 55 kDa in preparations from Wistar rats, and at 75-90 kDa in preparations from C57BL/6 mice. These were identified as gp96 by ECL Western blotting and, in the case of preparations from Wistar rats, by MS-MS. Although the lower molecular weight, gp96-related molecules might be partially degraded gp96, their reproducible presence, definition and characteristics suggest that they are alternative, species-specific isoforms of the molecule. A 55 kDa protein which exhibited a lower pI value than gp96 was present in all preparations and this was identified as calreticulin, another putative immunoregulatory molecule. This study confirms the reproducibility of the gp96 purification protocol and reveals the presence of multiple gp96 isoforms, some of which likely result from post-translational modifications such as differential glycosylation and

  14. Species identification of smoked and gravad fish products by sodium dodecylsulphate polyacrylamide gel electrophoresis, urea isoelectric focusing and native isoelectric focusing : a collaborative study

    DEFF Research Database (Denmark)

    Mackie, I.; Craig, A.; Etienne, M.

    2000-01-01

    A collaborative study on the use of sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), urea-isoelectric focusing (urea-IEF) and native isoelectric focusing for the identification of species of smoked salmonids, gravad salmonids and smoked eels was carried out by eight laborator......A collaborative study on the use of sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), urea-isoelectric focusing (urea-IEF) and native isoelectric focusing for the identification of species of smoked salmonids, gravad salmonids and smoked eels was carried out by eight...... laboratories. With SDS-PAGE, minor changes took place in the profiles of the processed salmonid species making it impossible or Very difficult to identify closely related species. With urea-IEF, there were fewer changes in the profiles due to processing and the system generally had greater species......-discriminating power for the processed salmonids than SDS-PAGE. The profiles of the eel species as obtained on SDS-PAGE or urea-IEF were not affected by smoking. Urea-IEF had greater species- discriminating power than SDS-PAGE for the eel species. Native IEF was useful in providing supplementary identification...

  15. Structural organization of the human glucocorticoid receptor determined by one- and two-dimensional gel electrophoresis of proteolytic receptor fragments

    International Nuclear Information System (INIS)

    Smith, A.C.; Harmon, J.M.

    1987-01-01

    The structural organization of the steroid-binding protein of the IM-9 cell glucocorticoid receptor was investigated by using one- and two-dimensional gel electrophoresis of proteolytic receptor fragments. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of receptor fragments isolated after trypsin digestion of immunopurified [ 3 H]dexamethasone 21-mesylate ([ 3 H]DM-) labeled receptor revealed the presence of a stable 26.5-kilodalton (kDa) steroid-containing non-DNA-binding fragment, derived from a larger, less stable, 29-kDa fragment. The 26.5-kDa tryptic fragment appeared to be completely contained within a 41-kDa, steroid-containing, DNA-binding species isolated after chymotrypsin digestion of the intact protein. Two-dimensional electrophoretic analysis of the [ 3 H]DM-labeled tryptic fragments resolved two 26.5-kDa and two 29-kDa components. This was the same number of isoforms seen in the intact protein, indicating that the charge heterogeneity of the steroid-binding protein is the result of modification within the steroid-containing, non-DNA-binding, 26.5-kDa tryptic fragment. Two-dimensional analysis of the 41-kDa [ 3 H]DM-labeled chymotryptic species revealed a pattern of isoforms more complex than that seen either in the intact protein or in the steroid-containing tryptic fragments. These results suggest that the 41-kDa [ 3 H]DM-labeled species resolved by one-dimensional SDS-PAGE after chymotrypsin digestion may be composed of several distinct proteolytic fragments

  16. Fluorescence of soil humic acids and their fractions obtained by tandem size exclusion chromatography-polyacrylamide gel electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Trubetskaya, O. [Russian Academy of Sciences, Moscow Region (Russian Federation). Shemyakin and Ovchinnikov Inst. of Bioorganic Chemistry; Trubetskoj, O. [Russian Academy of Sciences, Moscow Region (Russian Federation). Inst. of Basic Biological Problems; Guyot, G.; Richard, C. [UMR CNRS 6505, Aubiere (France). Lab. de Photochimie Moleculaire et Macromoleculaire; Andreux, F. [Centre des Sciences de la Terre, Dijon (France)

    2002-07-01

    Humic acids (HAs) extracted from soils of different origin (chernozem, ferralsol and ranker) and their fractions (A, B and C+D) obtained by tandem size exclusion chromatography-polyacrylamide gel electrophoresis were investigated by steady-state fluorescence spectroscopy in the emission mode. Independently of HA source, high molecular size fractions A and B are shown to be weakly fluorescent. The main fluorophores, especially those emitting at long wavelength (around 500-510 nm), are contained in the polar and low molecular size fractions C+D. As indicated by the observed pH effect, aromatic structures bearing carboxylate and OH substituents may be involved in these longer wavelength emissions. [author].

  17. Heparan sulfate proteoglycan from the extracellular matrix of human lung fibroblasts. Isolation, purification, and core protein characterization

    International Nuclear Information System (INIS)

    Heremans, A.; Cassiman, J.J.; Van den Berghe, H.; David, G.

    1988-01-01

    Confluent cultured human lung fibroblasts were labeled with 35SO4(2-). After 48 h of labeling, the pericellular matrix was prepared by Triton X-100 and deoxycholate extraction of the monolayers. Heparan sulfate proteoglycan (HSPG) accounted for nearly 80% of the total matrix [35S]proteoglycans. After solubilization in 6 M guanidinium HCl and cesium chloride density gradient centrifugation, the majority (78%) of these [35S] HSPG equilibrated at an average buoyant density of 1.35 g/ml. This major HSPG fraction was purified by ion-exchange chromatography on Mono Q and by gel filtration on Sepharose CL-4B, and further characterized by gel electrophoresis and immunoblotting. Intact [35S]HSPG eluted with Kav 0.1 from Sepharose CL-4B, whereas the protein-free [35S]heparan sulfate chains, obtained by alkaline borohydride treatment of the proteoglycan fractions, eluted with Kav 0.45 (Mr approximately 72,000). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, core (protein) preparations, obtained by heparitinase digestion of 125I-labeled HSPG fractions, yielded one major labeled band with apparent molecular mass of approximately 300 kDa. Reduction with beta-mercaptoethanol slightly increased the apparent Mr of the labeled band, suggesting a single polypeptide structure and the presence of intrachain disulfide bonds. Immunoadsorption experiments and immunostaining of electrophoretically separated heparitinase-digested core proteins with monoclonal antibodies raised against matrix and cell surface-associated HSPG suggested that the major matrix-associated HSPG of cultured human lung fibroblasts is distinct from the HSPG that are anchored in the membranes of these cells. Binding studies suggested that this matrix HSPG interacts with several matrix components, both through its glycosaminoglycan chains and through its heparitinase-resistant core. (Abstract Truncated)

  18. Detection of circular telomeric DNA without 2D gel electrophoresis.

    Science.gov (United States)

    Dlaska, Margit; Anderl, Conrad; Eisterer, Wolfgang; Bechter, Oliver E

    2008-09-01

    The end of linear chromosomes forms a lasso-like structure called the t-loop. Such t-loops resemble a DNA recombination intermediate, where the single-stranded 3' overhang is arrested in a stretch of duplex DNA. Presumably, such a t-loop can also be deleted via a recombination process. This would result in the occurrence of circular extrachromosomal telomeric DNA (t-circles), which are known to be abundantly present in immortal cells engaging the recombination-based alternative lengthening of telomeres pathway (ALT pathway). Little is known about the basic mechanism of telomeric recombination in these cells and what ultimately causes the generation of such t-circles. Current standard procedures for detecting these molecules involve 2D gel electrophoresis or electron microscopy. However, both methods are labor intense and sophisticated to perform. Here, we present a simpler, faster, and equally sensitive method for detecting t-circles. Our approach is a telomere restriction fragment assay that involves the enzymatic preservation of circular DNA with Klenow enzyme followed by Bal31 degradation of the remaining linear DNA molecules. We show that with this approach t-circles can be detected in ALT cell lines, whereas no t-circles are present in telomerase-positive cell lines. We consider our approach a valid method in which t-circle generation is the experimental readout.

  19. Two-dimensional gel electrophoresis pattern (pH 6-11) and identification of water-soluble barley seed and malt proteins by mass spectrometry

    DEFF Research Database (Denmark)

    Bak-Jensen, K.S.; Laugesen, S.; Roepstorff, P.

    2004-01-01

    A protocol was established for two-dimensional gel electrophoresis (2-DE) of barley seed and malt proteins in the pH range of 6-11. Proteins extracted from flour in a low-salt buffer were focused after cup-loading onto IPG strips. Successful separation in the second dimension was achieved using...... gradient gels in a horizontal SDS-PAGE system. Silver staining of gels visualized around 380 (seed) and 500 (malt) spots. Thirty-seven different proteins from seeds were identified in 60 spots, among these 46 were visualized also in the malt 2-D pattern. Proteins were identified by peptide mass...... in defence against pathogens (21 spots), 4 in storage, folding, and synthesis of proteins, and in nitrogen metabolism (5 spots), 6 in carbohydrate metabolism (11 spots), and 4 in stress and detoxification (9 spots). Six proteins (7 spots) were not grouped in these categories, and 3 were not ascribed...

  20. Reappraisal of quantitative gel zymography for matrix metalloproteinases.

    Science.gov (United States)

    Prescimone, Tommaso; Tognotti, Danika; Caselli, Chiara; Cabiati, Manuela; D'Amico, Andrea; Del Ry, Silvia; Giannessi, Daniela

    2014-09-01

    The determination of matrix metalloproteases (MMPs) is relevant in many pathophysiological conditions, especially if associated with extracellular matrix remodeling; however, the results obtained are closely linked to the method used and are not directly comparable. The aim of this study was to perform a reappraisal of quantitative gel zymography technique for MMPs in human plasma, to use for comparison with commercially available ELISA and in those experimental conditions where the MMP active form needs to be revealed. The critical methodological parameters of zymography were checked and a comparison with a routinely used ELISA was performed. Sensitivity and reproducibility levels of zymography are suitable for detection of MMP-9 in human plasma, providing results closely related to those obtained by ELISA. Analytical parameters of zymography were suitable for detection of MMPs in human plasma. Quantitative zymography for MMPs is an alternative method for comparing the results of ELISA widely employed for MMP determination, thus reducing the discrepancies between laboratories regarding gelatinase assay. © 2014 Wiley Periodicals, Inc.

  1. Nitroproteins in Human Astrocytomas Discovered by Gel Electrophoresis and Tandem Mass Spectrometry

    Science.gov (United States)

    Peng, Fang; Li, Jianglin; Guo, Tianyao; Yang, Haiyan; Li, Maoyu; Sang, Shushan; Li, Xuejun; Desiderio, Dominic M.; Zhan, Xianquan

    2015-12-01

    Protein tyrosine nitration is involved in the pathogenesis of highly fatal astrocytomas, a type of brain cancer. To understand the molecular mechanisms of astrocytomas and to discover new biomarkers/therapeutic targets, we sought to identify nitroproteins in human astrocytoma tissue. Anti-nitrotyrosine immunoreaction-positive proteins from a high-grade astrocytoma tissue were detected with two-dimensional gel electrophoresis (2DGE)-based nitrotyrosine immunoblots, and identified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Fifty-seven nitrotyrosine immunopositive protein spots were detected. A total of 870 proteins (nitrated and non-nitrated) in nitrotyrosine-immunopositive 2D gel spots were identified, and 18 nitroproteins and their 20 nitrotyrosine sites were identified with MS/MS analysis. These nitroproteins participate in multiple processes, including drug-resistance, signal transduction, cytoskeleton, transcription and translation, cell proliferation and apoptosis, immune response, phenotypic dedifferentiation, cell migration, and metastasis. Among those nitroproteins that might play a role in astrocytomas was nitro-sorcin, which is involved in drug resistance and metastasis and might play a role in the spread and treatment of an astrocytoma. Semiquantitative immune-based measurements of different sorcin expressions were found among different grades of astrocytomas relative to controls, and a semiquantitative increased nitration level in high-grade astrocytoma relative to control. Nitro-β-tubulin functions in cytoskeleton and cell migration. Semiquantitative immunoreactivity of β-tubulin showed increased expression among different grades of astrocytomas relative to controls and semiquantitatively increased nitration level in high-grade astrocytoma relative to control. Each nitroprotein was rationalized and related to the corresponding functional system to provide new insights into tyrosine nitration and its potential role in the

  2. Accounting for adjuvant-induced artifacts in the characterization of vaccine formulations by polyacrylamide gel electrophoresis.

    Science.gov (United States)

    Jakob, Virginie; Brunner, Livia; Barnier-Quer, Christophe; Blust, Molly; Collin, Nicolas; Carter, Lauren; Carter, Darrick; Rausch, Kelly M; Fox, Christopher B

    2017-04-01

    Several vaccine adjuvants comprise complex nano- or micro-particle formulations, such as oil-in-water emulsions. In order to characterize interactions and compatibility of oil-in-water emulsion adjuvants with protein antigens in vaccines, effective protein characterization methods that can accommodate potential interference from high concentrations of lipid-based particles are needed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a standard protein characterization technique which is affected by the presence of adjuvants such as oil-in-water emulsions. In this article, we investigate variations in SDS-PAGE methods that result in a reduction of adjuvant-induced staining artifacts. We have investigated whether the SDS method or the adjuvant composition were the reason for these artifacts and succeeded in reducing the artifacts with a modified sample preparation and different staining procedures. The best results were obtained by using gold staining or silver staining instead of a Coomassie Blue staining procedure. Moreover, the replacement of the dilution buffer (20% SDS to disrupt emulsion) by alternative detergents such as Tween® 80 and Triton® X-100 removed adjuvant-induced streaking artifacts at the top of the gel. These methods may be useful for improving characterization approaches of antigen-adjuvant mixtures by SDS-PAGE.

  3. Spot quantification in two dimensional gel electrophoresis image analysis: comparison of different approaches and presentation of a novel compound fitting algorithm

    Science.gov (United States)

    2014-01-01

    Background Various computer-based methods exist for the detection and quantification of protein spots in two dimensional gel electrophoresis images. Area-based methods are commonly used for spot quantification: an area is assigned to each spot and the sum of the pixel intensities in that area, the so-called volume, is used a measure for spot signal. Other methods use the optical density, i.e. the intensity of the most intense pixel of a spot, or calculate the volume from the parameters of a fitted function. Results In this study we compare the performance of different spot quantification methods using synthetic and real data. We propose a ready-to-use algorithm for spot detection and quantification that uses fitting of two dimensional Gaussian function curves for the extraction of data from two dimensional gel electrophoresis (2-DE) images. The algorithm implements fitting using logical compounds and is computationally efficient. The applicability of the compound fitting algorithm was evaluated for various simulated data and compared with other quantification approaches. We provide evidence that even if an incorrect bell-shaped function is used, the fitting method is superior to other approaches, especially when spots overlap. Finally, we validated the method with experimental data of urea-based 2-DE of Aβ peptides andre-analyzed published data sets. Our methods showed higher precision and accuracy than other approaches when applied to exposure time series and standard gels. Conclusion Compound fitting as a quantification method for 2-DE spots shows several advantages over other approaches and could be combined with various spot detection methods. The algorithm was scripted in MATLAB (Mathworks) and is available as a supplemental file. PMID:24915860

  4. 2-D Difference in gel electrophoresis combined with Pro-Q Diamond staining: a successful approach for the identification of kinase/phosphatase targets.

    Science.gov (United States)

    Orsatti, Laura; Forte, Eleonora; Tomei, Licia; Caterino, Marianna; Pessi, Antonello; Talamo, Fabio

    2009-07-01

    The protein tyrosine phosphatase PRL-3 is an appealing therapeutic cancer target for its well described involvement in the metastasis progression. Nevertheless, very little is known about PRL-3 role in tumorigenesis. In the attempt to identify the protein target of this phosphatase we have devised a model system based on the use of highly invasive HCT116 colon cancer cells over-expressing PRL-3. We used 2-D difference gel electrophoresis combined with the fluorescence staining Pro-Q Diamond selective for phosphorylated proteins to monitor changes in the phosphorylation status of possible substrates. Proteins whose phosphorylation level was negatively affected by PRL-3 over-expression were identified by MS. Two proteins were found to be significantly dephosphorylated in this condition, the cytoskeletal protein ezrin and elongation factor 2. Ezrin has already been described as having a proactive role in cancer metastasis through control of its phosphorylation status, and the PRL-3-induced modulation of ezrin phosphorylation in HCT116 and human umblical vascular endothelial cells is the subject of a separate paper by Forte et al. [Biochim. Biophys. Acta 2008, 1783, 334-344]. The combination of 2-D difference in gel electrophoresis and Pro-Q Diamond was hence confirmed successful in analyzing changes of protein phosphorylation which enable the identification of kinase/phosphatase targets.

  5. Single-cell microgel electrophoresis: an in vitro assay of radiosensitivity

    International Nuclear Information System (INIS)

    Deeley, J.O.T.; Moore, J.L.

    1993-01-01

    The results obtained by a microgel electrophoresis are comparable to conventional gel electrophoresis and elution techniques (Singh et al, 1989), DNA precipitation, alkali unwinding and cell clonogenicity assays (Olive et al, 1990). Since single cells are assessed, microgel electrophoresis is particularly appropriate for end-points such as the intercell variation in response. The simplicity, low cost and rapidity of microgel electrophoresis compared with other assays makes it particularly attractive for assessing the effects on DNA of radiation and other genotoxic agents on the general population. (Author)

  6. 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.

  7. Presence of a novel DNA methylation enzyme in methicillin-resistant Staphylococcus aureus isolates associated with pig farming leads to uninterpretable results in standard pulsed-field gel electrophoresis analysis.

    NARCIS (Netherlands)

    Bens, C.C.; Voss, A.; Klaassen, C.H.W.

    2006-01-01

    Genomic DNA from methicillin-resistant Staphylococcus aureus isolates recovered from pigs and their caretakers proved resistant to SmaI digestion, leading to uninterpretable results in standard pulsed-field gel electrophoresis. This is the result of a yet unknown restriction/methylation system in

  8. Quantitative two-dimensional gel electrophoresis analysis of human fibroblasts transformed by ras oncogenes.

    Science.gov (United States)

    Miller, M J; Maher, V M; McCormick, J J

    1992-11-01

    Quantitative two-dimensional gel electrophoresis was used to compare the cellular protein patterns of a normal foreskin-derived human fibroblasts cell line (LG1) and three immortal derivatives of LG1. One derivative, designated MSU-1.1 VO, was selected for its ability to grow in the absence of serum and is non-tumorigenic in athymic mice. The other two strains were selected for focus-formation following transfection with either Ha-ras or N-ras oncogenes and form high grade malignant tumors. Correspondence and cluster analysis provided a nonbiased estimate of the relative similarity of the different two-dimensional patterns. These techniques separated the gel patterns into three distinct classes: LG1, MSU-1.1 VO, and the ras transformed cell strains. The MSU-1.1 VO cells were more closely related to the parental LG1 than to the ras-transformed cells. The differences between the three classes were primarily quantitative in nature: 16% of the spots demonstrated statistically significant changes (P 2) in the rate of incorporation of radioactive amino acids. The patterns from the two ras-transformed cell strains were similar, and variations in the expression of proteins that occurred between the separate experiments obscured consistent differences between the Ha-ras and N-ras transformed cells. However, while only 9 out of 758 spots were classified as different (1%), correspondence analysis could consistently separate the two ras transformants. One of these spots was five times more intense in the Ha-ras transformed cells than the N-ras.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Matrix-assisted laser desorption fourier transform mass spectrometry for biological compounds

    Energy Technology Data Exchange (ETDEWEB)

    Hettich, R.; Buchanan, M.

    1990-01-01

    The recent development of matrix-assisted UV laser desorption (LD) mass spectrometry has made possible the ionization and detection of extremely large molecules (with molecular weights exceeding 100,000 Daltons). This technique has generated enormous interest in the biological community for the direct examination of large peptides and oligonucleotides. Although this matrix-assisted ionization method has been developed and used almost exclusively with time-of-flight (TOF) mass spectrometers, research is currently in progress to demonstrate this technique with trapped ion mass spectrometers, such as Fourier transform ion cyclotron resonance mass spectrometry (FTMS). The potential capabilities of FTMS for wide mass range, high resolution measurement, and ion trapping experiments suggest that this instrumental technique should be useful for the detailed structural characterization of large ions generated by the matrix-assisted technique. We have recently demonstrated that matrix-assisted ultraviolet laser desorption can be successfully used with FTMS for the ionization of small peptides. The objective of this report is to summarize the application and current limitations of matrix-assisted laser desorption FTMS for the characterization of peptides and oligonucleotides at the isomeric level. 4 refs., 3 figs., 2 tabs.

  10. Detection of heavy ion induced DNA double-strand breaks using static-field gel electrophoresis

    International Nuclear Information System (INIS)

    Taucher-Scholz, G.; Heilmann, J.; Schneider, G.; Kraft, G.

    1994-11-01

    Radiation induced DNA double-strand breaks (DSBs) were measured in Chinese hamster ovary cells (CHO-K1) using an experimental protocol involving static-field gel electrophoresis following exposure to various accelerated ions. Dose-effect curves were set up and relative biological efficiencies (RBEs) for DSB induction were determined for different radiation qualities. RBEs around 1 were obtained for low energy deuterons (6-7 keV/μm), while for high energy oxygen ions (20 keV/μm) an RBE value slightly greater than 1 was determined. Low energetic oxygen ions (LET ∼ 250 keV/μm) were found to show RBEs substantially below unity, and for higher LET particles (≥ 250 keV/μm) RBEs for DSB induction were generally found to be smaller than 1. The data presented here are in line with the generally accepted view that not induced DSBs, but misrepaired or unrepaired DNA-lesions are related to cellular inactivation. (orig.)

  11. Oxidizing gel formulation for nuclear decontamination: rheological and acidic properties of the organic matrix and its ozonolysis

    International Nuclear Information System (INIS)

    Rouy, E.

    2003-10-01

    An acidic and oxidizing gel was formulated with a purely organic matrix, xanthan gum, at low concentrations (1 to 2 wt %). This polymer gel was investigated in various media (aqueous, acidic and ceric) by means of rheology: shear thinning behaviour, thixotropy, yield stress... Evidences of unexpected rheological properties in highly concentrated media show that xanthan is quite convenient for industrial projection of this type of gel on metallic walls in nuclear plants, notwithstanding its time-limited resistance to oxidation (about a few hours). Complexation mechanisms between ceric species and polar sites of the polymer led us to characterise acidic properties of our xanthan sample by potentiometric titration and 1 H NMR techniques. The matrix was finally treated by ozonolysis to suppress organic residues, as required to handle nuclear wastes. In acidic medium, ozonolysis of the gel was achieved successfully while in acidic and ceric medium this process showed limited efficiency, needing further investigation to be clarified. (author)

  12. Molecular dynamics simulations of matrix assisted laser desorption ionization: Matrix-analyte interactions

    International Nuclear Information System (INIS)

    Nangia, Shivangi; Garrison, Barbara J.

    2011-01-01

    There is synergy between matrix assisted laser desorption ionization (MALDI) experiments and molecular dynamics (MD) simulations. To understand analyte ejection from the matrix, MD simulations have been employed. Prior calculations show that the ejected analyte molecules remain solvated by the matrix molecules in the ablated plume. In contrast, the experimental data show free analyte ions. The main idea of this work is that analyte molecule ejection may depend on the microscopic details of analyte interaction with the matrix. Intermolecular matrix-analyte interactions have been studied by focusing on 2,5-dihydroxybenzoic acid (DHB; matrix) and amino acids (AA; analyte) using Chemistry at HARvard Molecular Mechanics (CHARMM) force field. A series of AA molecules have been studied to analyze the DHB-AA interaction. A relative scale of AA molecule affinity towards DHB has been developed.

  13. Assessment of DNA damage in radiation workers by using single cell gel electrophoresis

    International Nuclear Information System (INIS)

    Jia Lili; Zhang Tao; Yang Yonghua; Wang Yan; Du Liqing; Cao Jia; Wang Hong; Liu Qiang; Fan Feiyue

    2010-01-01

    Objective: To assess the DNA damage of radiation workers in different grade hospitals, and to explore the correlation between the types of work or work time and the levels of DNA damage. Methods: DNA single strand break were detected by using alkaline single cell gel electrophoresis (SCGE), and the comet was analyzed with CASP (Comet Assay Software Project). TDNA%, TL, TM and OTM were calculated. Results: The parameters of SCGE in the radiation group were higher than those of control group (F=3.93, P<0.01). The significant difference was found not only among the different types of work or different work time, but also among the different grade hospitals (F=1.83, 1.91, P<0.05). Conclusions: Various levels of DNA damage could be detected in the radiation workers of the two hospitals. DNA damage of radiation workers is less serious in the higher-grade hospital than the lower grade one. Different types of work or work time might affect the DNA damage level. (authors)

  14. DNA double-strand break measurement in mammalian cells by pulsed-field gel electrophoresis: an approach using restriction enzymes and gene probing

    International Nuclear Information System (INIS)

    Loebrich, M.; Ikpeme, S.; Kiefer, J.

    1994-01-01

    DNA samples prepared from human SP 3 cells, which had not been exposed to various doses of X-ray, were treated with NotI restriction endonuclease before being run in a contour-clamped homogeneous electrophoresis system. The restriction enzyme cuts the DNA at defined positions delivering DNA sizes which can be resolved by pulsed-field gel electrophoresis (PFGE). In order to investigate only one of the DNA fragments, a human lactoferrin cDNA, pHL-41, was hybridized to the DNA separated by PFGE. As a result, only the DNA fragment which contains the hybridized gene was detected resulting in a one-band pattern. The decrease of this band was found to be exponential with increasing radiation dose. From the slope, a double-strand break induction rate of (6.3±0.7) x 10 -3 /Mbp/Gy was deduced for 80 kV X-rays. (Author)

  15. Open microfluidic gel electrophoresis: Rapid and low cost separation and analysis of DNA at the nanoliter scale.

    Science.gov (United States)

    Gutzweiler, Ludwig; Gleichmann, Tobias; Tanguy, Laurent; Koltay, Peter; Zengerle, Roland; Riegger, Lutz

    2017-07-01

    Gel electrophoresis is one of the most applied and standardized tools for separation and analysis of macromolecules and their fragments in academic research and in industry. In this work we present a novel approach for conducting on-demand electrophoretic separations of DNA molecules in open microfluidic (OM) systems on planar polymer substrates. The approach combines advantages of slab gel, capillary- and chip-based methods offering low consumable costs (<0.1$) circumventing cost-intensive microfluidic chip fabrication, short process times (5 min per analysis) and high sensitivity (4 ng/μL dsDNA) combined with reasonable resolution (17 bases). The open microfluidic separation system comprises two opposing reservoirs of 2-4 μL in volume, a semi-contact written gel line acting as separation channel interconnecting the reservoirs and sample injected into the line via non-contact droplet dispensing and thus enabling the precise control of the injection plug and sample concentration. Evaporation is prevented by covering aqueous structures with PCR-grade mineral oil while maintaining surface temperature at 15°C. The liquid gel line exhibits a semi-circular cross section of adaptable width (∼200-600 μm) and height (∼30-80 μm) as well as a typical length of 15-55 mm. Layout of such liquid structures is adaptable on-demand not requiring time consuming and repetitive fabrication steps. The approach was successfully demonstrated by the separation of a standard label-free DNA ladder (100-1000 bp) at 100 V/cm via in-line staining and laser induced fluorescent end-point detection using an automated prototype. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Comparison of protein patterns of xrs-5, a radiosensitive Chinese hamster ovary cell line, and CHO-K1, its radioresistant parent, using two-dimensional gel-electrophoresis

    International Nuclear Information System (INIS)

    Kramer, J.M.

    1991-01-01

    X-ray sensitive strains of Chinese hamster ovary cell lines have been used to analyze radiation repair mechanisms. One cell line, xrs-5, has been shown to be very sensitive to ionizing radiation and radical forming chemical mutagens. This sensitivity is thought to be a result a mutation in the DNA double strand break (DSB) repair mechanism, and its characterization has been a goal of several repair mechanism studies. Using two-dimensional gel electrophoresis, we have detected a protein (MW approximately 55KD) in the DNA/Nuclear Matrix (nucleoid) cell fraction of CHO-Kl cells that is absent in the nucleoid fraction of xrs-5. This protein is present, however, in both CHO-Kl and xrs-5 whole cell protein maps. To determine whether the 55KD protein is responsible for the radiosensitive and defective DSB repair phenotype of xrs-5 cells, studies are now underway to analyze revertants of xrs-5 that are proficient in DSB repair. Furthermore, an effort to sequence the protein in question is planned. 23 refs., 2 figs

  17. Comparison of Ribotyping, Randomly Amplified Polymorphic DNA Analysis, and Pulsed-Field Gel Electrophoresis in Typing of Lactobacillus rhamnosus and L. casei Strains

    OpenAIRE

    Tynkkynen, Soile; Satokari, Reetta; Saarela, Maria; Mattila-Sandholm, Tiina; Saxelin, Maija

    1999-01-01

    A total of 24 strains, biochemically identified as members of the Lactobacillus casei group, were identified by PCR with species-specific primers. The same set of strains was typed by randomly amplified polymorphic DNA (RAPD) analysis, ribotyping, and pulsed-field gel electrophoresis (PFGE) in order to compare the discriminatory power of the methods. Species-specific primers for L. rhamnosus and L. casei identified the type strain L. rhamnosus ATCC 7469 and the neotype strain L. casei ATCC 33...

  18. Investigating the fermentation of cocoa by correlating denaturing gradient gel electrophoresis profiles and near infrared spectra

    DEFF Research Database (Denmark)

    Nielsen, Dennis Sandris; Snitkjær, Pia; van der Berg, Franciscus Winfried J

    2008-01-01

    demonstrating the microbial succession taking place during the fermentation. Subsequently the DGGE spectra were correlated to the NIR spectra using Partial Least Squares regression models (PLS2). Correlations of 0.87 (bacterial derived DGGE spectra) and 0.81 (yeast derived DGGE spectra) were obtained indicating......Raw cocoa has an astringent, unpleasant taste and flavour, and has to be fermented, dried and roasted in order to obtain the characteristic cocoa flavour and taste. During the fermentation microbial activity outside the cocoa beans induces biochemical and physical changes inside the beans...... of the beans and the chemical processes inside the beans have been carried out previously. Recently it has been shown that Denaturing Gradient Gel Electrophoresis (DGGE) offers an efficient tool for monitoring the microbiological changes taking place during the fermentation of cocoa. Near Infrared (NIR...

  19. A Chip-Capillary Hybrid Device for Automated Transfer of Sample Pre-Separated by Capillary Isoelectric Focusing to Parallel Capillary Gel Electrophoresis for Two-Dimensional Protein Separation

    Science.gov (United States)

    Lu, Joann J.; Wang, Shili; Li, Guanbin; Wang, Wei; Pu, Qiaosheng; Liu, Shaorong

    2012-01-01

    In this report, we introduce a chip-capillary hybrid device to integrate capillary isoelectric focusing (CIEF) with parallel capillary sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) or capillary gel electrophoresis (CGE) toward automating two-dimensional (2D) protein separations. The hybrid device consists of three chips that are butted together. The middle chip can be moved between two positions to re-route the fluidic paths, which enables the performance of CIEF and injection of proteins partially resolved by CIEF to CGE capillaries for parallel CGE separations in a continuous and automated fashion. Capillaries are attached to the other two chips to facilitate CIEF and CGE separations and to extend the effective lengths of CGE columns. Specifically, we illustrate the working principle of the hybrid device, develop protocols for producing and preparing the hybrid device, and demonstrate the feasibility of using this hybrid device for automated injection of CIEF-separated sample to parallel CGE for 2D protein separations. Potentials and problems associated with the hybrid device are also discussed. PMID:22830584

  20. Quantification of DNA by Agarose Gel Electrophoresis and Analysis of the Topoisomers of Plasmid and M13 DNA Following Treatment with a Restriction Endonuclease or DNA Topoisomerase I

    Science.gov (United States)

    Tweedie, John W.; Stowell, Kathryn M.

    2005-01-01

    A two-session laboratory exercise for advanced undergraduate students in biochemistry and molecular biology is described. The first session introduces students to DNA quantification by ultraviolet absorbance and agarose gel electrophoresis followed by ethidium bromide staining. The second session involves treatment of various topological forms of…

  1. Reduction in beta-myosin heavy chains in stunned myocardium as assessed by nondenaturing gel electrophoresis.

    Science.gov (United States)

    Garcia, S C; Pomblum, V J; Gams, E; Rupp, H; Schipke, J D

    2007-09-01

    Myosin plays a key role in the structure and function of cardiac muscle. Three myosin isoenzymes (V(1), V(2), and V(3)) with different ATPase activities have been identified in mammalian ventricles based on their heavy chain constituents. The relative amount of myosin isoenzymes changes under physiological and pathological conditions. Until now, myosin isoenzymes have frequently been determined using either tube gel (nondenaturing) polyacrylamide gel electrophoresis (PAGE), or gradient or uniform sodium dodecyl sulfate (denaturing) PAGE. Both methods have disadvantages, e.g., a long running time. We developed, therefore, a uniform, nondenaturing PAGE with slab minigel format for analyzing the myosin isoenzymes in normoxic and stunned rabbit hearts. In normoxic hearts of adult rabbits, V(3) predominated over V(1) (46 vs 41%). In turn, in the stunned hearts, V(1) predominated over V(3) (70 vs 30%), and the heterodimeric V(2) was not anymore detectable. This alteration appears to result from a selective loss of myosin heavy chain (MHC)-beta. In parallel, the biochemical markers troponin I and creatine kinase were increased in the stunned hearts. We suggest that alterations of myosin isoenzymes in stunned myocardium can be monitored with native PAGE. The present analysis of myosin isoenzyme appears thus as a new tool for evaluating defects in MHC dimer formation in postischemic hearts.

  2. Comparison of ethanol-soluble proteins from different rye (Secale cereale) varieties by two-dimensional electrophoresis

    DEFF Research Database (Denmark)

    Radzikowski, Louise; Nesic, Ljiljana; Hansen, H.B.

    2002-01-01

    The major storage proteins from six rye varieties, grown under the same conditions in 1997 and 1998 in Ronhave, Denmark, were analyzed by two-dimensional (2-D) polyacrylamide gel electrophoresis. The proteins were extracted from ground rye kernels with 70% ethanol and separated by 2-D electrophor......The major storage proteins from six rye varieties, grown under the same conditions in 1997 and 1998 in Ronhave, Denmark, were analyzed by two-dimensional (2-D) polyacrylamide gel electrophoresis. The proteins were extracted from ground rye kernels with 70% ethanol and separated by 2-D...... electrophoresis. The gels were scanned, compared using ImageMaster(R) software and the data sets were analyzed by principal component analysis (PCA) using THE UNSCRAMBLER software. Afterwards MATLAB was used to make a cluster analysis of the varieties based on PCA. The analysis of the gels showed...... separately. When the results were combined from the two years five varieties could be differentiated. The results from the PCA confirmed the finding of the unique spots and cluster analysis was made in order to illustrate the results. The combination of the results from 2-D electrophoresis and other grain...

  3. ASTRO Research Fellow Presentation - A comparison of the comet assay and pulsed-field gel electrophoresis as a predictive assay for radiosensitivity in human fibroblasts

    International Nuclear Information System (INIS)

    Sarkaria, Jann N.; Eady, John J.; Peacock, John H.; Steel, G. Gordon

    1996-01-01

    Purpose/Objective: To determine whether neutral lysis single-cell gel electrophoresis (comet assay) or pulsed-field gel electrophoresis (PFGE) can be used as a predictive assay for tissue response to radiotherapy as an alternative to clonogenic survival measurements. Materials and Methods: The comet assay has been widely used to measure DNA double strand breaks (dsb) in individual cells, and it has been suggested that it could be used as an alternative to clonogenic assays to measure radiosensitivity. Previous studies in this lab have demonstrated the ability of pulsed-field gel electrophoresis, which also measures DNA dsb, to accurately predict the radiosensitivity of a panel of fibroblasts based on determination of residual DNA dsb. As part of an ongoing study examining the relationship between fibroblast radiosensitivity and normal-tissue radiation reactions, we have compared the sensitivity and accuracy of the comet assay and PFGE on a different panel of non-transformed fibroblasts derived from breast cancer patients who developed severe radiation late effects and from case-matched controls. For the measurement of initial damage, cells were suspended in PBS and irradiated on ice for the comet assay and irradiated in agarose plugs on ice for pFGE. Residual damage was measured following irradiation of confluent cultures at 37 degree sign C and subsequent incubation for four hours prior to preparation of agarose slides and plugs. All irradiations were performed with a 59 TBq 60 Co source at a dose rate of 1.7 Gy/min. Electrophoresis was performed following neutral pH cell lysis. Comet images were captured and analyzed using Optimas software with DNA damage quantitated by the comet moment. PFGE gels were analyzed using a phosphor-image analysis system and damage was quantitated based on the percent of activity released from the well. Results: The comet assay was able to detect initial DNA damage at a threshold of 5 Gy and exhibited a linear dose

  4. Electronic imaging systems for quantitative electrophoresis of DNA

    International Nuclear Information System (INIS)

    Sutherland, J.C.

    1989-01-01

    Gel electrophoresis is one of the most powerful and widely used methods for the separation of DNA. During the last decade, instruments have been developed that accurately quantitate in digital form the distribution of materials in a gel or on a blot prepared from a gel. In this paper, I review the various physical properties that can be used to quantitate the distribution of DNA on gels or blots and the instrumentation that has been developed to perform these tasks. The emphasis here is on DNA, but much of what is said also applies to RNA, proteins and other molecules. 36 refs

  5. Standardization and interlaboratory reproducibility assessment of pulsed-field gel electrophoresis-generated fingerprints of Acinetobacter baumannii.

    Science.gov (United States)

    Seifert, Harald; Dolzani, Lucilla; Bressan, Raffaela; van der Reijden, Tanny; van Strijen, Beppie; Stefanik, Danuta; Heersma, Herre; Dijkshoorn, Lenie

    2005-09-01

    A standard procedure for pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments of Acinetobacter baumannii was set up and validated for its interlaboratory reproducibility and its potential for use in the construction of an Internet-based database for international monitoring of epidemic strains. The PFGE fingerprints of strains were generated at three different laboratories with ApaI as the restriction enzyme and by a rigorously standardized procedure. The results were analyzed at the respective laboratories and also centrally at a national reference institute. In the first phase of the study, 20 A. baumannii strains, including 3 isolates each from three well-characterized hospital outbreaks and 11 sporadic strains, were distributed blindly to the participating laboratories. The local groupings of the isolates in each participating laboratory were identical and allowed the identification of the epidemiologically related isolates as belonging to three clusters and identified all unrelated strains as distinct. Central pattern analysis by using the band-based Dice coefficient and the unweighted pair group method with mathematical averaging as the clustering algorithm showed 95% matching of the outbreak strains processed at each local laboratory and 87% matching of the corresponding strains if they were processed at different laboratories. In the second phase of the study, 30 A. baumannii isolates representing 10 hospital outbreaks from different parts of Europe (3 isolates per outbreak) were blindly distributed to the three laboratories, so that each laboratory investigated 10 epidemiologically independent outbreak isolates. Central computer-assisted cluster analysis correctly identified the isolates according to their corresponding outbreak at an 87% clustering threshold. In conclusion, the standard procedure enabled us to generate PFGE fingerprints of epidemiologically related A. baumannii strains at different locations with sufficient

  6. A Comprehensive Quality Evaluation System for Complex Herbal Medicine Using PacBio Sequencing, PCR-Denaturing Gradient Gel Electrophoresis, and Several Chemical Approaches

    Directory of Open Access Journals (Sweden)

    Xiasheng Zheng

    2017-09-01

    Full Text Available Herbal medicine is a major component of complementary and alternative medicine, contributing significantly to the health of many people and communities. Quality control of herbal medicine is crucial to ensure that it is safe and sound for use. Here, we investigated a comprehensive quality evaluation system for a classic herbal medicine, Danggui Buxue Formula, by applying genetic-based and analytical chemistry approaches to authenticate and evaluate the quality of its samples. For authenticity, we successfully applied two novel technologies, third-generation sequencing and PCR-DGGE (denaturing gradient gel electrophoresis, to analyze the ingredient composition of the tested samples. For quality evaluation, we used high performance liquid chromatography assays to determine the content of chemical markers to help estimate the dosage relationship between its two raw materials, plant roots of Huangqi and Danggui. A series of surveys were then conducted against several exogenous contaminations, aiming to further access the efficacy and safety of the samples. In conclusion, the quality evaluation system demonstrated here can potentially address the authenticity, quality, and safety of herbal medicines, thus providing novel insight for enhancing their overall quality control.Highlight: We established a comprehensive quality evaluation system for herbal medicine, by combining two genetic-based approaches third-generation sequencing and DGGE (denaturing gradient gel electrophoresis with analytical chemistry approaches to achieve the authentication and quality connotation of the samples.

  7. A Comprehensive Quality Evaluation System for Complex Herbal Medicine Using PacBio Sequencing, PCR-Denaturing Gradient Gel Electrophoresis, and Several Chemical Approaches.

    Science.gov (United States)

    Zheng, Xiasheng; Zhang, Peng; Liao, Baosheng; Li, Jing; Liu, Xingyun; Shi, Yuhua; Cheng, Jinle; Lai, Zhitian; Xu, Jiang; Chen, Shilin

    2017-01-01

    Herbal medicine is a major component of complementary and alternative medicine, contributing significantly to the health of many people and communities. Quality control of herbal medicine is crucial to ensure that it is safe and sound for use. Here, we investigated a comprehensive quality evaluation system for a classic herbal medicine, Danggui Buxue Formula, by applying genetic-based and analytical chemistry approaches to authenticate and evaluate the quality of its samples. For authenticity, we successfully applied two novel technologies, third-generation sequencing and PCR-DGGE (denaturing gradient gel electrophoresis), to analyze the ingredient composition of the tested samples. For quality evaluation, we used high performance liquid chromatography assays to determine the content of chemical markers to help estimate the dosage relationship between its two raw materials, plant roots of Huangqi and Danggui. A series of surveys were then conducted against several exogenous contaminations, aiming to further access the efficacy and safety of the samples. In conclusion, the quality evaluation system demonstrated here can potentially address the authenticity, quality, and safety of herbal medicines, thus providing novel insight for enhancing their overall quality control. Highlight : We established a comprehensive quality evaluation system for herbal medicine, by combining two genetic-based approaches third-generation sequencing and DGGE (denaturing gradient gel electrophoresis) with analytical chemistry approaches to achieve the authentication and quality connotation of the samples.

  8. A Comprehensive Quality Evaluation System for Complex Herbal Medicine Using PacBio Sequencing, PCR-Denaturing Gradient Gel Electrophoresis, and Several Chemical Approaches

    Science.gov (United States)

    Zheng, Xiasheng; Zhang, Peng; Liao, Baosheng; Li, Jing; Liu, Xingyun; Shi, Yuhua; Cheng, Jinle; Lai, Zhitian; Xu, Jiang; Chen, Shilin

    2017-01-01

    Herbal medicine is a major component of complementary and alternative medicine, contributing significantly to the health of many people and communities. Quality control of herbal medicine is crucial to ensure that it is safe and sound for use. Here, we investigated a comprehensive quality evaluation system for a classic herbal medicine, Danggui Buxue Formula, by applying genetic-based and analytical chemistry approaches to authenticate and evaluate the quality of its samples. For authenticity, we successfully applied two novel technologies, third-generation sequencing and PCR-DGGE (denaturing gradient gel electrophoresis), to analyze the ingredient composition of the tested samples. For quality evaluation, we used high performance liquid chromatography assays to determine the content of chemical markers to help estimate the dosage relationship between its two raw materials, plant roots of Huangqi and Danggui. A series of surveys were then conducted against several exogenous contaminations, aiming to further access the efficacy and safety of the samples. In conclusion, the quality evaluation system demonstrated here can potentially address the authenticity, quality, and safety of herbal medicines, thus providing novel insight for enhancing their overall quality control. Highlight: We established a comprehensive quality evaluation system for herbal medicine, by combining two genetic-based approaches third-generation sequencing and DGGE (denaturing gradient gel electrophoresis) with analytical chemistry approaches to achieve the authentication and quality connotation of the samples. PMID:28955365

  9. Drawing a different picture with pencil lead as matrix-assisted laser desorption/ionization matrix for fullerene derivatives.

    Science.gov (United States)

    Nye, Leanne C; Hungerbühler, Hartmut; Drewello, Thomas

    2018-02-01

    Inspired by reports on the use of pencil lead as a matrix-assisted laser desorption/ionization matrix, paving the way towards matrix-free matrix-assisted laser desorption/ionization, the present investigation evaluates its usage with organic fullerene derivatives. Currently, this class of compounds is best analysed using the electron transfer matrix trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene] malononitrile (DCTB), which was employed as the standard here. The suitability of pencil lead was additionally compared to direct (i.e. no matrix) laser desorption/ionization-mass spectrometry. The use of (DCTB) was identified as the by far gentler method, producing spectra with abundant molecular ion signals and much reduced fragmentation. Analytically, pencil lead was found to be ineffective as a matrix, however, appears to be an extremely easy and inexpensive method for producing sodium and potassium adducts.

  10. Quantitation of yeast total proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer for uniform loading.

    Science.gov (United States)

    Sheen, Hyukho

    2016-04-01

    Proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer are difficult to quantitate due to SDS and reducing agents being in the buffer. Although acetone precipitation has long been used to clean up proteins from detergents and salts, previous studies showed that protein recovery from acetone precipitation varies from 50 to 100% depending on the samples tested. Here, this article shows that acetone precipitates proteins highly efficiently from SDS-PAGE sample buffer and that quantitative recovery is achieved in 5 min at room temperature. Moreover, precipitated proteins are resolubilized with urea/guanidine, rather than with SDS. Thus, the resolubilized samples are readily quantifiable with Bradford reagent without using SDS-compatible assays. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Time-based distribution of Staphylococcus saprophyticus pulsed field gel-electrophoresis clusters in community-acquired urinary tract infections

    Directory of Open Access Journals (Sweden)

    Viviane Santos de Sousa

    2013-02-01

    Full Text Available The epidemiology of urinary tract infections (UTI by Staphylococcus saprophyticus has not been fully characterised and strain typing methods have not been validated for this agent. To evaluate whether epidemiological relationships exist between clusters of pulsed field gel-electrophoresis (PFGE genotypes of S. saprophyticus from community-acquired UTI, a cross-sectional surveillance study was conducted in the city of Rio de Janeiro, Brazil. In total, 32 (16% female patients attending two walk-in clinics were culture-positive for S. saprophyticus. Five PFGE clusters were defined and evaluated against epidemiological data. The PFGE clusters were grouped in time, suggesting the existence of community point sources of S. saprophyticus. From these point sources, S. saprophyticus strains may spread among individuals.

  12. A subtle calculation method for nanoparticle’s molar extinction coefficient: The gift from discrete protein-nanoparticle system on agarose gel electrophoresis

    Science.gov (United States)

    Zhong, Ruibo; Yuan, Ming; Gao, Haiyang; Bai, Zhijun; Guo, Jun; Zhao, Xinmin; Zhang, Feng

    2016-03-01

    Discrete biomolecule-nanoparticle (NP) conjugates play paramount roles in nanofabrication, in which the key is to get the precise molar extinction coefficient of NPs. By making best use of the gift from a specific separation phenomenon of agarose gel electrophoresis (GE), amphiphilic polymer coated NP with exact number of bovine serum albumin (BSA) proteins can be extracted and further experimentally employed to precisely calculate the molar extinction coefficient of the NPs. This method could further benefit the evaluation and extraction of any other dual-component NP-containing bio-conjugates.

  13. Presence of a Novel DNA Methylation Enzyme in Methicillin-Resistant Staphylococcus aureus Isolates Associated with Pig Farming Leads to Uninterpretable Results in Standard Pulsed-Field Gel Electrophoresis Analysis

    OpenAIRE

    Bens, Corina C. P. M.; Voss, Andreas; Klaassen, Corné H. W.

    2006-01-01

    Genomic DNA from methicillin-resistant Staphylococcus aureus isolates recovered from pigs and their caretakers proved resistant to SmaI digestion, leading to uninterpretable results in standard pulsed-field gel electrophoresis. This is the result of a yet unknown restriction/methylation system in the genus Staphylococcus with the recognition sequence CCNGG.

  14. Allozyme comparison of three Trypanosoma species (Kinetoplastida: Trypanosomatidae) of toads and frogs by starch-gel electrophoresis.

    Science.gov (United States)

    Martin, D S; Desser, S S; Hong, H

    1992-04-01

    Six metabolic enzymes, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, isocitrate dehydrogenase, malate dehydrogenase, phosphoglucomutase, and purine nucleoside phosphorylase, from clonal isolates of 3 presumptive species of Trypanosoma (T. fallisi, T. ranarum, and T. rotatorium) from 3 anuran hosts (Bufo americanus, Rana clamitans, and Rana catesbeiana) were compared using starch-gel electrophoresis. Although bands were shared among the different zymodemes of isolates of the same host genus, low genetic polymorphism of the enzyme loci was observed with few apparent shared bands between samples isolated from frogs and toads. A distance value calculated between toad and frog trypanosome isolates suggests the likelihood of long-time separation of species. Cluster analysis based on overall similarity distinguished the trypanosomes of toads and frogs as separate taxa, suggesting that host specificity and observed morphological differences are consistent with heritable allozyme differences.

  15. An improved method for detecting genetic variation in DNA using denaturing gradient gel electrophoresis

    International Nuclear Information System (INIS)

    Takahashi, Norio; Hiyama, Keiko; Kodaira, Mieko; Satoh, Chiyoko.

    1990-05-01

    We have examined the feasibility of denaturing gradient gel electrophoresis (DGGE) of RNA:DNA duplexes to detect variations in genomic and cloned DNAs. The result has demonstrated that use of RNA:DNA duplexes makes DGGE much more practical for screening a large number of samples than use of DNA:DNA heteroduplexes, because preparation of RNA probes is easier than that of DNA probes. Three different 32 P-labeled RNA probes were produced. Genomic or cloned DNAs were digested with restriction enzymes and hybridized to labeled RNA probes, and resulting RNA:DNA duplexes were examined by DGGE. The presence of a mismatch(es) was detected as a difference in the mobility of bands on the gel. The experimental conditions were determined using DNA segments from cloned normal and three thalassemic human β-globin genes. The results from experiments on the cloned DNAs suggest that DGGE of RNA:DNA duplexes will detect nucleotide substitutions and deletions in DNA. In the course of these studies, a polymorphism due to a single-base substitution at position 666 of IVS2 (IVS2-666) of the human β-globin gene was directly identified using genomic DNA samples. A study of 59 unrelated Japanese from Hiroshima was undertaken in which the frequency of the allele with C at IVS2-666 was 0.48 and that of the allele with T was 0.52. This approach was found to be very effective for detecting heritable variation and should be a powerful tool for detecting fresh mutations in DNA, which occur outside the known restriction sites. (author)

  16. Application of preparative disk gel electrophoresis for antigen purification from inclusion bodies.

    Science.gov (United States)

    Okegawa, Yuki; Koshino, Masanori; Okushima, Teruya; Motohashi, Ken

    2016-02-01

    Specific antibodies are a reliable tool to examine protein expression patterns and to determine the protein localizations within cells. Generally, recombinant proteins are used as antigens for specific antibody production. However, recombinant proteins from mammals and plants are often overexpressed as insoluble inclusion bodies in Escherichia coli. Solubilization of these inclusion bodies is desirable because soluble antigens are more suitable for injection into animals to be immunized. Furthermore, highly purified proteins are also required for specific antibody production. Plastidic acetyl-CoA carboxylase (ACCase: EC 6.4.1.2) from Arabidopsis thaliana, which catalyzes the formation of malonyl-CoA from acetyl-CoA in chloroplasts, formed inclusion bodies when the recombinant protein was overexpressed in E. coli. To obtain the purified protein to use as an antigen, we applied preparative disk gel electrophoresis for protein purification from inclusion bodies. This method is suitable for antigen preparation from inclusion bodies because the purified protein is recovered as a soluble fraction in electrode running buffer containing 0.1% sodium dodecyl sulfate that can be directly injected into immune animals, and it can be used for large-scale antigen preparation (several tens of milligrams). Copyright © 2015 Elsevier Inc. All rights reserved.

  17. The protective effect of caffeine on DNA photosensitive damage: a gel electrophoresis

    International Nuclear Information System (INIS)

    Huang Liping; Ma Jianhua

    2009-01-01

    Agarose gel electrophoresis was performed to study interaction effect of caffeine on photosensitive injury of DNA caused by anthraquinone-2-sulphonic acid disodium (AQS), a model compound of strong photosensitizer, under 254 nm or 365nm UV irradiation Photosensitive injury of DNA induced by AQS under deoxidized condition was used as control. The results show that caffeine may resist effectively the injury effect of photosensitive damage and strong UV irradiation on DNA. The effects depend on the caffeine and AQS concentration, and irradiation time. Caffeine in concentration of 0.01-3.0 μg/μL, may prevent DNA from damage induced by UV light, but caffeine in concentration of >5.0 μg/μL accelerates the DNA damage. In particular, in the aqueous solution system of DNA, caffeine and AQS, at pH 6.25-7.35, the caffeine in concentration of 2.5-4.50 μg/μL may resist the photosensitive injury of DNA caused by AQS under the deoxidized condition and exposure by 254 nm UV for 10 min. And caffeine in concentration of 5 μg/μL would present a synergetic effect on the photosensitive injury of DNA. Possible molecular mechanism also is discussed. (authors)

  18. Determination of damage and In vivo DNA repairing through the unicellular in gel electrophoresis technique

    International Nuclear Information System (INIS)

    Mendiola C, M.T.; Morales R, P.

    1997-01-01

    The experimental conditions were standardized for the unicellular in gel electrophoresis technique setting up (EUG) at the Cellular Radiobiology laboratory. Preliminary experiments were realized with human cells and mouse which were exposed to ionizing radiation or hydroxide peroxide (H 2 O 2 ) to induce DNA damage and to verify the technique performance. It was analysed the In vivo repairing kinetics of induced damage by gamma radiation in mouse leukocytes which were exposed to 137 Cs source and taking samples of peripheric blood of the tail of each mouse at different exposure times and processing them for EUG. In function of the cells proportion with damage in each time it was determined the existence of fast repairing mechanism at the first 15 minutes followed by a slight increase in the damage and a late repairing stage between 30 and 90 minutes. It was analysed this behavior and the potentiality of this In vivo system. (Author)

  19. DNA agarose gel electrophoresis for antioxidant analysis: Development of a quantitative approach for phenolic extracts.

    Science.gov (United States)

    Silva, Sara; Costa, Eduardo M; Vicente, Sandra; Veiga, Mariana; Calhau, Conceição; Morais, Rui M; Pintado, Manuela E

    2017-10-15

    Most of the fast in vitro assays proposed to determine the antioxidant capacity of a compound/extract lack either biological context or employ complex protocols. Therefore, the present work proposes the improvement of an agarose gel DNA electrophoresis in order to allow for a quantitative estimation of the antioxidant capacity of pure phenolic compounds as well as of a phenolic rich extract, while also considering their possible pro-oxidant effects. The result obtained demonstrated that the proposed method allowed for the evaluation of the protection of DNA oxidation [in the presence of hydrogen peroxide (H 2 O 2 ) and an H 2 O 2 /iron (III) chloride (FeCl 3 ) systems] as well as for the observation of pro-oxidant activities, with the measurements registering interclass correlation coefficients above 0.9. Moreover, this method allowed for the characterization of the antioxidant capacity of a blueberry extract while demonstrating that it had no perceived pro-oxidant effect. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Large abnormal peak on capillary zone electrophoresis due to contrast agent.

    Science.gov (United States)

    Wheeler, Rachel D; Zhang, Liqun; Sheldon, Joanna

    2017-01-01

    Background Some iodinated radio-contrast media absorb ultraviolet light and can therefore be detected by capillary zone electrophoresis. If seen, these peaks are typically small with 'quantifications' of below 5 g/L. Here, we describe the detection of a large peak on capillary zone electrophoresis that was due to the radio-contrast agent, Omnipaque™. Methods Serum from a patient was analysed by capillary zone electrophoresis, and the IgG, IgA, IgM and total protein concentrations were measured. The serum sample was further analysed by gel electrophoresis and immunofixation. Results Capillary zone electrophoresis results for the serum sample showed a large peak with a concentration high enough to warrant urgent investigation. However, careful interpretation alongside the serum immunoglobulin concentrations and total protein concentration showed that the abnormal peak was a pseudoparaprotein rather than a monoclonal immunoglobulin. This was confirmed by analysis with gel electrophoresis and also serum immunofixation. The patient had had a CT angiogram with the radio-contrast agent Omnipaque™; addition of Omnipaque™ to a normal serum sample gave a peak with comparable mobility to the pseudoparaprotein in the patient's serum. Conclusions Pseudoparaproteins can appear as a large band on capillary zone electrophoresis. This case highlights the importance of a laboratory process that detects significant electrophoretic abnormalities promptly and interprets them in the context of the immunoglobulin concentrations. This should avoid incorrect reporting of pseudoparaproteins which could result in the patient having unnecessary investigations.

  1. Molecular Typing of Salmonella Isolates in Poultry by Pulsed-Field Gel Electrophoresis in Iran

    Directory of Open Access Journals (Sweden)

    Narges Golab

    2014-11-01

    Full Text Available Background: Salmonella is one of the most widespread zoonotic enter pathogenic microorganisms found in the global food chain. Poultryand Poultry products have been identified as one of the important foodborne sources of Salmonella. Pulsed-Field Gel Electrophoresis (PFGE is a gold standard typing method for identification of Salmonella isolates during outbreaks and epidemiological investigations. Objectives: The aim of this study was to carry out molecular typing of Salmonella enterica spp. by PFGE technique. Materials and Methods: All 47 Salmonella isolates were serotyped and then subjected to PFGE. Total isolates were analyzed by means of the molecular technique XbaI PFGE. Results: In the current work, PFGE and serotyping were used to subtype 47 Salmonella isolates belonging to 22 different serotypes and derived from poultry. Thirty-nine PFGE patterns out of 47 isolates were obtained. The Discrimination Index (DI by serotyping (0.93 was lower than PFGE (DI = 0.99. Conclusions: In conclusion, molecular methods such as PFGE can be used for epidemiological characterization of Salmonella serotypes.

  2. Fluorographic detection of tritiated glycopeptides and oligosaccharides separated on polyacrylamide gels: analysis of glycans from Dictyostelium discoideum glycoproteins

    International Nuclear Information System (INIS)

    Prem Das, O.; Henderson, E.J.

    1986-01-01

    Previous workers have shown that oligosaccharides and glycopeptides can be separated by electrophoresis in buffers containing borate ions. However, normal fluorography of tritium-labeled structures cannot be performed because the glycans are soluble and can diffuse during equilibration with scintillants. This problem has been circumvented by equilibration of the gel with 2,5-diphenyloxazole (PPO) prior to electrophoresis. The presence of PPO in the gel during electrophoresis does not alter mobility of the glycopeptides and oligosaccharides. After electrophoresis, the gel is simply dried and fluorography performed. This allows sensitive and precise comparisons of labeled samples in parallel lanes of a slab gel and, since mobilities are highly reproducible, between different gels. The procedure is preparative in that after fluorography the gel bands can be quantitatively eluted for further study, without any apparent modification by the procedure. In this report, the procedure is illustrated by fractionation of both neutral and anionic glycopeptides produced by the cellular slime mold Dictyostelium discoideum

  3. Lipovitellin-phosvitin crystals with orthorhombic features: thin-section electron microscopy, gel electrophoresis, and microanalysis in teleost and amphibian yolk platelets and a comparison with other vertebrates

    International Nuclear Information System (INIS)

    Lange, R.H.; Richter, H.P.; Riehl, R.; Zierold, K.; Trandaburu, T.; Magdowski, G.

    1983-01-01

    Yolk-platelet crystals in the teleosts Pelvicachromis pulcher and Noemacheilus barbatulus and the amphibians Xenopus laevis, Rana temporaria, R. esculenta, and Triturus sp. have been studied by electron diffraction and imaging using a standardized processing (glutaraldehyde-osmium tetroxide fixation, glutaraldehyde-urea embedding, thin-section staining), by X-ray microanalysis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of their constituents. The crystal lattice is orthorhombic having. Crystal images in the three axial projections point to the presence of space group P212121 (or an approximation of it since the lipovitellin dimers cannot be fully equivalent in some cases), to differences between the phosvitins of the two teleosts, and to a highly unusual stain exclusion from large crystal constituents interpreted as representing lipovitellin dimers. Microanalysis in ultrathin cryosections and other preparations revealed K and Cl to be the prominent ions in the crystals (and in the superficial layer of the platelet). Gel electrophoresis (including data of cyclostomes) showed considerable molecular variations despite a closely similar crystal architecture, emphasizing a physiological significance of the architecture, which may have remained conserved for nearly 400 million years according to paleontologic views

  4. DNA migration mechanism analyses for applications in capillary and microchip electrophoresis

    Science.gov (United States)

    Forster, Ryan E.; Hert, Daniel G.; Chiesl, Thomas N.; Fredlake, Christopher P.; Barron, Annelise E.

    2009-01-01

    In 2009, electrophoretically driven DNA separations in slab gels and capillaries have the sepia tones of an old-fashioned technology in the eyes of many, even while they remain ubiquitously used, fill a unique niche, and arguably have yet to reach their full potential. For comic relief, what is old becomes new again: agarose slab gel separations are used to prepare DNA samples for “next-gen” sequencing platforms (e.g., the Illumina and 454 machines)—dsDNA molecules within a certain size range are “cut out” of a gel and recovered for subsequent “massively parallel” pyrosequencing. In this review, we give a Barron lab perspective on how our comprehension of DNA migration mechanisms in electrophoresis has evolved, since the first reports of DNA separations by CE (∼1989) until now, 20 years later. Fused silica capillaries, and borosilicate glass and plastic microchips, quietly offer increasing capacities for fast (and even “ultra-fast”), efficient DNA separations. While the channel-by-channel scaling of both old and new electrophoresis platforms provides key flexibility, it requires each unique DNA sample to be prepared in its own micro- or nanovolume. This Achille's heel of electrophoresis technologies left an opening through which pooled-sample, next-gen DNA sequencing technologies rushed. We shall see, over time, whether sharpening understanding of transitions in DNA migration modes in crosslinked gels, nanogel solutions, and uncrosslinked polymer solutions will allow electrophoretic DNA analysis technologies to flower again. Microchannel electrophoresis, after a quiet period of metamorphosis, may emerge sleeker and more powerful, to claim its own important niche applications. PMID:19582705

  5. Amino Acid Composition, Molecular Weight Distribution and Gel Electrophoresis of Walnut (Juglans regia L. Proteins and Protein Fractionations

    Directory of Open Access Journals (Sweden)

    Xiaoying Mao

    2014-01-01

    Full Text Available As a by-product of oil production, walnut proteins are considered as an additional source of plant protein for human food. To make full use of the protein resource, a comprehensive understanding of composition and characteristics of walnut proteins are required. Walnut proteins have been fractionated and characterized in this study. Amino acid composition, molecular weight distribution and gel electrophoresis of walnut proteins and protein fractionations were analyzed. The proteins were sequentially separated into four fractions according to their solubility. Glutelin was the main component of the protein extract. The content of glutelin, albumin, globulin and prolamin was about 72.06%, 7.54%, 15.67% and 4.73% respectively. Glutelin, albumin and globulin have a balanced content of essential amino acids, except for methionine, with respect to the FAO pattern recommended for adults. SDS-PAGE patterns of albumin, globulin and glutelin showed several polypeptides with molecular weights 14.4 to 66.2 kDa. The pattern of walnut proteins in two-dimension electrophoresis (2-DE showed that the isoelectric point was mainly in the range of 4.8–6.8. The results of size exclusion chromatogram indicated molecular weight of the major components of walnut proteins were between 3.54 and 81.76 kDa.

  6. Tracing outbreaks of Streptococcus equi infection (strangles) in horses using sequence variation in the seM gene and pulsed-field gel electrophoresis.

    Science.gov (United States)

    Lindahl, Susanne; Söderlund, Robert; Frosth, Sara; Pringle, John; Båverud, Viveca; Aspán, Anna

    2011-11-21

    Strangles is a serious respiratory disease in horses caused by Streptococcus equi subspecies equi (S. equi). Transmission of the disease occurs by direct contact with an infected horse or contaminated equipment. Genetically, S. equi strains are highly homogenous and differentiation of strains has proven difficult. However, the S. equi M-protein SeM contains a variable N-terminal region and has been proposed as a target gene to distinguish between different strains of S. equi and determine the source of an outbreak. In this study, strains of S. equi (n=60) from 32 strangles outbreaks in Sweden during 1998-2003 and 2008-2009 were genetically characterized by sequencing the SeM protein gene (seM), and by pulsed-field gel electrophoresis (PFGE). Swedish strains belonged to 10 different seM types, of which five have not previously been described. Most were identical or highly similar to allele types from strangles outbreaks in the UK. Outbreaks in 2008/2009 sharing the same seM type were associated by geographic location and/or type of usage of the horses (racing stables). Sequencing of the seM gene generally agreed with pulsed-field gel electrophoresis profiles. Our data suggest that seM sequencing as a epidemiological tool is supported by the agreement between seM and PFGE and that sequencing of the SeM protein gene is more sensitive than PFGE in discriminating strains of S. equi. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. Two distinct clones of methicillin-resistant Staphylococcus aureus (MRSA) with the same USA300 pulsed-field gel electrophoresis profile: a potential pitfall for identification of USA300 community-associated MRSA

    DEFF Research Database (Denmark)

    Larsen, Anders Rhod; Goering, Richard; Stegger, Marc

    2009-01-01

    Analysis of methicillin-resistant Staphylococcus aureus (MRSA) characterized as USA300 by pulsed-field gel electrophoresis identified two distinct clones. One was similar to community-associated USA300 MRSA (ST8-IVa, t008, and Panton-Valentine leukocidin positive). The second (ST8-IVa, t024...

  8. Conformational studies of the (+)-trans, (-)-trans, (+)-cis, and (-)-cis adducts of anti-benzo[a]pyrene diolepoxide to N2-dG in duplex oligonucleotides using polyacrylamide gel electrophoresis and low-temperature fluorescence spectroscopy

    NARCIS (Netherlands)

    Suh, Myungkoo; Ariese, Freek; Small, Gerald J.; Jankowiak, Ryszard; Liu, Tong Ming; Geacintov, Nicholas E.

    1995-01-01

    Using polyacrylamide gel electrophoresis (PAGE) and low-temperature, laser-induced fluorescence line narrowing (FLN) and non-line narrowing (NLN) spectroscopic methods, the conformational characteristics of stereochemically defined and site-specific adducts derived from the binding of

  9. A comparative analysis of phloem exudate proteins from Cucumis melo, Cucumis sativus and Cucurbita maxima by polyacrylamide gel electrophoresis and isoelectric focusing.

    Science.gov (United States)

    Sabnis, D D; Hart, J W

    1976-01-01

    Proteins in sieve tube exudate from Cucumis melo L., Cucumis sativus L. and Cucurbita maxima Duch. were analysed by gel electrophoresis and isoelectric focusing. Estimated molecular weights and isoelectric points for the major and minor proteins from each plant species are presented. Electrophoresis revealed striking differences between the protein complements of exudatc from the two genera investigated. Similarly, although a few exudate proteins from the two species of Cucumis possessed identical molecular weights, several major proteins were peculiar to each species. Isoelectric focusing of proteins in exudate samples from the three plants confirmed the marked differences in their protein complements. Furthermore, focusing also revealed differences between cultivars of Cucumis sativus. Both Cucumis sativus and Cucurbita maxima possessed relatively large amounts of basic proteins; these were absent in exudate from Cucumis melo. The implications of these results are discussed in relation to present concepts regarding the interrelationships and possible functional roles of P-proteins.

  10. Characterization of organic matrix extracted from fresh water pearls

    International Nuclear Information System (INIS)

    Ma Yufei; Gao Yonghua; Feng Qingling

    2011-01-01

    Aragonite pearl and vaterite pearl from cultured Hyriopsis cumingii in Zhuji (Zhejiang province, China) were chosen for the study. The matrix proteins were extracted using water and weak acid, and classified as water soluble matrix (WSM), acid soluble matrix (ASM) and acid insoluble matrix (AIM). The proteins from both pearls were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), X-ray diffraction (XRD) and Fourier transformation infrared spectra (FTIR). The results showed that, AIM of aragonite pearl and vaterite pearl had an ordered structure of α-helix. ASM conformations of these two pearls were different from each other. WSM differed the most between these two pearls. - Research Highlights: → We use a specific method for extracting matrix proteins from aragonite pearl and vaterite pearl respectively. → The matrix proteins are extracted by water and weak acid, and classified as water soluble matrix (WSM), acid soluble matrix (ASM) and acid insoluble matrix (AIM). → AIM of aragonite pearl and vaterite pearl have an ordered structure. ASM conformations of the two pearls are different from each other. WSM differ the most between these two pearls.

  11. Gel structure has an impact on pericellular and extracellular matrix deposition, which subsequently alters metabolic activities in chondrocyte-laden PEG hydrogels.

    Science.gov (United States)

    Nicodemus, G D; Skaalure, S C; Bryant, S J

    2011-02-01

    While designing poly(ethylene glycol) hydrogels with high moduli suitable for in situ placement is attractive for cartilage regeneration, the impact of a tighter crosslinked structure on the organization and deposition of the matrix is not fully understood. The objectives of this study were to characterize the composition and spatial organization of new matrix as a function of gel crosslinking and study its impact on chondrocytes in terms of anabolic and catabolic gene expression and catabolic activity. Bovine articular chondrocytes were encapsulated in hydrogels with three crosslinking densities (compressive moduli 60, 320 and 590 kPa) and cultured for 25 days. Glycosaminoglycan production increased with culture time and was greatest in the gels with lowest crosslinking. Collagens II and VI, aggrecan, link protein and decorin were localized to pericellular regions in all gels, but their presence decreased with increasing gel crosslinking. Collagen II and aggrecan expression were initially up-regulated in gels with higher crosslinking, but increased similarly up to day 15. Matrix metalloproteinase (MMP)-1 and MMP-13 expression were elevated (∼25-fold) in gels with higher crosslinking throughout the study, while MMP-3 was unaffected by gel crosslinking. The presence of aggrecan and collagen degradation products confirmed MMP activity. These findings indicate that chondrocytes synthesized the major cartilage components within PEG hydrogels, however, gel structure had a significant impact on the composition and spatial organization of the new tissue and on how chondrocytes responded to their environment, particularly with respect to their catabolic expression. Copyright © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  12. Attempt to run urinary protein electrophoresis using capillary technique.

    Science.gov (United States)

    Falcone, Michele

    2014-10-01

    The study of urinary protein has a predominant place in the diagnosis of kidney disease. The most common technique is agarose gel electrophoresis (AGE). For several years, the technique of choice applied to the analysis of serum proteins has been CE, a system that uses capillary fused silica, subjected to high voltage to separate and measure serum proteins. The purpose of this paper was to perform capillary electrophoresis on urinary proteins which, at present, are not interpretable due to the many nonspecific peaks visible when using gel electrophoresis. In order to carry out our research, we used a capillary V8 analyzer together with an agarose gel system from the same company. AGE was taken as the reference method, for which urine was used without any pretreatment. For the V8 system, urine was subjected to purification on granular-activated carbon and then inserted into the V8 analyzer, selecting a program suitable for liquids with low protein content. We examined 19 urine samples collected over 24 hrs from both hospitalized and external patients with different types of proteinuria plus a serum diluted 1/61 considered as a control to recognize the bands. Both methods showed the same protein fractions and classified the proteinuria in a similar way. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Effects of Storage and Granary Weevil Infestation on Gel Electrophoresis and Protein Solubility Properties of Hard and Soft Wheat Flours.

    Science.gov (United States)

    Keskin, Sule; Yalçin, Erkan; Özkaya, Hazim

    2018-02-24

    The objective of this study was to investigate the effects of storage and granary weevil, Sitophilus granarius (L.; Coleoptera: Curculionidae), infestation on pH, protein solubility (PS) and gel electrophoresis properties of meal and roller-milled flours of hard (Ceyhan-99 cv.) and soft (Eser cv.) wheat cultivars, respectively, after 6 mo of storage. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) technique was applied for studying the electrophoretic properties. Hard and soft wheats were infested with non-sexed S. granarius at a rate of two adults/ kg, and stored for 6 mo at 30 ± 1°C and 70 ± 5% RH. The pest-free wheat samples were used as control. The infested and its control samples were collected monthly, and after cleaning the granary weevils, they were hammer-milled or roller-milled in order to get meal flours and roller-milled flours, respectively. The effect of infestation on the storage proteins was more obvious in meal flours than that of the roller-milled flours. Granary weevil feeding resulted secreting of hydrolyzing enzymes and increased the acidity of flours; subsequently the breaking and releasing of some storage proteins generally caused a decrease in pH and an increase in PS values of the meal flours of wheat cultivars. SDS-PAGE results generally indicated that towards the end of storage, the insect population, that greatly increased, caused minor protein depletions resulting decreasing protein band intensities between 113 and 58 kDa of hard wheat meal flour and 101 and 40 kDa of soft wheat roller-milled flour. Consequently, the potential effect of changes probably occurred in high molecular weight glutenin subunits of both wheat cultivars.

  14. Culture-based and denaturing gradient gel electrophoresis analysis of the bacterial community from Chungkookjang, a traditional Korean fermented soybean food.

    Science.gov (United States)

    Hong, Sung Wook; Choi, Jae Young; Chung, Kun Sub

    2012-10-01

    The bacterial community of Chungkookjang and raw rice-straw collected from various areas in South Korea was investigated using both culture-dependent and culture-independent methods. Pure cultures were isolated from Chungkookjang and raw rice-straw on tryptic soy agar plates with 72 to 121 colonies and identified by 16S rDNA gene sequence analysis, respectively. The traditional culture-based method and denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rDNA confirmed that Pantoea agglomerans and B. subtilis were identified as predominant in the raw rice-straw and Chungkookjang, respectively, from Iljuk district of Gyeonggi province, P. ananatis and B. licheniformis were identified as predominant in the raw rice-straw and Chungkookjang from Wonju district of Gangwon province, and Microbacterium sp. and B. licheniformis were identified as predominant in the raw rice-straw and Chungkookjang from Sunchang district of Jeolla province. Other strains, such as Bacillus, Enterococcus, Pseudomonas, Rhodococcus, and uncultured bacteria were also present in raw rice-straw and Chungkookjang. A comprehensive analysis of these microorganisms would provide a more detailed understanding of the biologically active components of Chungkookjang and help improve its quality. Polymerase chain reaction-denaturing gradient gel electrophoresis analysis can be successfully applied to a fermented food to detect unculturable or more species than the culture-dependent method. This technique is an effective and convenient culture-independent method for studying the bacterial community in Chungkookjang. In this study, the bacterial community of Chungkookjang collected from various areas in South Korea was investigated using both culture-dependent and culture-independent methods. © 2012 Institute of Food Technologists®

  15. Identification of Increased Amounts of Eppin Protein Complex Components in Sperm Cells of Diabetic and Obese Individuals by Difference Gel Electrophoresis*

    Science.gov (United States)

    Paasch, Uwe; Heidenreich, Falk; Pursche, Theresia; Kuhlisch, Eberhard; Kettner, Karina; Grunewald, Sonja; Kratzsch, Jürgen; Dittmar, Gunnar; Glander, Hans-Jürgen; Hoflack, Bernard; Kriegel, Thomas M.

    2011-01-01

    Metabolic disorders like diabetes mellitus and obesity may compromise the fertility of men and women. To unveil disease-associated proteomic changes potentially affecting male fertility, the proteomes of sperm cells from type-1 diabetic, type-2 diabetic, non-diabetic obese and clinically healthy individuals were comparatively analyzed by difference gel electrophoresis. The adaptation of a general protein extraction procedure to the solubilization of proteins from sperm cells allowed for the resolution of 3187 fluorescent spots in the difference gel electrophoresis image of the master gel, which contained the entirety of solubilized sperm proteins. Comparison of the pathological and reference proteomes by applying an average abundance ratio setting of 1.6 and a p ≤ 0.05 criterion resulted in the identification of 79 fluorescent spots containing proteins that were present at significantly changed levels in the sperm cells. Biometric evaluation of the fluorescence data followed by mass spectrometric protein identification revealed altered levels of 12, 71, and 13 protein species in the proteomes of the type-1 diabetic, type-2 diabetic, and non-diabetic obese patients, respectively, with considerably enhanced amounts of the same set of one molecular form of semenogelin-1, one form of clusterin, and two forms of lactotransferrin in each group of pathologic samples. Remarkably, β-galactosidase-1-like protein was the only protein that was detected at decreased levels in all three pathologic situations. The former three proteins are part of the eppin (epididymal proteinase inhibitor) protein complex, which is thought to fulfill fertilization-related functions, such as ejaculate sperm protection, motility regulation and gain of competence for acrosome reaction, whereas the putative role of the latter protein to function as a glycosyl hydrolase during sperm maturation remains to be explored at the protein/enzyme level. The strikingly similar differences detected in the

  16. Disparity between Multilocus Enzyme Electrophoresis, Microsatellite Markers and Pulsed-Field Gel Electrophoresis in epidemiological tracking of Candida albicans.

    Science.gov (United States)

    Boriollo, Marcelo Fabiano Gomes; Dias, Ricardo Antunes; Fiorini, João Evangelista; Oliveira, Nelma de Mello Silva; Spolidório, Denise Madalena Palomari; de Souza, Henrique Marques Barbosa; Figueira, Antonio Vargas de Oliveira; Pizzirani-Kleiner, Aline Aparecida

    2010-09-01

    Various molecular systems are available for epidemiological, genetic, evolutionary, taxonomic and systematic studies of innumerable fungal infections, especially those caused by the opportunistic pathogen C. albicans. A total of 75 independent oral isolates were selected in order to compare Multilocus Enzyme Electrophoresis (MLEE), Electrophoretic Karyotyping (EK) and Microsatellite Markers (Simple Sequence Repeats - SSRs), in their abilities to differentiate and group C. albicans isolates (discriminatory power), and also, to evaluate the concordance and similarity of the groups of strains determined by cluster analysis for each fingerprinting method. Isoenzyme typing was performed using eleven enzyme systems: Adh, Sdh, M1p, Mdh, Idh, Gdh, G6pdh, Asd, Cat, Po, and Lap (data previously published). The EK method consisted of chromosomal DNA separation by pulsed-field gel electrophoresis using a CHEF system. The microsatellite markers were investigated by PCR using three polymorphic loci: EF3, CDC3, and HIS3. Dendrograms were generated by the SAHN method and UPGMA algorithm based on similarity matrices (S(SM)). The discriminatory power of the three methods was over 95%, however a paired analysis among them showed a parity of 19.7-22.4% in the identification of strains. Weak correlation was also observed among the genetic similarity matrices (S(SM)(MLEE)xS(SM)(EK)xS(SM)(SSRs)). Clustering analyses showed a mean of 9+/-12.4 isolates per cluster (3.8+/-8 isolates/taxon) for MLEE, 6.2+/-4.9 isolates per cluster (4+/-4.5 isolates/taxon) for SSRs, and 4.1+/-2.3 isolates per cluster (2.6+/-2.3 isolates/taxon) for EK. A total of 45 (13%), 39 (11.2%), 5 (1.4%) and 3 (0.9%) clusters pairs from 347 showed similarity (S(J)) of 0.1-10%, 10.1-20%, 20.1-30% and 30.1-40%, respectively. Clinical and molecular epidemiological correlation involving the opportunistic pathogen C. albicans may be attributed dependently of each method of genotyping (i.e., MLEE, EK, and SSRs) supplemented

  17. The Make 2D-DB II package: conversion of federated two-dimensional gel electrophoresis databases into a relational format and interconnection of distributed databases.

    Science.gov (United States)

    Mostaguir, Khaled; Hoogland, Christine; Binz, Pierre-Alain; Appel, Ron D

    2003-08-01

    The Make 2D-DB tool has been previously developed to help build federated two-dimensional gel electrophoresis (2-DE) databases on one's own web site. The purpose of our work is to extend the strength of the first package and to build a more efficient environment. Such an environment should be able to fulfill the different needs and requirements arising from both the growing use of 2-DE techniques and the increasing amount of distributed experimental data.

  18. Automated image alignment for 2D gel electrophoresis in a high-throughput proteomics pipeline.

    Science.gov (United States)

    Dowsey, Andrew W; Dunn, Michael J; Yang, Guang-Zhong

    2008-04-01

    The quest for high-throughput proteomics has revealed a number of challenges in recent years. Whilst substantial improvements in automated protein separation with liquid chromatography and mass spectrometry (LC/MS), aka 'shotgun' proteomics, have been achieved, large-scale open initiatives such as the Human Proteome Organization (HUPO) Brain Proteome Project have shown that maximal proteome coverage is only possible when LC/MS is complemented by 2D gel electrophoresis (2-DE) studies. Moreover, both separation methods require automated alignment and differential analysis to relieve the bioinformatics bottleneck and so make high-throughput protein biomarker discovery a reality. The purpose of this article is to describe a fully automatic image alignment framework for the integration of 2-DE into a high-throughput differential expression proteomics pipeline. The proposed method is based on robust automated image normalization (RAIN) to circumvent the drawbacks of traditional approaches. These use symbolic representation at the very early stages of the analysis, which introduces persistent errors due to inaccuracies in modelling and alignment. In RAIN, a third-order volume-invariant B-spline model is incorporated into a multi-resolution schema to correct for geometric and expression inhomogeneity at multiple scales. The normalized images can then be compared directly in the image domain for quantitative differential analysis. Through evaluation against an existing state-of-the-art method on real and synthetically warped 2D gels, the proposed analysis framework demonstrates substantial improvements in matching accuracy and differential sensitivity. High-throughput analysis is established through an accelerated GPGPU (general purpose computation on graphics cards) implementation. Supplementary material, software and images used in the validation are available at http://www.proteomegrid.org/rain/.

  19. Two Dimensional Electrophoresis of Proteins from Cultures of Erysiphe graminis f.sp. hordei

    DEFF Research Database (Denmark)

    Torp, J.; Andersen, Brian

    1982-01-01

    Conidial proteins from barley powdery mildew, Erysiphe graminis f. sp. hordei, were separated by 2-dimensional electrophoresis in polyacrylamide slab gels. Isoelectric focusing was used in the first dimension and separation according to molecular weight in a gel containing sodium dodecyl sulphate...

  20. Characterization of biological macromolecules by electrophoresis and neutron activation

    International Nuclear Information System (INIS)

    Stone, S.F.; Hancock, D.; Zeisler, R.

    1987-01-01

    A procedure combining polyacrylamide gel electrophoresis (PAGE) with INAA and autoradiography was developed to study biological macromolecules and their associated trace elements. Results from the application of this method to several metalloproteins are presented. (author)

  1. The application of single cell gel electrophoresis or comet assay to human monitoring studies Aplicacion de la electroforesis unicelular o ensayo cometa en estudios de monitoreo humano

    Directory of Open Access Journals (Sweden)

    Mahara Valverde

    1999-11-01

    Full Text Available Objective. In the search of new human genotoxic biomarkers, the single cell gel electrophoresis assay has been proposed as a sensible alternative. Material and methods. This technique detects principally single strand breaks as well as alkali-labile and repair-retarded sites. Results. Herein we present our experience using the single cell gel electrophoresis assay in human population studies, both occupationally and environmentally exposed. Conclusions. We discuss the assay feasibility as a genotoxic biomarker.Objetivo. En la búsqueda de nuevos marcadores genotóxicos aplicables a estudios de poblaciones humanas expuestos a xenobióticos, la utilización del ensayo de electroforesis en una sola célula se ha propuesto como un método sensible y una buena alternativa. Material y métodos. Esta técnica detecta rompimientos en el ADN de cadena sencilla, así como sitios álcali lábiles y sitios retardados de reparación. Resultados. En este trabajo, presentamos nuestra experiencia utilizando este ensayo en poblaciones humanas expuestas ocupacionalmente o ambientalmente a diferentes xenobióticos. Conclusiones. Se discute la posible utilidad de este ensayo como un biomarcador de efecto genotóxico.

  2. Changes in protein abundance between tender and tough meat from bovine Longissimus thoracis muscle assessed by isobaric Tag for Relative and Absolute Quantitation (iTRAQ) and 2-dimensional gel electrophoresis analysis

    DEFF Research Database (Denmark)

    Bjarnadóttir, S G; Hollung, K; Høy, M

    2012-01-01

    The aim of this study was to find potential biomarkers for meat tenderness in bovine Longissimus thoracis muscle and to compare results from isobaric Tag for Relative and Absolute Quantitation (iTRAQ) and 2-dimensional gel electrophoresis (2-DE) analysis. The experiment included 4 tender and 4...

  3. Study of ionization process of matrix molecules in matrix-assisted laser desorption ionization

    Energy Technology Data Exchange (ETDEWEB)

    Murakami, Kazumasa; Sato, Asami; Hashimoto, Kenro; Fujino, Tatsuya, E-mail: fujino@tmu.ac.jp

    2013-06-20

    Highlights: ► Proton transfer and adduction reaction of matrix in MALDI were studied. ► Hydroxyl group forming intramolecular hydrogen bond was related to the ionization. ► Intramolecular proton transfer in the electronic excited state was the initial step. ► Non-volatile analytes stabilized protonated matrix in the ground state. ► A possible mechanism, “analyte support mechanism”, has been proposed. - Abstract: Proton transfer and adduction reaction of matrix molecules in matrix-assisted laser desorption ionization were studied. By using 2,4,6-trihydroxyacetophenone (THAP), 2,5-dihydroxybenzoic acid (DHBA), and their related compounds in which the position of a hydroxyl group is different, it was clarified that a hydroxyl group forming an intramolecular hydrogen bond is related to the ionization of matrix molecules. Intramolecular proton transfer in the electronic excited state of the matrix and subsequent proton adduction from a surrounding solvent to the charge-separated matrix are the initial steps for the ionization of matrix molecules. Nanosecond pump–probe NIR–UV mass spectrometry confirmed that the existence of analyte molecules having large dipole moment in their structures is necessary for the stabilization of [matrix + H]{sup +} in the electronic ground state.

  4. Two Dimensional Electrophoresis of Galactosidase Relating to the Disappearance of Bombyx Lectin Activity

    OpenAIRE

    カトウ, ヤスオ; Yasuo, Kato

    2004-01-01

    "Two dimensional polyacrylamide gel electroporesis (2 D-PAGE) analysis on the haemolymph of Bombyx mori was performed using the Mini-PROTEAN mini tube gel two dimensional polyacrylamide gel electrophoresis system (Bio-Rad Laboratories, Inc.). The result on various electrophoretical conditions using the haemolymph-protein showed the possibility that the haemolymph-protein was separated actually by means of this method. Moreover, the result of 2 D-PAGE analysis on Fraction II obtained by gel fi...

  5. Synaptic vesicle proteins under conditions of rest and activation: analysis by 2-D difference gel electrophoresis.

    Science.gov (United States)

    Burré, Jacqueline; Beckhaus, Tobias; Corvey, Carsten; Karas, Michael; Zimmermann, Herbert; Volknandt, Walter

    2006-09-01

    Synaptic vesicles are organelles of the nerve terminal that secrete neurotransmitters by fusion with the presynaptic plasma membrane. Vesicle fusion is tightly controlled by depolarization of the plasma membrane and a set of proteins that may undergo post-translational modifications such as phosphorylation. In order to identify proteins that undergo modifications as a result of synaptic activation, we induced massive exocytosis and analysed the synaptic vesicle compartment by benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE and difference gel electrophoresis (DIGE) followed by MALDI-TOF-MS. We identified eight proteins that revealed significant changes in abundance following nerve terminal depolarization. Of these, six were increased and two were decreased in abundance. Three of these proteins were phosphorylated as detected by Western blot analysis. In addition, we identified an unknown synaptic vesicle protein whose abundance increased on synaptic activation. Our results demonstrate that depolarization of the presynaptic compartment induces changes in the abundance of synaptic vesicle proteins and post-translational protein modification.

  6. Development of two dimensional electrophoresis method using single chain DNA

    International Nuclear Information System (INIS)

    Ikeda, Junichi; Hidaka, So

    1998-01-01

    By combining a separation method due to molecular weight and a method to distinguish difference of mono-bases, it was aimed to develop a two dimensional single chain DNA labeled with Radioisotope (RI). From electrophoretic pattern difference of parent and variant strands, it was investigated to isolate the root module implantation control gene. At first, a Single Strand Conformation Polymorphism (SSCP) method using concentration gradient gel was investigated. As a result, it was formed that intervals between double chain and single chain DNAs expanded, but intervals of both single chain DNAs did not expand. On next, combination of non-modified acrylic amide electrophoresis method and Denaturing Gradient-Gel Electrophoresis (DGGE) method was examined. As a result, hybrid DNA developed by two dimensional electrophoresis arranged on two lines. But, among them a band of DNA modified by high concentration of urea could not be found. Therefore, in this fiscal year's experiments, no preferable result could be obtained. By the used method, it was thought to be impossible to detect the differences. (G.K.)

  7. Synthesis of gels with basis of titanium tungstates as matrixes of radioactive generators

    International Nuclear Information System (INIS)

    Galico C, L.

    2005-01-01

    The heteropolyanions, compounds formed by the union of molybdates or tungstates polyanions with atoms of metals like zirconium, titanium, cerium, thorium, tin, etc., have been used as generator matrixes of 99m Tc or 188 Re. Particularly they have been studied and produced successfully in our laboratory, generators of 99 Mo/ 99 m Tc at basis of gels zirconium molybdates and titanium molybdates. Considering that the molybdenum and tungsten, as well as the technetium and the rhenium, its belong to the same groups of transition metals, it is feasible that gels can be synthesized at basis of titanium tungstates, continuing a methodology similar to that of the gels titanium molybdates or zirconium molybdates, to produce generators 188 W/ 188 Re. The 188 Re possess nuclear characteristics that make it attractive for therapeutic applications, since, it emits β - particles of a great energy (2.12 MeV); joined to the possibility of being able to unite to different ligands (bifunctional agents) and biomolecules (antibodies or fragments of proteins), as it makes the 99m Tc, useful in radioimmunotherapy. Commercially the 188 Re generators use a chromatographic column loaded with alumina where the 188 Re, it is adsorbed and eluted the 188 ReO 4 - by means of a saline solution The alumina adsorbs around 0.2% of the 188 Re, situation that forces to use 188 Re of a high specific activity. The use of the gels technology, allows to work with medium or low specific activities of 188 Re, opening the possibility of their production in countries whose nuclear capacity is medium or low. In particular, the synthesized gels with basis of titanium offer the possibility of being synthesized with non active material, for later on to be irradiated and directly produce the generator, since, the titanium 51 Ti, unique radioisotope produced by the titanium, has a half life of 5.79 min. This synthesis method avoids the manipulation of radioactive material during the synthesis of the gels, process

  8. Analysis of electrophoresis performance

    Science.gov (United States)

    Roberts, G. O.

    1984-01-01

    The SAMPLE computer code models electrophoresis separation in a wide range of conditions. Results are included for steady three dimensional continuous flow electrophoresis (CFE), time dependent gel and acetate film experiments in one or two dimensions and isoelectric focusing in one dimension. The code evolves N two dimensional radical concentration distributions in time, or distance down a CFE chamber. For each time or distance increment, there are six stages, successively obtaining the pH distribution, the corresponding degrees of ionization for each radical, the conductivity, the electric field and current distribution, and the flux components in each direction for each separate radical. The final stage is to update the radical concentrations. The model formulation for ion motion in an electric field ignores activity effects, and is valid only for low concentrations; for larger concentrations the conductivity is, therefore, also invalid.

  9. Molecular Analysis of Mycobacterium avium Isolates by Using Pulsed-Field Gel Electrophoresis and PCR

    Science.gov (United States)

    Pestel-Caron, Martine; Graff, Gabriel; Berthelot, Gilles; Pons, Jean-Louis; Lemeland, Jean-François

    1999-01-01

    Genetic relationships among 46 isolates of Mycobacterium avium recovered from 37 patients in a 2,500-bed hospital from 1993 to 1998 were assessed by pulsed-field gel electrophoresis (PFGE) and PCR amplification of genomic sequences located between the repetitive elements IS1245 and IS1311. Each technique enabled the identification of 27 to 32 different patterns among the 46 isolates, confirming that the genetic heterogeneity of M. avium strains is high in a given community. Furthermore, this retrospective analysis of sporadic isolates allowed us (i) to suggest the existence of two remanent strains in our region, (ii) to raise the question of the possibility of nosocomial acquisition of M. avium strains, and (iii) to document laboratory contamination. The methods applied in the present study were found to be useful for the typing of M. avium isolates. In general, both methods yielded similar results for both related and unrelated isolates. However, the isolates in five of the six PCR clusters were distributed among two to three PFGE patterns, suggesting that this PCR-based method may have limitations for the analysis of strains with low insertion sequence copy numbers or for resolution of extended epidemiologic relationships. PMID:10405383

  10. Imaging of Selenium by Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) in 2-D Electrophoresis Gels and Biological Tissues.

    Science.gov (United States)

    Cruz, Elisa Castañeda Santa; Susanne Becker, J; Sabine Becker, J; Sussulini, Alessandra

    2018-01-01

    Selenium and selenoproteins are important components of living organisms that play a role in different biological processes. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is a powerful analytical technique that has been employed to obtain distribution maps of selenium in biological tissues in a direct manner, as well as in selenoproteins, previously separated by their molecular masses and isoelectric points using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). In this chapter, we present the protocols to perform LA-ICP-MS imaging experiments, allowing the distribution visualization and determination of selenium and/or selenoproteins in biological systems.

  11. Proteomic changes in Debaryomyces hansenii upon exposure to NaCl stress

    DEFF Research Database (Denmark)

    Gori, Klaus; Hébraud, Michel; Chambon, Christophe

    2007-01-01

    The proteome of the highly NaCl-tolerant yeast Debaryomyces hansenii was investigated by two-dimensional polyacrylamide gel electrophoresis (2D PAGE), and 47 protein spots were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) followed by mass spectrometry (MS...... 7% and 4% of the rate in medium without NaCl. In addition, the number of protein spots detected on 2D gels prepared from cells exposed to 8% and 12% (w/v) NaCl exceeded less than 28% of the number of protein spots detected on 2D gels prepared from cells without added NaCl. Several proteins were...

  12. Spectroscopic and electrochemical characterization of cytochrome c encapsulated in a bio sol-gel matrix.

    Science.gov (United States)

    Deriu, Daniela; Pagnotta, Sara Emanuela; Santucci, Roberto; Rosato, Nicola

    2008-08-01

    Sol-gel technique represents a remarkably versatile method for protein encapsulation. To enhance sol-gel biocompatibility, systems envisaging the presence of calcium and phosphates in the sol-gel composition were recently prepared and investigated. Unfortunately, the low pH at which solutions were prepared (pH proteins, because the acidic environment induces protein denaturation. In this paper we apply a new protocol based on the introduction of calcium nitrate to the inorganic phase, with formation of a binary bioactive system. In this case protein encapsulation results versatile and secure, being achieved at a pH close to neutrality (pH 6.0); also, the presence of calcium is expected to enhance system biocompatibility. To determine the properties of the salt-doped sol-gel and the influence exerted on entrapped biosystems, the structural and functional properties of embedded cytochrome c have been investigated. Data obtained indicate that the salt-doped sol-gel induces no significant change in the structure and the redox properties of the embedded protein; also, the matrix increases protein stability. Interestingly, the presence of calcium nitrate appears determinant for refolding of the acid-denatured protein. This is of interest in the perspective of future applications in biosensoristic area.

  13. Initial proteome analysis of caffeine-induced proteins in Aspergillus tamarii using two-dimensional fluorescence difference gel electrophoresis.

    Science.gov (United States)

    Gutiérrez-Sánchez, Gerardo; Atwood, James; Kolli, V S Kumar; Roussos, Sévastianos; Augur, Christopher

    2012-04-01

    Caffeine is toxic to most microorganisms. However, some filamentous fungi, such as Aspergillus tamarii, are able to metabolize this alkaloid when fed caffeine as the sole nitrogen source. The aim of the present work was to identify intracellular A. tamarii proteins, regulated by caffeine, using fluorescence difference two-dimensional gel electrophoresis. Specific proteins from two culture media of A. tamarii grown either on ammonium sulfate or caffeine as the sole nitrogen source were analysed by mass spectrometry. Thirteen out of a total of 85 differentially expressed spots were identified after database search. Identified up-regulated proteins include phosphoglycerate kinase, malate dehydrogenase, dyp-type peroxidase family protein, heat shock protein, Cu, Zn superoxidase dismutase and xanthine dehydrogenase. Some of the proteins identified in this study are involved in the caffeine degradation pathway as well as in stress response, suggesting that stress proteins could be involved in caffeine metabolism in filamentous fungi.

  14. Quantification of DNA damage by single-cell electrophoresis

    International Nuclear Information System (INIS)

    Ikushima, Takaji

    1990-01-01

    A simple technique of micro-agarose gel electrophoresis has been developed to quantify DNA damage in individual cells. Cells are embedded in agarose gel on microscope slides, lysed by detergents and then electrophoresed for a short time under neutral or alkaline condition. In irradiated cells, DNA migrates from the nucleus toward the anode, displaying commet-like pattern by staining with DNA-specific fluorescence dye. DNA damage is evaluated by measuring the distance of DNA migration. The technique was applied for measuring DNA damage in single cells exposed to 60 Co γ-rays, or to KUR radiation in the presence or absence of 10 B-enriched boric acid. The enhanced production of double-stranded DNA breaks by 10 B(n,α) 7 Li reaction was demonstrated here. The significant increase in the length of DNA migration was observed in single cells exposed to such a low dose as 20 cGy after alkaline micro electrophoresis. (author)

  15. Unfolding of hemoglobin variants--insights from urea gradient gel electrophoresis photon correlation spectroscopy and zeta potential measurements

    International Nuclear Information System (INIS)

    Bhattacharya, Jaydeep; GhoshMoulick, Ranjita; Choudhuri, Utpal; Chakrabarty, Prantar; Bhattacharya, Pranab K.; Lahiri, Prabir; Chakraborti, Bikas; Dasgupta, Anjan Kr.

    2004-01-01

    The unfolding pattern of crystal human hemoglobin and variants of hemoglobin obtained from hemolysate were studied using transverse urea gradient gel electrophoresis (TUGGE). A smooth sigmoid like increase of electrophoretic mobility was observed with increasing urea concentrations. A decrease in electrophoretic mobility resulted, if the protein was unfolded with guanidium hydrochloride (GdnHCl). The anomaly was resolved after the Stoke's radii (obtained using the photon correlation spectroscopy) and zeta potential (measured using laser Doppler velocimetry) measurements were made at different denaturant concentrations. Addition of denaturant led to formation of extended structure, irrespective of the nature of the denaturant, as indicated by increase in Stoke's radii in both cases (urea and GdnHCl). The unexpected increase in electrophoretic mobility in case of urea could be explained in terms of a critical redistribution of negative charge at intermediate stages of the unfolding process. In case of GdnHCl, the higher ionic strength masked the charge effect. The mobility, being solely dependent on size, decreased at higher denaturant concentration. Incidentally, folding loci of other hemoglobin variants (e.g. HbE) or that of post-translationally modified hemoglobin (e.g. HbA1c) could be determined by studying the charge distribution and hydrodynamic radius at varying denaturing stress and in each case the gel migration profile could be approximately scaled by the ratio of charge and hydrodynamic diameter of the protein. While unfolding induced charge effect was most pronounced in HbA0 (and crystal ferrous hemoglobin), the unfolding induced aggregation (manifested by the increase in Stoke's radii) was predominantly observed in the variant forms HbE and HbA1c. Representing the proteins by a plot, in which charge and hydrodynamic diameter are on independent axes, may be a useful way of characterizing protein variants having similar migration profiles on native gels

  16. DNA Sequencing by Capillary Electrophoresis

    Science.gov (United States)

    Karger, Barry L.; Guttman, Andras

    2009-01-01

    Sequencing of human and other genomes has been at the center of interest in the biomedical field over the past several decades and is now leading toward an era of personalized medicine. During this time, DNA sequencing methods have evolved from the labor intensive slab gel electrophoresis, through automated multicapillary electrophoresis systems using fluorophore labeling with multispectral imaging, to the “next generation” technologies of cyclic array, hybridization based, nanopore and single molecule sequencing. Deciphering the genetic blueprint and follow-up confirmatory sequencing of Homo sapiens and other genomes was only possible by the advent of modern sequencing technologies that was a result of step by step advances with a contribution of academics, medical personnel and instrument companies. While next generation sequencing is moving ahead at break-neck speed, the multicapillary electrophoretic systems played an essential role in the sequencing of the Human Genome, the foundation of the field of genomics. In this prospective, we wish to overview the role of capillary electrophoresis in DNA sequencing based in part of several of our articles in this journal. PMID:19517496

  17. The Cutting Edge of Affinity Electrophoresis Technology.

    Science.gov (United States)

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

    2015-03-18

    Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years.

  18. Ceramic matrix composites by microwave assisted CVI

    International Nuclear Information System (INIS)

    Currier, R.P.; Devlin, D.J.

    1993-01-01

    Chemical vapor infiltration (CVI) processes for producing continuously reinforced ceramic composites are reviewed. Potential advantages of microwave assisted CVI are noted and numerical studies of microwave assisted CVI are reviewed. The models predict inverted thermal gradients in fibrous ceramic preforms subjected to microwave radiation and suggest processing strategies for achieving uniformly dense composites. Comparisons are made to experimental results on silicon-based composite systems. The role played by the relative ability of fiber and matrix to dissipate microwave energy is noted. Results suggest that microwave induced inverted gradients can be exploited to promote inside-out densification. 10 refs., 2 figs

  19. Undergraduate physics laboratory: Electrophoresis in chromatography paper

    Science.gov (United States)

    Hyde, Alexander; Batishchev, Oleg

    2015-12-01

    An experiment studying the physical principles of electrophoresis in liquids was developed for an undergraduate laboratory. We have improved upon the standard agarose gel electrophoresis experimental regime with a straightforward and cost-effective procedure, in which drops of widely available black food coloring were separated by electric field into their dye components on strips of chromatography paper soaked in a baking soda/water solution. Terminal velocities of seven student-safe dyes were measured as a function of the electric potential applied along the strips. The molecular mobility was introduced and calculated by analyzing data for a single dye. Sources of systematic and random errors were investigated.

  20. [Autogenous platelet-rich plasma gel with acellular xenogeneic dermal matrix for treatment of deep II degree burns].

    Science.gov (United States)

    Hao, Tianzhi; Zhu, Jingmin; Hu, Wenbo; Zhang, Hua; Gao, Zhenhui; Wen, Xuehui; Zhou, Zhi; Lu, Gang; Liu, Jingjie; Li, Wen

    2010-06-01

    To investigate the effectiveness of autogenous platelet-rich plasma (PRP) gel with acellular xenogeneic dermal matrix in the treatment of deep II degree burns. From January 2007 to December 2009, 30 cases of deep II degree burns were treated. There were 19 males and 11 females with an average age of 42.5 years (range, 32-57 years). The burn area was 10% to 48% of total body surface area. The time from burn to hospitalization was 30 minutes to 8 hours. All patients were treated with tangential excision surgery, one side of the wounds were covered with autogenous PRP gel and acellular xenogeneic dermal matrix (PRP group), the other side of the wounds were covered with acellular xenogeneic dermal matrix only (control group). The healing rate, healing time, infection condition, and scar formation were observed. At 7 days after operation, the infection rate in PRP group (6.7%, 2/30) was significantly lower than that in control group (16.7%, 5/30, P deep II degree burns as well as alleviate the scar proliferation.

  1. Western Blotting using Capillary Electrophoresis

    OpenAIRE

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

    2011-01-01

    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 a...

  2. Efficient method of protein extraction from Theobroma cacao L. roots for two-dimensional gel electrophoresis and mass spectrometry analyses.

    Science.gov (United States)

    Bertolde, F Z; Almeida, A-A F; Silva, F A C; Oliveira, T M; Pirovani, C P

    2014-07-04

    Theobroma cacao is a woody and recalcitrant plant with a very high level of interfering compounds. Standard protocols for protein extraction were proposed for various types of samples, but the presence of interfering compounds in many samples prevented the isolation of proteins suitable for two-dimensional gel electrophoresis (2-DE). An efficient method to extract root proteins for 2-DE was established to overcome these problems. The main features of this protocol are: i) precipitation with trichloroacetic acid/acetone overnight to prepare the acetone dry powder (ADP), ii) several additional steps of sonication in the ADP preparation and extractions with dense sodium dodecyl sulfate and phenol, and iii) adding two stages of phenol extractions. Proteins were extracted from roots using this new protocol (Method B) and a protocol described in the literature for T. cacao leaves and meristems (Method A). Using these methods, we obtained a protein yield of about 0.7 and 2.5 mg per 1.0 g lyophilized root, and a total of 60 and 400 spots could be separated, respectively. Through Method B, it was possible to isolate high-quality protein and a high yield of roots from T. cacao for high-quality 2-DE gels. To demonstrate the quality of the extracted proteins from roots of T. cacao using Method B, several protein spots were cut from the 2-DE gels, analyzed by tandem mass spectrometry, and identified. Method B was further tested on Citrus roots, with a protein yield of about 2.7 mg per 1.0 g lyophilized root and 800 detected spots.

  3. Evaluation of genotoxicity after application of Listerine(R) on human lymphocytes by micronucleus and single cell gel electrophoresis assays.

    Science.gov (United States)

    Türkez, Hasan; Togar, Basak; Arabaci, Taner

    2012-04-01

    Listerine (LN) is one of the most commonly used mouth rinses worldwide although very limited information is available concerning its genotoxicity. In another view, the biological safety profile of oral care products is frequently assumed on the basis of simplistic test models. Therefore, the present study was undertaken to investigate the in vitro genotoxic potential of LN using micronucleus and single cell gel electrophoresis tests as genetic endpoints. Different concentrations of LN (0-100% of ml/culture, v/v) were applied to whole human blood cultures (n = 5). The result of the present study showed that there were no statistically significant differences (p > 0.05) between the control group and the groups treated with LN alone in both analysed endpoints. In conclusion, our result first demonstrated the absence of genotoxicity of LN on human lymphocytes.

  4. The use of time-resolved fluorescence in gel-based proteomics for improved biomarker discovery

    Science.gov (United States)

    Sandberg, AnnSofi; Buschmann, Volker; Kapusta, Peter; Erdmann, Rainer; Wheelock, Åsa M.

    2010-02-01

    This paper describes a new platform for quantitative intact proteomics, entitled Cumulative Time-resolved Emission 2-Dimensional Gel Electrophoresis (CuTEDGE). The CuTEDGE technology utilizes differences in fluorescent lifetimes to subtract the confounding background fluorescence during in-gel detection and quantification of proteins, resulting in a drastic improvement in both sensitivity and dynamic range compared to existing technology. The platform is primarily designed for image acquisition in 2-dimensional gel electrophoresis (2-DE), but is also applicable to 1-dimensional gel electrophoresis (1-DE), and proteins electroblotted to membranes. In a set of proof-of-principle measurements, we have evaluated the performance of the novel technology using the MicroTime 100 instrument (PicoQuant GmbH) in conjunction with the CyDye minimal labeling fluorochromes (GE Healthcare, Uppsala, Sweden) to perform differential gel electrophoresis (DIGE) analyses. The results indicate that the CuTEDGE technology provides an improvement in the dynamic range and sensitivity of detection of 3 orders of magnitude as compared to current state-of-the-art image acquisition instrumentation available for 2-DE (Typhoon 9410, GE Healthcare). Given the potential dynamic range of 7-8 orders of magnitude and sensitivities in the attomol range, the described invention represents a technological leap in detection of low abundance cellular proteins, which is desperately needed in the field of biomarker discovery.

  5. Characterization of Trichuris skrjabini by isoenzyme gel electrophoresis: comparative study with Trichuris ovis.

    Science.gov (United States)

    Cutillas, C; German, P; Arias, P; Guevara, D

    1996-10-01

    Morphological and biometric studies were performed in Trichuris skrjabini (Baskakov, 1924) collected from the caecum of Capra hircus. The LDH (EC 1.1.1.27.), G6PD (EC 1.1.1.49.), GPI (EC 5.3.1.9.), MDH (EC 1.1.1.37) and malic enzyme (ME) (EC 1.1.1.40) isoenzymatic patterns of T. skrjabini were determined by starch gel electrophoresis. The G6PD and GPI isoenzymatic patterns of T. skrjabini displayed two anodic bands for both enzymes: one fast migration band and one band near the origin. This isoenzymatic pattern was interpreted as two gene loci encoding both enzymes. The LDH isoenzymatic pattern of T. skrjabini was characterized by the presence of a cathodically migrating band, while the MDH isoenzymatic pattern showed a very slow cathodic band. These two phenotypes were interpreted as the expression of a homozygous state of a gene locus for LDH and MDH in T. skrjabini. The ME isoenzymatic pattern was characterized by the presence of a single anodic band. Further, comparative isoenzymatic studies were carried out between T. skrjabini and T. ovis. The different G6PD, GPI, LDH, MDH and ME isoenzymatic patterns observed for both species allowed us to distinguish them and therefore to use isoenzymatic patterns as a diagnostic tool to differentiate species of Trichuris.

  6. Separation and identification of DNA-carcinogen adduct conformers by polyacrylamide gel electrophoresis with laser-induced fluorescence detection

    Energy Technology Data Exchange (ETDEWEB)

    Marsch, G.A.; Jankowiak, R.; Farhat, J.H.; Small, G.J. (Ames Lab., IA (United States) Iowa State Univ., Ames (United States))

    1992-12-01

    The authors have developed a separation protocol utilizing high-resolution polyacrylamide gel electrophoresis (PAGE) to isolate stable anti-benzo[a]pyrene diol epoxide adducts of oligodeoxynucleotides. Both enantiomers produced multiple adduct species. The distribution of adduct types could be quantitated by densitometry of autoradiograms or Cerenkov counting of eluted oligomers modified by anti-BPDE isomers. Laser-induced fluorescence (LIF) spectra of eluted adducts at 4.2 K (fluorescence line-narrowing spectroscopy) and 77 K revealed that bands corresponded to pure conformers of pyrene chromophore. Carcinogen-modified oligodeoxynucleotides were single-stranded, but there were often considerable stacking interactions between the pyrenyl residues and the oligonucleotide bases, indicating that electrophoresed oligomers were single-stranded but in a native, versus random-coil conformation. The ability to identify and quantitate adducts by PAGE-LIF, coupled with the high resolution and sensitivity of both techniques, makes PAGE and LIF in tandem a potentially powerful tool in the study of chemical carcinogenesis or other ligand-DNA interactions. 43 refs., 7 figs., 1 tab.

  7. Capillary array electrophoresis using laser-excited confocal fluorescence detection

    Energy Technology Data Exchange (ETDEWEB)

    Huang, X.C.; Quesada, M.A.; Mathies, R.A. [Univ. of California, Berkeley, CA (United States)

    1992-04-15

    Capillary electrophoresis (CE) has found widespread application in analytical and biomedical research, and the scope and sophistication of CE is still rapidly advancing. Gel-filled capillaries have been employed for the rapid separation and analysis of synthetic polynucleotides, DNA sequencing fragments, and DNA restriction fragments. Open-tube capillary electrophoresis has attained subattomole detection levels in amino acid separations 14 and proven its utility for the separation of proteins, viruses, and bacteria. Separation of the optical isomers of dansyl amino acids has also been successfully demonstrated. Micellar electrokinetic capillary chromatography, isoelectric focusing, and on-column derivatization can all be performed on CE columns, demonstrating the utility of capillary electrophoresis as an analytical and micropreparative tool. 29 refs., 6 figs., 1 tab.

  8. In vitro study of prebiotic properties of levan-type exopolysaccharides from Lactobacilli and non-digestible carbohydrates using denaturing gradient gel electrophoresis.

    Science.gov (United States)

    Bello, F D; Walter, J; Hertel, C; Hammes, W P

    2001-07-01

    Batch cultures inoculated with human faeces were used to study the prebiotic properties of levan-type exopolysaccharides (EPS) from Lactobacillus sanfranciscensis as well as levan, inulin, and fructooligosaccharide (FOS). Denaturing gradient gel electrophoresis of 16S rDNA fragments generated by PCR with universal primers was used to analyse the cultures. Characteristic changes were revealed in the composition of the gut bacteria during fermentation of the carbohydrates. An enrichment of Bifidobacterium spp. was found for the EPS and inulin but not for levan and FOS. The bifidogenic effect of the EPS was confirmed by culturing on selective medium. In addition, the use of EPS and FOS resulted in enhanced growth of Eubacterium biforme and Clostridium perfringens, respectively.

  9. Ultraviolet absorption detection of DNA in gels

    International Nuclear Information System (INIS)

    Mahon, A.R.

    1998-01-01

    A method and apparatus for the detection and quantification of large fragments of unlabelled deoxyribonucleic acid (DNA) in agarose gels is presented. The technique is based on ultra-violet (UV) absorption by nucleotides. A deuterium lamp was used to illuminate regions of an electrophoresis gel. As DNA bands passed through the illuminated region of the gel the amount of UV light transmitted was reduced due to DNA absorption. Two detection systems were investigated. In the first system, synthetic chemical vapour deposition (CVD) diamond strip detectors were used to locate regions of DNA in the gels by detecting the transmitted light. CVD diamond has a high indirect band gap of 5.45 eV and is therefore sensitive to UV photons of wavelengths < 224 nm. A number of CVD diamond samples were characterised to investigate their suitability as detectors for this application. The detectors' quantum efficiency, UV response and time response were measured. DNA bands containing as little as 20 ng were detected by the diamond. In a second system, a deuterium lamp was used to illuminate individual sample lanes of an electrophoresis gel via an array of optical fibres. During electrophoresis the regions of DNA were detected with illumination at 260 nm, using a UV-sensitive charge coupled device (CCD). As the absorption coefficient of a DNA sample is approximately proportional to its mass, the technique is inherently quantitative. This system had a detection limit of 0.25 ng compared with 2-10 ng for the most popular conventional technique, ethidium bromide (EtBr) staining. Using this detection technique, the DNA sample remains in its native state. The removal of carcinogenic dyes from the detection procedure greatly reduces associated biological hazards. (author)

  10. Agarose Gel Electrophoresis Reveals Structural Fluidity of a Phage T3 DNA Packaging Intermediate

    Science.gov (United States)

    Serwer, Philip; Wright, Elena T.

    2012-01-01

    We find a new aspect of DNA packaging-associated structural fluidity for phage T3 capsids. The procedure is (1) glutaraldehyde cross-linking of in vivo DNA packaging intermediates for stabilization of structure and then (2) determining of effective radius by two-dimensional agarose gel electrophoresis (2d-AGE). The intermediates are capsids with incompletely packaged DNA (ipDNA) and without an external DNA segment; these intermediates are called ipDNA-capsids. We initially increase production of ipDNA-capsids by raising NaCl concentration during in vivo DNA packaging. By 2d-AGE, we find a new state of contracted shell for some particles of one previously identified ipDNA-capsid. The contracted shell-state is found when ipDNA length/mature DNA length (F) is above 0.17, but not at lower F. Some contracted-shell ipDNA-capsids have the phage tail; others do not. The contracted-shell ipDNA-capsids are explained by premature DNA maturation cleavage that makes accessible a contracted-shell intermediate of a cycle of the T3 DNA packaging motor. The analysis of ipDNA-capsids, rather than intermediates with uncleaved DNA, provides a simplifying strategy for a complete biochemical analysis of in vivo DNA packaging. PMID:22222979

  11. Molecular identification of the microbiota of French sourdough using temporal temperature gradient gel electrophoresis.

    Science.gov (United States)

    Ferchichi, Mounir; Valcheva, Rosica; Prévost, Hervé; Onno, Bernard; Dousset, Xavier

    2007-01-01

    The microbiota of four industrial French sourdoughs (BF, GO, VB and RF) was characterized by PCR temporal temperature gel electrophoresis (TTGE). The TTGE technique reveals differences in the 16S rDNA V6-V8 regions of these bacteria. DNA was extracted directly from sourdough samples. A specific TTGE fingerprint was determined for 30 bacterial species, including members of the genera Lactobacillus, Leuconostoc and Weissella, all known to be present in sourdough. These sourdoughs contain different species of lactic acid bacteria (LAB) depending on ecological conditions prevailing in the different sourdough fermentations. Only a few LAB species were found to be competitive and became dominant. Lactobacillus sanfranciscensis was observed as the most frequently found species. In sourdough GO, L. sanfranciscensis, Lactobacillus panis and two new species, Lactobacillus nantensis and Lactobacillus hammesii, were detected. Sourdough BF contain L. sanfranciscensis, Lactobacillus spicheri and Lactobacillus pontis. In sourdough VB, which differed in the process temperature, we identified exclusively L. sanfranciscensis and Leuconostoc mesenteroïdes subsp. mesenteroïdes. Lactobacillus frumenti, L. hammesii and Lacobacillus paralimentarius became the predominant species in sourdough RF. Compared with conventional bacteriological methods, the use of this new molecular approach to analyze the sourdough ecosystem should therefore allow a more complete and rapid assessment of its specific microbiota.

  12. Pulsed-field gel electrophoresis of chromosomal DNA of methicillin-resistant Staphylococcus aureus associated with nosocomial infections.

    Science.gov (United States)

    Hanifah, Y A; Hiramatsu, K

    1994-12-01

    Methicillin-resistant Staphylococcus aureus (MRSA) infection has been endemic in the University Hospital, Kuala Lumpur since the late 1970s. Fifty isolates of MRSA obtained from clinical specimens of patients with nosocomial infections associated with this organism have been studied by pulsed-field gel electrophoresis (PFGE) of its chromosomal DNA fragments to discrimate between strains and to identify the predominant strain. Twenty-one chromosomal patterns were observed which could be further grouped into nine types. The predominant strain was Type 9-b (40% of isolates) found mainly in the Orthopaedic and Surgical Units. Outbreak strains found in the Special Care Nursery were of Type 1, entirely different from those of the surgical ward S2, which were of Type 9-b. Type 8 strains were found mainly at one end of the hospital building where the maternity, paediatric and orthopaedic units were situated. Genomic DNA fingerprinting by PFGE is recommended as a useful and effective tool for the purpose of epidemiological studies of MSRA infections, particularly for nosocomial infections.

  13. Model creation of moving redox reaction boundary in agarose gel electrophoresis by traditional potassium permanganate method.

    Science.gov (United States)

    Xie, Hai-Yang; Liu, Qian; Li, Jia-Hao; Fan, Liu-Yin; Cao, Cheng-Xi

    2013-02-21

    A novel moving redox reaction boundary (MRRB) model was developed for studying electrophoretic behaviors of analytes involving redox reaction on the principle of moving reaction boundary (MRB). Traditional potassium permanganate method was used to create the boundary model in agarose gel electrophoresis because of the rapid reaction rate associated with MnO(4)(-) ions and Fe(2+) ions. MRB velocity equation was proposed to describe the general functional relationship between velocity of moving redox reaction boundary (V(MRRB)) and concentration of reactant, and can be extrapolated to similar MRB techniques. Parameters affecting the redox reaction boundary were investigated in detail. Under the selected conditions, good linear relationship between boundary movement distance and time were obtained. The potential application of MRRB in electromigration redox reaction titration was performed in two different concentration levels. The precision of the V(MRRB) was studied and the relative standard deviations were below 8.1%, illustrating the good repeatability achieved in this experiment. The proposed MRRB model enriches the MRB theory and also provides a feasible realization of manual control of redox reaction process in electrophoretic analysis.

  14. Bacteria community study of combined periodontal-endodontic lesions using denaturing gradient gel electrophoresis and sequencing analysis.

    Science.gov (United States)

    Li, Hong; Guan, Rui; Sun, Jinghua; Hou, Benxiang

    2014-10-01

    The entire microbial population and predominant microflora of root canals (RCs) and adjacent periodontal pockets (PPs) from teeth with combined periodontal-endodontic lesions were determined and compared. Pooled RC and PP samples were collected from the molars of 20 patients diagnosed with combined periodontal-endodontic lesions. DNA was extracted for polymerase chain reaction-based denaturing gradient gel electrophoresis (PCR-DGGE), cloning, and sequence analysis. A coefficient of similarity (Cs) was used to determine the similarity of the bacterial profiles from RCs and PPs. Significantly fewer bands were produced by PCR-DGGE from RCs (5.9 ± 1.7) than from PPs (8.0 ± 1.8) (P bacteria in both the RC and PP samples were (in descending order) Filifactor alocis, Parvimonas micra, Porphyromonas gingivalis, and Tannerella forsythia. The high similarity in the sets of organisms present in both RC and PP samples in this study suggests that the pocket could be a source of RC infection. The data also demonstrate that combined periodontal-endodontic lesions consist of a diverse and complex microbial community.

  15. Immobilization of Mo(IV) complex in hybrid matrix obtained via sol-gel technique

    Energy Technology Data Exchange (ETDEWEB)

    Marques, C.; Sousa, A.M.; Freire, C.; Neves, I.C.; Fonseca, A.M.; Silva, C.J.R

    2003-10-06

    A molybdenum(IV) complex, trans-bis-[1,2-bis(diphenylphosphino)ethane]-fluoro-(diazopropano) -molybdenum tetraphenylborate, [MoF(DIAZO)(dppe){sub 2}][BPh{sub 4}], was prepared and immobilized in a hybrid matrix synthesized by the sol-gel process. The host matrix, designated as U(500), is an organic-inorganic network material, classed as ureasil, that combines a reticulated siliceous backbone linked by short polyether-based segments. Urea bridges make the link between these two components, and the polymerization of silicate substituted terminal groups generates the inorganic network. The free Mo(IV) complex and all new materials were characterized by spectroscopic techniques (FT-IR and UV-Vis) and thermal analysis (DSC). The ionic conductivity of the resulting material was also studied. The results indicate that immobilized Mo(IV) complex has kept its solid-state structure, although there is evidence of inter-molecular interactions between the Mo(IV) complex and some groups/atoms of the hybrid host matrix.

  16. The Cutting Edge of Affinity Electrophoresis Technology

    Science.gov (United States)

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

    2015-01-01

    Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years. PMID:28248262

  17. Approach to analysis of single nucleotide polymorphisms by automated constant denaturant capillary electrophoresis

    International Nuclear Information System (INIS)

    Bjoerheim, Jens; Abrahamsen, Torveig Weum; Kristensen, Annette Torgunrud; Gaudernack, Gustav; Ekstroem, Per O.

    2003-01-01

    Melting gel techniques have proven to be amenable and powerful tools in point mutation and single nucleotide polymorphism (SNP) analysis. With the introduction of commercially available capillary electrophoresis instruments, a partly automated platform for denaturant capillary electrophoresis with potential for routine screening of selected target sequences has been established. The aim of this article is to demonstrate the use of automated constant denaturant capillary electrophoresis (ACDCE) in single nucleotide polymorphism analysis of various target sequences. Optimal analysis conditions for different single nucleotide polymorphisms on ACDCE are evaluated with the Poland algorithm. Laboratory procedures include only PCR and electrophoresis. For direct genotyping of individual SNPs, the samples are analyzed with an internal standard and the alleles are identified by co-migration of sample and standard peaks. In conclusion, SNPs suitable for melting gel analysis based on theoretical thermodynamics were separated by ACDCE under appropriate conditions. With this instrumentation (ABI 310 Genetic Analyzer), 48 samples could be analyzed without any intervention. Several institutions have capillary instrumentation in-house, thus making this SNP analysis method accessible to large groups of researchers without any need for instrument modification

  18. Preliminary study on biological dosimetry using alkaline single cell gel electrophoresis of human peripheral lymphocytes

    International Nuclear Information System (INIS)

    Liu Qingjie; Lu Xue; Feng Jiangbing; Chen Deqing; Chen Xiaosui

    2006-01-01

    Objective: To explore the feasibility of alkaline single cell gel electrophoresis (SCGE) in biological dosimetry of ionizing radiation. Methods: Normal peripheral blood samples from two healthy males were exposed to different doses coblat-60 gamma-rays, ranged from 0 to 5 Gy, and the tail length (TL) and Oliver tail moment (TM) of the lymphocytes were analyzed with SCGE. The dose-effect curves of TL and TM were fitted respectively. The TL and TM of lymphocytes for eight radiation workers were analyzed with SCGE, cumulative doses were estimated using the fitted TL and TM equations, and then compared with the recorded monitoring doses. Results: The TLs or TMs of normal human lymphocytes were increased with the irradiation doses, and its relationship can be fitted with a linear-quadratic equations: Y=13.59 + 20.87X - 2.27 X 2 for TL, and Y = 8.50 + 15.04X - 1.43X 2 for TM, respectively (Y denotes TL or TM value, X is radiation dose). The doses estimated with TM equation were closer to the recorded monitoring doses than that with TL equation. Conclusions: The TM in lymphocytes analyzed with SCGE is a promising radiation biological dosimeter. (authors)

  19. Subtyping of Salmonella enterica isolated from humans and food animals using Pulsed-Field Gel Electrophoresis

    Directory of Open Access Journals (Sweden)

    Golab, N.

    2016-07-01

    Full Text Available Salmonella infections are the second leading cause of zoonotic bacterial foodborne illness. Main source of infection in human is contaminated food products. The aim of this study was sub typing isolates of Salmonella enterica obtained during our previous study by Pulsed Field Gel Electrophoresis (PFGE technique. All 46 Salmonella isolates were serotyped and then subjected to PFGE. Total isolates were analyzed by means of the molecular technique XbaI PFGE. In this study, PFGE and serotyping were used to subtype 46 Salmonella isolates belonging to 27different serovars and derived from human and different food origins. Among these isolates, S. Typhimurium was found to be the most predominant serovar. 40 PFGE patterns out of 46 isolates were obtained. The Discrimination Index obtained by serotyping (DI = 0.93 was lower than PFGE (DI = 0.99. Subtyping of Salmonella enterica is very important and shows that animal origin can be one of a reservoir that potentially could be transferred to human through the food chain. In addition, results of this study also revealed that this procedure is a golden standard for genotyping of such salmonella serotypes.

  20. The minimum amount of "matrix " needed for matrix-assisted pulsed laser deposition of biomolecules

    DEFF Research Database (Denmark)

    Tabetah, Marshall; Matei, Andreea; Constantinescu, Catalin

    2014-01-01

    The ability of matrix-assisted pulsed laser evaporation (MAPLE) technique to transfer and deposit high-quality thin organic, bioorganic, and composite films with minimum chemical modification of the target material has been utilized in numerous applications. One of the outstanding problems in MAPLE...

  1. Protein electrophoretic migration data from custom and commercial gradient gels

    Directory of Open Access Journals (Sweden)

    Andrew J. Miller

    2016-12-01

    Full Text Available This paper presents data related to the article “A method for easily customizable gradient gel electrophoresis” (A.J. Miller, B. Roman, E.M. Norstrom, 2016 [1]. Data is presented on the rate of electrophoretic migration of proteins in both hand-poured and commercially acquired acrylamide gradient gels. For each gel, migration of 9 polypeptides of various masses was measured upon completion of gel electrophoresis. Data are presented on the migration of proteins within separate lanes of the same gel as well as migration rates from multiple gels.

  2. Correlation between γ-ray-induced DNA double-strand breakage and cell killing after biologically relevant doses: analysis by pulsed-field gel electrophoresis

    International Nuclear Information System (INIS)

    Murray, D.

    1994-01-01

    We examined the degree of correlation between γ-ray-induced lethality and DNA double-strand breaks (dsbs) after biologically relevant doses of radiation. Radiation lethality was modified by treating 14 C-labelled Chinese hamster ovary cells with either of two aminothiols (WR-1065 or WR-255591) and the associated effect on dsb induction was determined by pulsed-field gel electrophoresis (PFGE). The use of phosphorimaging to analyse the distribution of 14 C-activity in the gel greatly improved the low-dose resolution of the PFGE assay. Both WR-1065 and WR-255591 protected against dsb induction and lethality to a similar extent after low doses of radiation. although this correlation broke down when supralethal doses were used to induce dsbs. Thus, the level of dsbs induced in these cells as measured by PFGE after survival-curve doses of γ-radiation is consistently predictive of the degree of lethality obtained, implying a cause-effect relationship between these two parameters and confirming previous results obtained using the neutral filter elution assay for dsbs. (author)

  3. The new horizon in 2D electrophoresis: new technology to increase resolution and sensitivity.

    Science.gov (United States)

    Moche, Martin; Albrecht, Dirk; Maaß, Sandra; Hecker, Michael; Westermeier, Reiner; Büttner, Knut

    2013-06-01

    A principally new type of an electrophoresis setup for the second dimension of 2DE named HPE (high performance electrophoresis) has recently become available that provides excellent reproducibility much superior to traditional 2DE. It takes up ideas from early beginnings of 2DE which could not be satisfactory realized at that time. The new HPE system is in contrast to all other established systems a horizontal electrophoresis that employs a new type of precast polyacrylamide gels on film-backing and runs on a multilevel flatbed electrophoresis apparatus. In a systematic approach we compared its features to traditional 2DE for the cytosolic proteome of Bacillus subtilis. Not only the reproducibility is enhanced, but also nearly all qualitative parameters as resolution, sensitivity, the number of protein spots (25% more), and the number of different proteins (also additional 25%) are markedly increased. More than 200 proteins were exclusively found in HPE. This new electrophoresis system does not use buffer tanks. No glass plates are needed. Therefore handling of gels is greatly facilitated and very simple to use even for personnel with low technical skills. The new HPE system is technically at the beginnings and further development with increased performance can be expected. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Deposition of matrix-free fullerene films with improved morphology by matrix-assisted pulsed laser evaporation (MAPLE)

    DEFF Research Database (Denmark)

    Canulescu, Stela; Schou, Jørgen; Fæster, Søren

    2013-01-01

    Thin films of C60 were deposited by matrix-assisted pulsed laser evaporation (MAPLE) from a frozen target of anisole with 0.67 wt% C60. Above a fluence of 1.5 J/cm2 the C60 films are strongly non-uniform and are resulting from transfer of matrix-droplets containing fullerenes. At low fluence...... the fullerene molecules in the films are intact, the surface morphology is substantially improved and there are no measurable traces of the matrix molecules in the film. This may indicate a regime of dominant evaporation at low fluence which merges into the MAPLE regime of liquid ejection of the host matrix...

  5. Ghost peaks observed after atmospheric pressure matrix-assisted laser desorption/ionization experiments may disclose new ionization mechanism of matrix-assisted hypersonic velocity impact ionization.

    Science.gov (United States)

    Moskovets, Eugene

    2015-08-30

    Understanding the mechanisms of matrix-assisted laser desorption/ionization (MALDI) promises improvements in the sensitivity and specificity of many established applications in the field of mass spectrometry. This paper reports a serendipitous observation of a significant ion yield in a post-ionization experiment conducted after the sample had been removed from a standard atmospheric pressure (AP)-MALDI source. This post-ionization is interpreted in terms of collisions of microparticles moving with a hypersonic velocity into a solid surface. Calculations show that the thermal energy released during such collisions is close to that absorbed by the top matrix layer in traditional MALDI. The microparticles, containing both the matrix and analytes, could be detached from a film produced inside the inlet capillary during the sample ablation and accelerated by the flow rushing through the capillary. These observations contribute some new perspective to ion formation in both laser and laser-less matrix-assisted ionization. An AP-MALDI ion source hyphenated with a three-stage high-pressure ion funnel system was utilized for peptide mass analysis. After the laser had been turned off and the MALDI sample removed, ions were detected during a gradual reduction of the background pressure in the first funnel. The constant-rate pressure reduction led to the reproducible appearance of different singly and doubly charged peptide peaks in mass spectra taken a few seconds after the end of the MALDI analysis of a dried-droplet spot. The ion yield as well as the mass range of ions observed with a significant delay after a completion of the primary MALDI analysis depended primarily on the background pressure inside the first funnel. The production of ions in this post-ionization step was exclusively observed during the pressure drop. A lower matrix background and significant increase in relative yield of double-protonated ions are reported. The observations were partially consistent

  6. Comparative Proteome Analysis of Oil Palm (Elaeis guineensis Jacq.) Floret and Fruit Tissues

    Institute of Scientific and Technical Information of China (English)

    Kok S.Y.; Ong-Abdullah M.

    2012-01-01

    Marked changes are expected when flowers are fertilized and fruit formation is initiated.In this study,differentially expressed proteins between florets (day before pollination and day after pollination) and fruit (2 weeks after pollination) tissues of oil palm (Elaeis guineensis Jacq.) were identified and characterized.Proteins were extracted using phenol-based method and subjected to one-and two-dimensional gel electrophoresis.One-dimensional gel electrophoresis of protein extracts showed a number of protein bands common to both types of floret and fruit tissues.Subsequently,protein extracts resolved by two-dimensional gel electrophoresis in the 4 to 7 pH range revealed about 53 expressed proteins showed significant differences during fertilization and fruit formation.The differentially expressed proteins were excised and digested with trypsin.Proteins were then identified with the matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry (MALDI-TOF/TOF-MS),resulting in a 73.5% success in protein identification.The identified proteins were mainly involved in protein metabolism,biological regulation,stress response and oxidationreduction process.Further verification of the results are underway but nonetheless this study has provided some valuable information on fertilization and early fruit formation in oil palm.

  7. Highly sensitive and selective spectrofluorimetric determination of metoclopramide hydrochloride in pharmaceutical tablets and serum samples using Eu3+ ion doped in sol-gel matrix.

    Science.gov (United States)

    Attia, M S; Aboaly, M M

    2010-06-30

    A simple, sensitive and selective spectrofluorimetric method for the determination of Metoclopramide hydrochloride (MCP) is developed. The MCP can remarkably enhances the luminescence intensity of the Eu(3+) ion doped in sol-gel matrix at lambda(ex)=380 nm in DMSO at pH 8.7. The intensity of the emission band of Eu(3+) ion doped in sol-gel matrix increases due to energy transfer from MCP to Eu(3+) in the excited state. The enhancement of the emission band of Eu(3+) ion doped in sol-gel matrix at 617 nm was found to be directly proportional to the concentration of MCP with a dynamic range of 5 x 10(-9) - 1.0 x 10(-6) mol L(-1) and detection limit of 2.2 x10(-11) mol L(-1). Copyright 2010 Elsevier B.V. All rights reserved.

  8. Characterization of the proteins comprising the integral matrix of Strongylocentrotus purpuratus embryonic spicules

    Science.gov (United States)

    Killian, C. E.; Wilt, F. H.

    1996-01-01

    In the present study, we enumerate and characterize the proteins that comprise the integral spicule matrix of the Strongylocentrotus purpuratus embryo. Two-dimensional gel electrophoresis of [35S]methionine radiolabeled spicule matrix proteins reveals that there are 12 strongly radiolabeled spicule matrix proteins and approximately three dozen less strongly radiolabeled spicule matrix proteins. The majority of the proteins have acidic isoelectric points; however, there are several spicule matrix proteins that have more alkaline isoelectric points. Western blotting analysis indicates that SM50 is the spicule matrix protein with the most alkaline isoelectric point. In addition, two distinct SM30 proteins are identified in embryonic spicules, and they have apparent molecular masses of approximately 43 and 46 kDa. Comparisons between embryonic spicule matrix proteins and adult spine integral matrix proteins suggest that the embryonic 43-kDa SM30 protein is an embryonic isoform of SM30. An adult 49-kDa spine matrix protein is also identified as a possible adult isoform of SM30. Analysis of the SM30 amino acid sequences indicates that a portion of SM30 proteins is very similar to the carbohydrate recognition domain of C-type lectin proteins.

  9. Two-dimensional gel electrophoresis data in support of leaf comparative proteomics of two citrus species differing in boron-tolerance

    Directory of Open Access Journals (Sweden)

    Wen Sang

    2015-09-01

    Full Text Available Here, we provide the data from a comparative proteomics approach used to investigate the response of boron (B-tolerant ‘Xuegan’ (Citrus sinensis and B-intolerant ‘Sour pummelo’ (Citrus grandis leaves to B-toxicity. Using two-dimensional gel electrophoresis (2-DE technique, we identified 50 and 45 protein species with a fold change of more than 1.5 and a P-value of less than 0.05 from B-toxic C. sinensis and C. grandis leaves. These B-toxicity-responsive protein species were mainly involved in carbohydrate and energy metabolism, antioxidation and detoxification, stress responses, coenzyme biosynthesis, protein and amino acid metabolism, signal transduction, cell transport, cytoskeleton, nucleotide metabolism, and cell cycle and DNA processing. A detailed analysis of this data may be obtained from Sang et al. (J. Proteomics 114 (2015[1].

  10. Dehumidifier assisted drying of a model fruit pulp-based gel and sensory attributes.

    Science.gov (United States)

    Tiwari, Shipra; Ravi, Ramasamy; Bhattacharya, Suvendu

    2012-07-01

    Model fruit pulp-based gels were prepared by varying mango pulp (0% to 50%), sucrose (0% to 20%), and agar (1% to 3%) and according to a response surface experimental design followed by drying at a low temperature of 40 °C upto 15 h in a tray dryer assisted by a dehumidifier. The moisture content, shrinkage (SHR), and rheological parameters (failure strain, failure stress (FS), firmness, and energy for compression) were determined as a function of drying time. The composition of gel, particularly the agar content had a prominent effect on the characteristics of the dried gel. Detailed descriptive sensory analysis employing principle component analysis (PCA) biplot indicated two distinct groups of attributes; the first group comprised initial and final moisture contents, extent of moisture removal (EMR), and shrinkage. The fracture stress and energy formed the second group. The analysis of variance for failure stress showed that it depended only on the positive linear and quadratic effects of agar (significant at P ≤ 0.01 and 0.05, respectively). The theoretically predicted extent of moisture removal at 95.6% could be achieved when the level of agar was 1.2%; pulp and sucrose levels were also close to their lowest levels of 3.6% and 0.04%, respectively. Scope exists to develop gel-based fruit analogues wherein an appropriate hydrocolloid can be employed along with fruit juice/pulp. To provide a reasonable shelf-life of the developed intermediate moisture containing product, dehumidifier assisted drying is a pragmatic approach that affects sensory and rheological attributes of the dried fruit analogue. © 2012 Institute of Food Technologists®

  11. Comparative gel-based phosphoproteomics in response to signaling molecules

    KAUST Repository

    Marondedze, Claudius; Lilley, Kathryn S.; Thomas, Ludivine

    2013-01-01

    The gel-based proteomics approach is a valuable technique for studying the characteristics of proteins. This technique has diverse applications ranging from analysis of a single protein to the study of the total cellular proteins. Further, protein quality and to some extent distribution can be first assessed by means of one-dimensional gel electrophoresis and then more informatively, for comparative analysis, using the two-dimensional gel electrophoresis technique. Here, we describe how to take advantage of the availability of fluorescent dyes to stain for a selective class of proteins on the same gel for the detection of both phospho- and total proteomes. This enables the co-detection of phosphoproteins as well as total proteins from the same gel and is accomplished by utilizing two different fluorescent stains, the ProQ-Diamond, which stains only phosphorylated proteins, and Sypro Ruby, which stains the entire subset of proteins. This workflow can be applied to gain insights into the regulatory mechanisms induced by signaling molecules such as cyclic nucleotides through the quantification and subsequent identification of responsive phospho- and total proteins. © Springer Science+Business Media New York 2013.

  12. Comparative gel-based phosphoproteomics in response to signaling molecules

    KAUST Repository

    Marondedze, Claudius

    2013-09-03

    The gel-based proteomics approach is a valuable technique for studying the characteristics of proteins. This technique has diverse applications ranging from analysis of a single protein to the study of the total cellular proteins. Further, protein quality and to some extent distribution can be first assessed by means of one-dimensional gel electrophoresis and then more informatively, for comparative analysis, using the two-dimensional gel electrophoresis technique. Here, we describe how to take advantage of the availability of fluorescent dyes to stain for a selective class of proteins on the same gel for the detection of both phospho- and total proteomes. This enables the co-detection of phosphoproteins as well as total proteins from the same gel and is accomplished by utilizing two different fluorescent stains, the ProQ-Diamond, which stains only phosphorylated proteins, and Sypro Ruby, which stains the entire subset of proteins. This workflow can be applied to gain insights into the regulatory mechanisms induced by signaling molecules such as cyclic nucleotides through the quantification and subsequent identification of responsive phospho- and total proteins. © Springer Science+Business Media New York 2013.

  13. DESIGN OF ELECTROPHORESIS DEVICE FOR OPTIMATION OF DNA VISUALIZATION AND DNA CONCENTRATION USING SOFTWARE

    Directory of Open Access Journals (Sweden)

    H.P. Kusumaningrum

    2014-07-01

    Full Text Available Molekul DNA menunjukkan polarisasi yang kuat sehingga memungkinkan baik gerak elektroforesis berdasarkan muatan negatifnya maupun gerak dielektroforesis berdasarkan induksi polarisasi. Perancangan alat menggunakan kombinasi prinsip elektroforesis dan dielektroforesis dilengkapi perangkat lunak untuk mengukur konsentrasinya sangat diperlukan. Utamanya mengingat uji kualitatif DNA berbasis visualisasi pada gel elektroforesis bersifat sangat subyektif dan kurang terukur. Pengukuran konsentrasi DNA menggunakan spektrofotometer UV/VIS sangat tergantung oleh ketersediaannya di laboratorium. Penelitian bertujuan untuk mendesain piranti untuk mengukur konsentrasi DNA berdasarkan visualisasinya pada gel elektroforesis menggunakan perangkat lunak berbasis MatLab. Pengukuran konsentrasi DNA didasarkan visualisasinya pada gel elektroforesis lalu dibandingkan dengan hasil penghitungan spektrofotometer UV/VIS. Hasil penelitian menggunakan piranti tersebut memperlihatkan visualisasi DNA yang lebih optimal. Hasil pengukuran jumlah DNA menggunakan spektrofotometer memiliki kecenderungan yang sama dengan hasil pengukuran menggunakan perangkat lunak berbasis MatLab meskipun terdapat perbedaan nilai kuantitatif.ABSTRACTMolecules of deoxyribo nucleic acid (DNA show a strong polarization allowing for both motions of the dielectrophoresis induced by polarization and electrophoresis based on its negative charge. Considering high subjective and less quantifiable result of the visualization based qualitative test of DNA on gel electrophoresis, designing the tool using a combination of the principles of electrophoresis and dielectrophoresis completed with a software for optimization of DNA visualization and to measure the concentration of small and large–sized DNA fragment is very needed. Accuracy of measurement of DNA concentration using a spectrophotometer UV /VIS is depend on its availability in the laboratory. The aim of this study was to design device for

  14. 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.

  15. Oil field and freshwater isolates of Shewanella putrefaciens have lipopolysaccharide polyacrylamide gel profiles characteristic of marine bacteria

    International Nuclear Information System (INIS)

    Pickard, C.; Foght, J.M.; Pickard, M.A.; Westlake, D.W.S.

    1993-01-01

    The lipopolysaccharide structure of oil field and freshwater isolates of bacteria that reduce ferric iron, recently classified as strains of Shewanella putrefaciens, was analyzed using polyacrylamide gel electrophoresis and a lipopolysaccharide-specific silver-staining procedure. The results demonstrate that all the oil field and freshwater isolates examined exhibited the more hydrophobic R-type lipopolysaccharide, which has been found to be characteristic of Gram-negative marine bacteria. This hydrophobic lipopolysaccharide would confer an advantage on bacteria involved in hydrocarbon degradation by assisting their association with the surface of oil droplets. 15 refs., 1 fig

  16. Agarose gel shift assay reveals that calreticulin favors substrates with a quaternary structure in solution

    DEFF Research Database (Denmark)

    Boelt, Sanne Grundvad; Houen, Gunnar; Højrup, Peter

    2015-01-01

    Here we present an agarose gel shift assay that, in contrast to other electrophoresis approaches, is loaded in the center of the gel. This allows proteins to migrate in either direction according to their isoelectric points. Therefore, the presented assay enables a direct visualization, separation...... structure. It is also demonstrated that the agarose gel shift assay is useful in the study of other protein interactions and can be used as an alternative method to native polyacrylamide gel electrophoresis....... measure of interactions. Therefore, no interaction studies between calreticulin and substrates in solution have been investigated previously. The results presented here indicate that calreticulin has a preference for substrates with a quaternary structure and primarily β-sheets in their secondary...

  17. Decreasing the amount of trypsin in in-gel digestion leads to diminished chemical noise and improved protein identifications.

    Science.gov (United States)

    Hu, Mo; Liu, Yanhua; Yu, Kaiwen; Liu, Xiaoyun

    2014-09-23

    Pre-fractionation by gel electrophoresis is often combined with liquid chromatography-mass spectrometry (LC-MS) for large-scale profiling of complex protein samples. An essential component of this widely applied proteomic platform is in-gel protein digestion. In nearly two decades of practicing this approach, an extremely high level of trypsin has been utilized due to the consideration of slow enzyme diffusion into the gel matrix. Here we report that trypsin autolysis products contribute to the bulk of chemical noise in in-gel digestion and remarkably we found evidence that the amount of trypsin can be slashed by an order of magnitude with comparable digestion performance. By revising perhaps the most critical element of this decade-old digestion protocol, the proteomics community relying on gel separation prior to LC-MS analysis will benefit instantly from much lowered cost due to enzyme expenditure. More importantly, substantially reduced chemical noise (i.e., trypsin self-cleavage products) as a result of less enzyme usage translates into more protein identifications when limited amounts of samples are the interest of interrogation. In-gel digestion is one of the most widely used methods in proteomics. An exceedingly high level of trypsin has been utilized due to the consideration of slow enzyme diffusion into the gel matrix. This requirement has been faithfully kept in nearly two decades of practicing this approach. Here we report that trypsin concentration can be slashed by at least an order of magnitude while still providing comparable digestion performance. Thus the proteomics community relying on gel separation prior to LC-MS analysis will benefit instantly from much lowered enzyme cost. More importantly, substantially reduced chemical noise (i.e., trypsin autolysis products) due to less enzyme usage translates into ~30% more protein identifications when limited amounts of protein samples are analyzed. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Polyacrylamide gel electrophoretic preparation of labelled and non-labelled peptides for radioimmunoassay

    International Nuclear Information System (INIS)

    Besch, W.; Woltanski, K.P.; Keilacker, H.; Kohnert, K.D.

    1986-01-01

    Radioiodinated polypeptide hormones, such as insulin, glucagon, human growth hormone, and human C-peptide are employed for radioimmunoassays for investigation of hormonal alterations in states of disturbed carbohydrate metabolism. Iodination was performed using chloramine T. Iodination products of these polypeptide hormones and, for preparation of standard material, native human C-peptide from cadaver pancreases were fractionated by polyacrylamide gel electrophoresis at pH 8.9. Disc electrophoresis in 24 cm long gel rods resulted in stable tracers with high specific activity as well as homogeneous standard material being highly suitable for radioimmunoassays. (author)

  19. Bacterial community structure in Apis florea larvae analyzed by denaturing gradient gel electrophoresis and 16S rRNA gene sequencing.

    Science.gov (United States)

    Saraithong, Prakaimuk; Li, Yihong; Saenphet, Kanokporn; Chen, Zhou; Chantawannakul, Panuwan

    2015-10-01

    This study characterizes the colonization and composition of bacterial flora in dwarf Asian honeybee (Apis florea) larvae and compares bacterial diversity and distribution among different sampling locations. A. florea larvae were collected from 3 locations in Chiang Mai province, Thailand. Bacterial DNA was extracted from each larva using the phenol-chloroform method. Denaturing gradient gel electrophoresis was performed, and the dominant bands were excised from the gels, cloned, and sequenced for bacterial species identification. The result revealed similarities of bacterial community profiles in each individual colony, but differences between colonies from the same and different locations. A. florea larvae harbor bacteria belonging to 2 phyla (Firmicutes and Proteobacteria), 5 classes (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacilli, and Clostridia), 6 genera (Clostridium, Gilliamella, Melissococcus, Lactobacillus, Saccharibacter, and Snodgrassella), and an unknown genus from uncultured bacterial species. The classes with the highest abundance of bacteria were Alphaproteobacteria (34%), Bacilli (25%), Betaproteobacteria (11%), Gammaproteobacteria (10%), and Clostridia (8%), respectively. Similarly, uncultured bacterial species were identified (12%). Environmental bacterial species, such as Saccharibacter floricola, were also found. This is the first study in which sequences closely related to Melissococcus plutonius, the causal pathogen responsible for European foulbrood, have been identified in Thai A. florea larvae. © 2014 Institute of Zoology, Chinese Academy of Sciences.

  20. Unfolding of hemoglobin variants--insights from urea gradient gel electrophoresis photon correlation spectroscopy and zeta potential measurements

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharya, Jaydeep; GhoshMoulick, Ranjita; Choudhuri, Utpal; Chakrabarty, Prantar; Bhattacharya, Pranab K.; Lahiri, Prabir; Chakraborti, Bikas; Dasgupta, Anjan Kr

    2004-09-27

    The unfolding pattern of crystal human hemoglobin and variants of hemoglobin obtained from hemolysate were studied using transverse urea gradient gel electrophoresis (TUGGE). A smooth sigmoid like increase of electrophoretic mobility was observed with increasing urea concentrations. A decrease in electrophoretic mobility resulted, if the protein was unfolded with guanidium hydrochloride (GdnHCl). The anomaly was resolved after the Stoke's radii (obtained using the photon correlation spectroscopy) and zeta potential (measured using laser Doppler velocimetry) measurements were made at different denaturant concentrations. Addition of denaturant led to formation of extended structure, irrespective of the nature of the denaturant, as indicated by increase in Stoke's radii in both cases (urea and GdnHCl). The unexpected increase in electrophoretic mobility in case of urea could be explained in terms of a critical redistribution of negative charge at intermediate stages of the unfolding process. In case of GdnHCl, the higher ionic strength masked the charge effect. The mobility, being solely dependent on size, decreased at higher denaturant concentration. Incidentally, folding loci of other hemoglobin variants (e.g. HbE) or that of post-translationally modified hemoglobin (e.g. HbA1c) could be determined by studying the charge distribution and hydrodynamic radius at varying denaturing stress and in each case the gel migration profile could be approximately scaled by the ratio of charge and hydrodynamic diameter of the protein. While unfolding induced charge effect was most pronounced in HbA0 (and crystal ferrous hemoglobin), the unfolding induced aggregation (manifested by the increase in Stoke's radii) was predominantly observed in the variant forms HbE and HbA1c. Representing the proteins by a plot, in which charge and hydrodynamic diameter are on independent axes, may be a useful way of characterizing protein variants having similar migration profiles on

  1. Thermal stability of octadecylsilane hybrid silicas prepared by grafting and sol-gel methods

    International Nuclear Information System (INIS)

    Brambilla, Rodrigo; Santos, Joao H.Z. dos; Miranda, Marcia S.L.; Frost, Ray L.

    2008-01-01

    Hybrid silicas bearing octadecylsilane groups were prepared by grafting and sol-gel (SG) methods. The effect of the preparative route on the thermal stability was evaluated by means of thermal gravimetric analysis (TGA), infrared emission spectroscopy (IRES) and, complementary, by 13 C solid-state nuclear magnetic resonance ( 13 C NMR) and matrix assisted laser deionization time of flight mass spectroscopy (MALDI-TOF-MS). Silicas prepared by the grafting route seem to be slightly more stable than those produced by the sol-gel method. This behavior seems to be associated to the preparative route, since grafting affords a liquid-like conformation, while in the case of sol-gel a highly organized crystalline chain conformation was observed

  2. The detection of Mycoplasma (formerly Eperythrozoon) wenyonii by 16S rDNA PCR and denaturing gradient gel electrophoresis.

    Science.gov (United States)

    McAuliffe, Laura; Lawes, Joanna; Bell, Suzanna; Barlow, Alex; Ayling, Roger; Nicholas, Robin

    2006-10-31

    Although the role of Mycoplasma wenyonii in disease is still subject to some debate, infections have been reported to result in parasitaemia, anaemia, scrotal and hind limb oedema, tachycardia, pyrexia, infertility, swollen teats, prefemoral lymphadenopathy and decreased milk production. Previously, diagnosis of M. wenyonii has been based on blood smears but is not specific for M. wenyonii and can be difficult to interpret. We have previously described the use of PCR and denaturing gradient gel electrophoresis (DGGE) for the detection and differentiation of Mycoplasma species. DGGE enables the rapid and specific identification of Mycoplasma species and is ideally suited to detecting both mixed infections and new and unusual species. In this study, we have used DGGE with universal primers to detect M. wenyonii DNA from blood samples. DGGE can be used on blood samples as a rapid and specific test for M. wenyonii and can also be used as a screening test for other blood borne pathogens.

  3. Investigations into the oxygen effect during the radiolysis of serum albumin with the help of SDS-polyacrylamide gel electrophoresis

    International Nuclear Information System (INIS)

    Schilling, K.B.G.

    1984-01-01

    In this work the radiation induced structural changes of serum albumin were researched with the help of SDS-polyacrylamide gel electrophoresis. Cow serum albumin (bSA) at a concentration of 1 mg/ml was dissolved in a solution of 10 -2 M sodium phosphate buffer and in an air saturated solution with and without formiate (10 -1 M) and irradiated with 200 kV X-rays up to doses of 1500 Gy in a nitrogen atmosphere. The irradiation products were separated, with and without reduction, to 7.5 and 10% polyacrylamide gel, their molecular weights measured and the quantitative decrease of the monomeric serum albumin with increasing dose estimated using staining methods. With increasing dose in an air atmosphere the amount of bSA decreases, after reduction even more so, and at least 14 different protein fragments whose molecular weights were determined result from this. In the formiate-containing solution a bSA decrease of up to 20% was observed, but only in the reduced solutions. The nitrogen atmosphere during the irradiation resulted in protein aggregate formation (2, 3 and 4 times the molecular weight of the monomeric bSA). Changes in the protein size, structure, and net charge as reasons for the electrophoretic behavior are discussed as well as the importance of the primary radicals during the radiation chemical reactions. (orig./RB) [de

  4. Factors affecting the separation performance of proteins in capillary electrophoresis.

    Science.gov (United States)

    Zhu, Yueping; Li, Zhenqing; Wang, Ping; Shen, Lisong; Zhang, Dawei; Yamaguchi, Yoshinori

    2018-04-15

    Capillary electrophoresis (CE) is an effective tool for protein separation and analysis. Compared with capillary gel electrophoresis (CGE), non-gel sieving capillary electrophoresis (NGSCE) processes the superiority on operation, repeatability and automaticity. Herein, we investigated the effect of polymer molecular weight and concentration, electric field strength, and the effective length of the capillary on the separation performance of proteins, and find that (1) polymer with high molecular weight and concentration favors the separation of proteins, although concentrated polymer hinders its injection into the channel of the capillary due to its high viscosity. (2) The resolution between the adjacent proteins decreases with the increase of electric field strength. (3) When the effective length of the capillary is long, the separation performance improves at the cost of separation time. (4) 1.4% (w/v) hydroxyethyl cellulose (HEC), 100 V/cm voltage and 12 cm effective length offers the best separation for the proteins with molecular weight from 14,400 Da to 97,400 Da. Finally, we employed the optimal electrophoretic conditions to resolve Lysozyme, Ovalbumin, BSA and their mixtures, and found that they were baseline resolved within 15 min. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. Molecular Typing of Acinetobacter Baumannii Clinical Strains in Tehran by Pulsed-Field Gel Electrophoresis

    Directory of Open Access Journals (Sweden)

    Neda Farahani

    2013-03-01

    Full Text Available Background & Objective : Currently, Acinetobacter baumannii is an important nosocomial pathogen insofar as its hospital outbreaks have been described from various geographical areas. Since the discrimination of strains within a species is important for delineating nosocomial outbreaks, this study was conducted with the aim of genotyping the A. baumannii clinical strains in Tehran via the pulsed-field gel electrophoresis (PFGE method, which is the most accurate method used for the typing of bacterial species.   Materials & methods: This study was performed on 70 isolates of acinetobacter baumannii isolated from patients from Baqiyatallah, Rasoole Akram, and Milad hospitals in Tehran. Cultural and biochemical methods were used for the identification of the isolates in species level, and then susceptibility tests were carried out on 50 isolates of A. baumannii using the disk diffusion method. The PFGE method was performed on the isolates by Apa I restriction enzyme. Finally, the results of the PFGE were analyzed. Result: Acinetobacter baumannii strains isolated from hospitals in Tehran showed seven different genetic patterns, two of which were sporadic . Also, genotypic profiles were different in each hospital, and different patterns of genetic resistance to common antibiotics were observed. Conclusion: A lthough diversity was observed among the strains of A. baumannii by the PFGE method in Tehran, no epidemic strains were found among them.  

  6. Difference gel electrophoresis (DiGE) identifies differentially expressed proteins in endoscopically-collected pancreatic fluid

    Science.gov (United States)

    Paulo, Joao A.; Lee, Linda S.; Banks, Peter A.; Steen, Hanno; Conwell, Darwin L.

    2012-01-01

    Alterations in the pancreatic fluid proteome of individuals with chronic pancreatitis may offer insights into the development and progression of the disease. The endoscopic pancreas function test (ePFT) can safely collect large volumes of pancreatic fluid that are potentially amenable to proteomic analyses using difference gel electrophoresis (DiGE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Pancreatic fluid was collected endoscopically using the ePFT method following secretin stimulation from three individuals with severe chronic pancreatitis and three chronic abdominal pain controls. The fluid was processed to minimize protein degradation and the protein profiles of each cohort, as determined by DiGE and LC-MS/MS, were compared. This DiGE-LC-MS/MS analysis reveals proteins that are differentially expressed in chronic pancreatitis compared to chronic abdominal pain controls. Proteins with higher abundance in pancreatic fluid from chronic pancreatitis individuals include: actin, desmoplankin, alpha-1-antitrypsin, SNC73, and serotransferrin. Those of relatively lower abundance include carboxypeptidase B, lipase, alpha-1-antichymotrypsin, alpha-2-macroglobulin, Arp2/3 subunit 4, glyceraldehyde-3-phosphate dehydrogenase, and protein disulfide isomerase. Endoscopic collection (ePFT) in tandem with DiGE-LC-MS/MS is a suitable approach for pancreatic fluid proteome analysis, however, further optimization of our protocol, as outlined herein, may improve proteome coverage in future analyses. PMID:21792986

  7. Agarose gel electrophoresis reveals structural fluidity of a phage T3 DNA packaging intermediate.

    Science.gov (United States)

    Serwer, Philip; Wright, Elena T

    2012-01-01

    We find a new aspect of DNA packaging-associated structural fluidity for phage T3 capsids. The procedure is (i) glutaraldehyde cross-linking of in vivo DNA packaging intermediates for the stabilization of structure and then (ii) determining effective radius by two-dimensional agarose gel electrophoresis (2D-AGE). The intermediates are capsids with incompletely packaged DNA (ipDNA) and without an external DNA segment; these intermediates are called ipDNA-capsids. We initially increase the production of ipDNA-capsids by raising NaCl concentration during in vivo DNA packaging. By 2D-AGE, we find a new state of contracted shell for some particles of one previously identified ipDNA-capsid. The contracted shell-state is found when the ipDNA length/mature DNA length (F) is above 0.17, but not at lower F. Some contracted-shell ipDNA-capsids have the phage tail; others do not. The contracted-shell ipDNA-capsids are explained by premature DNA maturation cleavage that makes accessible a contracted-shell intermediate of a cycle of the T3 DNA packaging motor. The analysis of ipDNA-capsids, rather than intermediates with uncleaved DNA, provides a simplifying strategy for a complete biochemical analysis of in vivo DNA packaging. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Epidemiologic Genotyping of Methicillin-Resistant Staphylococcus aureus (MRSA by Pulsed-Field Gel Electrophoresis (PFGE

    Directory of Open Access Journals (Sweden)

    Maja Ostojić

    2008-08-01

    Full Text Available čćStaphylococcus aureus has long been recognized as one of the leading cause of hospital infections all over the world. Increased frequency of methicillin-resistant Staphylococcus aureus (MRSA in hospitalized patients and possibility of vancomycin resistance requires rapid and reliable characterization of isolates and control of MRSA spread in hospitals. Typing of isolates helps to understand pathogenesis and route of the hospital pathogen spread. In this study in the analysis of an outbreak of MRSA infections in one surgical ward, we used pulsed-field gel electrophoresis (PFGE as a method of typing. PFGE revealed one epidemic strain type A in 13 out of 16 patients, and another two types (type B in two patients and type C in one patient. Discussing the typing results in the ward has changed the admission policy of patients with infected vascular ulcers who were then cured as outpatients, and admitted for surgery after that. This policy resulted with the stopping of the outbreak; during next 2,5 year there was no further MRSA outbreak in the ward. PFGE also showed subtypes which enabled the insight into dynamics of MRSA strain changes during the outbreak. PFGE could be recommended as a screening method in the MRSA outbreak analysis. Because of it’s high discriminatory power still remains the gold standard for MRSA typing

  9. Newborn screening by matrix-assisted laser desorption/ionization mass spectrometry based on parylene-matrix chip.

    Science.gov (United States)

    Kim, Jo-Il; Noh, Joo-Yoon; Kim, Mira; Park, Jong-Min; Song, Hyun-Woo; Kang, Min-Jung; Pyun, Jae-Chul

    2017-08-01

    Newborn screening for diagnosis of phenylketonuria, homocystinuria, and maple syrup urine disease have been conducted by analyzing the concentration of target amino acids using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) based on parylene-matrix chip. Parylene-matrix chip was applied to MALDI-ToF MS analysis reducing the matrix peaks significantly at low mass-to-charge ratio range (m/z  0.98) and the LODs were ranging from 9.0 to 22.9 μg/mL. Effect of proteins in serum was estimated by comparing MALDI-ToF mass spectra of amino acids-spiked serum before and after the methanol extraction. Interference of other amino acids on analysis of target analyte was determined to be insignificant. From these results, MALDI-ToF MS based on parylene-matrix chip could be applicable to medical diagnosis of neonatal metabolic disorders. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Sodium Dedecyl Suphate Polyacrilamide Gel Electrophosis of ...

    African Journals Online (AJOL)

    polyacrilamide gel electrophoresis (SDS-PAGE). The isolates were obtained from the faeces of diarrhoeic children with the age range of 0 t 36 months attending paediatric clinic at the Lagos University Teaching Hospital (LUTH) and Obafemi Awolowo ...

  11. Variations among Japanese of the factor IX gene (F9) detected by PCR-denaturing gradient gel electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Satoh, Chiyoko; Takahashi, Norio; Asakawa, Junichi; Hiyama, Keiko; Kodaira, Meiko (Radiation Effects Research Foundation, Hiroshima (Japan))

    1993-01-01

    In the course of feasibility studies to examine the efficiencies and practicalities of various techniques for screening for genetic variations, the human coagulation factor IX (F9) genes of 63 Japanese families were examined by PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Four target sequences with lengths of 983-2,891 bp from the F9 genes of 126 unrelated individuals from Hiroshima and their 100 children were amplified by PCR, digested with restriction enzymes to approximately 500-bp fragments, and examined by DGGE - a total of 6,724 bp being examined per individual. GC-rich sequences (GC-clamps) of 40 bp were attached to both ends of the target sequences, as far as was feasible. Eleven types of new nucleotide substitutions were detected in the population, none of which produced RFLPs or caused hemophilia B. By examining two target sequences in a single lane, approximately 8,000 bp in a diploid individual could be examined. This approach is very effective for the detection of variations in DNA and is applicable to large-scale population studies. 46 refs., 3 figs., 1 tab.

  12. Denaturing gradient gel electrophoresis (DGGE as a powerful novel alternative for differentiation of epizootic ISA virus variants.

    Directory of Open Access Journals (Sweden)

    Marisela Carmona

    Full Text Available Infectious Salmon Anemia is a devastating disease critically affecting world-wide salmon production. Chile has been particularly stricken by this disease which in all cases has been directly related with its causative agent, a novel orthomyxovirus which presents specific and distinctive infective features. Among these, two molecular markers have been directly associated with pathogenicity in two of the eight RNA sub genomic coding units of the virus: an insertion hot spot region present in viral segment 5 and a Highly Polymorphic Region (HPR located in viral segment 6. Here we report the successful adaptation of a PCR-dependent denaturing gel electrophoresis technique (DGGE, which enables differentiation of selected reported HPR epizootic variants detected in Chile. At the same time, the technique allows us to distinguish one nucleotide differences in sequences associated with the intriguing, and still not well-understood, insertion events which tend to occur on RNA Segment 5. Thus, the versatility of the technique opens new opportunities for improved understanding of the complex biology of all ISA variants as well as possible applications to other highly variable pathogens.

  13. Mapping and identification of HeLa cell proteins separated by immobilized pH-gradient two-dimensional gel electrophoresis and construction of a two-dimensional polyacrylamide gel electrophoresis database

    DEFF Research Database (Denmark)

    Shaw, AC; Rossel Larsen, M; Roepstorff, P

    1999-01-01

    The HeLa cell line, a human adenocarcinoma, is used in many research fields, since it can be infected with a wide range of viruses and intracellular bacteria. Therefore, the mapping of HeLa cell proteins is useful for the investigation of parasite host cell interactions. Because of the recent imp...... these and future data accessible for interlaboratory comparison, we constructed a 2-D PAGE database on the World Wide Web....... the mapping of [35S]methionine/cysteine-labeled HeLa cell proteins with the 2-D PAGE (IPG)-system, using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and N-terminal sequencing for protein identification. To date 21 proteins have been identified and mapped. In order to make...

  14. Development of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis reference method for the analysis and identification of fish species in raw and heat-processed samples : A collaborative study

    DEFF Research Database (Denmark)

    Pineiro, C.; Barros-Velazquez, J.; Perez-Martin, R.I.

    1999-01-01

    A collaborative study was carried out in seven European labs with the aim of achieving a sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) standard operation procedure to identify fish species in raw and cooked samples. Urea and SDS-containing solutions were evaluated...... silver stained, yielded good results and afforded higher reproducibility, thus allowing a better matching of results among the laboratories participating in this collaborative study. Under the optimized technical conditions described above, all the fish species tested, either raw and cooked, yielded...... seemed not to be influenced so much by the state of the sample (raw, cooked at 60 degrees C, cooked at 85 degrees C). Desalting, ultrafiltration or treatment with RNase/DNase did not improve the discriminatory power of the protein patterns. Commercial homogeneous 15% ExcelGels, especially when they were...

  15. Characterization of carbohydrates using highly fluorescent 2-aminobenzoic acid tag following gel electrophoresis of glycoproteins.

    Science.gov (United States)

    Anumula, K R; Du, P

    1999-11-15

    Application of the most sensitive fluorescent label 2-aminobenzoic acid (anthranilic acid, AA) for characterization of carbohydrates from the glycoproteins ( approximately 15 pmol) separated by polyacrylamide gel electrophoresis is described. AA label is used for the determination of both monosaccharide composition and oligosaccharide map. For the monosaccharide determination, bands containing the glycoprotein of interest are excised from the polyvinylidene fluoride (PVDF) membrane blots, hydrolyzed in 20% trifluoroacetic acid, derivatized, and analyzed by C-18 reversed-phase high-performance liquid chromatography. For the oligosaccharide mapping, bands were digested with peptide N-glycosidase F (PNGase F) in order to release the N-linked oligosaccharides, derivatized, and analyzed by normal-phase anion-exchange chromatography. For convenience, the PNGase F digestion was performed in 1:100 diluted ammonium hydroxide overnight. The oligosaccharide yield from ammonium hydroxide-PNGase F digestion was better or equal to all the other reported procedures, and the presumed "oligosaccharide-amine" product formed in the reaction mixture did not interfere with labeling of the oligosaccharides under the conditions used for derivatization. Sequencing of oligosaccharides can be performed using the same mapping method following treatment with an array of glycosidases. In addition, the mapping method is useful for determining the relative and simultaneous distribution of sialic acid and fucose. Copyright 1999 Academic Press.

  16. Indirect fluorometric detection techniques on thin layer chromatography and effect of ultrasound on gel electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Yinfa, Ma.

    1990-12-10

    Thin-layer chromatography (TLC) is a broadly applicable separation technique. It offers many advantages over high performance liquid chromatography (HPLC), such as easily adapted for two-dimensional separation, for whole-column'' detection and for handling multiple samples, etc. However, due to its draggy development of detection techniques comparing with HPLC, TLC has not received the attention it deserves. Therefore, exploring new detection techniques is very important to the development of TLC. It is the principal of this dissertation to present a new detection method for TLC -- indirect fluorometric detection method. This detection technique is universal sensitive, nondestructive, and simple. This will be described in detail from Sections 1 through Section 5. Section 1 and 3 describe the indirect fluorometric detection of anions and nonelectrolytes in TLC. In Section 2, a detection method for cations based on fluorescence quenching of ethidium bromide is presented. In Section 4, a simple and interesting TLC experiment is designed, three different fluorescence detection principles are used for the determination of caffeine, saccharin and sodium benzoate in beverages. A laser-based indirect fluorometric detection technique in TLC is developed in Section 5. Section 6 is totally different from Sections 1 through 5. An ultrasonic effect on the separation of DNA fragments in agarose gel electrophoresis is investigated. 262 refs.

  17. Use of denaturing gradient gel electrophoresis to detect Actinobacteria associated with the human faecal microbiota.

    Science.gov (United States)

    Hoyles, Lesley; Clear, Jessica A; McCartney, Anne L

    2013-08-01

    With the exceptions of the bifidobacteria, propionibacteria and coriobacteria, the Actinobacteria associated with the human gastrointestinal tract have received little attention. This has been due to the seeming absence of these bacteria from most clone libraries. In addition, many of these bacteria have fastidious growth and atmospheric requirements. A recent cultivation-based study has shown that the Actinobacteria of the human gut may be more diverse than previously thought. The aim of this study was to develop a denaturing gradient gel electrophoresis (DGGE) approach for characterizing Actinobacteria present in faecal samples. Amount of DNA added to the Actinobacteria-specific PCR used to generate strong PCR products of equal intensity from faecal samples of five infants, nine adults and eight elderly adults was anti-correlated with counts of bacteria obtained using fluorescence in situ hybridization probe HGC69A. A nested PCR using Actinobacteria-specific and universal PCR-DGGE primers was used to generate profiles for the Actinobacteria. Cloning of sequences from the DGGE bands confirmed the specificity of the Actinobacteria-specific primers. In addition to members of the genus Bifidobacterium, species belonging to the genera Propionibacterium, Microbacterium, Brevibacterium, Actinomyces and Corynebacterium were found to be part of the faecal microbiota of healthy humans. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Development of an in situ evaluation system for neural cells using extracellular matrix-modeled gel culture.

    Science.gov (United States)

    Nagai, Takayuki; Ikegami, Yasuhiro; Mizumachi, Hideyuki; Shirakigawa, Nana; Ijima, Hiroyuki

    2017-10-01

    Two-dimensional monolayer culture is the most popular cell culture method. However, the cells may not respond as they do in vivo because the culture conditions are different from in vivo conditions. However, hydrogel-embedding culture, which cultures cells in a biocompatible culture substrate, can produce in vivo-like cell responses, but in situ evaluation of cells in a gel is difficult. In this study, we realized an in vivo-like environment in vitro to produce cell responses similar to those in vivo and established an in situ evaluation system for hydrogel-embedded cell responses. The extracellular matrix (ECM)-modeled gel consisted of collagen and heparin (Hep-col) to mimic an in vivo-like environment. The Hep-col gel could immobilize growth factors, which is important for ECM functions. Neural stem/progenitor cells cultured in the Hep-col gel grew and differentiated more actively than in collagen, indicating an in vivo-like environment in the Hep-col gel. Second, a thin-layered gel culture system was developed to realize in situ evaluation of the gel-embedded cells. Cells in a 200-μm-thick gel could be evaluated clearly by a phase-contrast microscope and immunofluorescence staining through reduced optical and diffusional effects. Finally, we found that the neural cells cultured in this system had synaptic connections and neuronal action potentials by immunofluorescence staining and Ca 2+ imaging. In conclusion, this culture method may be a valuable evaluation system for neurotoxicity testing. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  19. Improved protocol for isolation of Campylobacter spp. from retail broiler meat and use of pulsed field gel electrophoresis for the typing of isolates.

    Science.gov (United States)

    Oyarzabal, Omar A; Williams, Aretha; Zhou, Ping; Samadpour, Mansour

    2013-10-01

    To improve the detection of Campylobacter spp. in retail broiler meat, a reference method (R subsamples) based on the enrichment of 25 g of meat in Bolton broth at 42°C under microaerobiosis was compared with an alternative method (A subsamples) consisting in the rinsing of meat samples for 30s in buffered peptone water with antimicrobials with incubation at 42°C under aerobiosis. One piece of meat (breasts, tenderloins and thighs) was rinse in experiment 1 (A1) and two pieces in experiment 2 (A2). Campylobacter spp. were isolated on agar plates and identified by PCR. Retail samples in Alabama had less prevalence (P ≤ 0.05) than samples in the state of Washington. The percentage of positive was higher (P ≤ 0.05) in A than in R subsamples and rinsing two pieces of meat yielded the highest percentage of positive subsamples. R subsamples showed variations in the prevalence by product. However, A subsamples had similar prevalence of positives among products compare to the result from reference method. More Campylobacter coli isolates were collected in A2 subsamples. Pulse field gel electrophoresis (PFGE) was used as subtyping method to study the genome similarity among the isolates from all methods. A larger diversity of isolates were detected by PFGE in A2 subsamples. Denaturing gradient gel electrophoresis analysis suggested that the initial bacterial populations of the meat samples impact the final bacterial profile after enrichment. Rinsing broiler meats was less time consuming, required less sample preparation and was more sensitive than the reference method for the isolation of naturally occurring Campylobacter spp. This new method could help with epidemiological and intervention studies to control Campylobacter spp. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Electrophoretic deposition of sol-gel-derived ceramic coatings

    International Nuclear Information System (INIS)

    Zhang, Y.; Crooks, R.M.

    1992-01-01

    In this paper the physical, optical, and chemical characteristics of electrophoretically and dip-coated sol-gel ceramic films are compared. The results indicate that electrophoresis may allow a higher level of control over the chemistry and structure of ceramic coatings than dip-coating techniques. For example, controlled-thickness sol-gel coatings can be prepared by adjusting the deposition time or voltage. Additionally, electrophoretic coatings can be prepared in a four-component alumino-borosilicate sol display interesting optical characteristics. For example, the ellipsometrically-measured refractive indices of electrophoretic coatings are higher than the refractive indices of dip-coated films cast from identical sols, and they are also higher than any of the individual sol components. This result suggests that there are physical and/or chemical differences between films prepared by dip-coating and electrophoresis

  1. Adipose Extracellular Matrix/Stromal Vascular Fraction Gel Secretes Angiogenic Factors and Enhances Skin Wound Healing in a Murine Model

    Directory of Open Access Journals (Sweden)

    Mingliang Sun

    2017-01-01

    Full Text Available Mesenchymal stem cells are an attractive cell type for cytotherapy in wound healing. The authors recently developed a novel, adipose-tissue-derived, injectable extracellular matrix/stromal vascular fraction gel (ECM/SVF-gel for stem cell therapy. This study was designed to assess the therapeutic effects of ECM/SVF-gel on wound healing and potential mechanisms. ECM/SVF-gel was prepared for use in nude mouse excisional wound healing model. An SVF cell suspension and phosphate-buffered saline injection served as the control. The expression levels of vascular endothelial growth factor (VEGF, basic fibroblast growth factor (bFGF, and monocyte chemotactic protein-1 (MCP-1 in ECM/SVF-gel were analyzed at different time points. Angiogenesis (tube formation assays of ECM/SVF-gel extracts were evaluated, and vessels density in skin was determined. The ECM/SVF-gel extract promoted tube formation in vitro and increased the expression of the angiogenic factors VEGF and bFGF compared with those in the control. The expression of the inflammatory chemoattractant MCP-1 was high in ECM/SVF-gel at the early stage and decreased sharply during the late stage of wound healing. The potent angiogenic effects exerted by ECM/SVF-gel may contribute to the improvement of wound healing, and these effects could be related to the enhanced inflammatory response in ECM/SVF-gel during the early stage of wound healing.

  2. 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.

  3. Typing of Ochrobactrum anthropi clinical isolates using automated repetitive extragenic palindromic-polymerase chain reaction DNA fingerprinting and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

    Science.gov (United States)

    Quirino, Angela; Pulcrano, Giovanna; Rametti, Linda; Puccio, Rossana; Marascio, Nadia; Catania, Maria Rosaria; Matera, Giovanni; Liberto, Maria Carla; Focà, Alfredo

    2014-03-22

    Ochrobactrum anthropi (O. anthropi), is a non-fermenting gram-negative bacillus usually found in the environment. Nevertheless, during the past decade it has been identified as pathogenic to immunocompromised patients. In this study, we assessed the usefulness of the automated repetitive extragenic palindromic-polymerase chain reaction (rep-PCR-based DiversiLab™ system, bioMèrieux, France) and of matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF MS) for typing of twentythree O. anthropi clinical isolates that we found over a four-months period (from April 2011 to August 2011) in bacteriemic patients admitted in the same operative unit of our hospital. Pulsed-field gel electrophoresis (PFGE), commonly accepted as the gold standard technique for typing, was also used. Analysis was carried out using the Pearson correlation coefficient to determine the distance matrice and the unweighted pair group method with arithmetic mean (UPGMA) to generate dendogram. Rep-PCR analysis identified four different patterns: three that clustered together with 97% or more pattern similarity, and one whose members showed < 95% pattern similarity. Interestingly, strains isolated later (from 11/06/2011 to 24/08/2011) displayed a pattern with 99% similarity. MALDI-TOF MS evaluation clustered the twentythree strains of O. anthropi into a single group containing four distinct subgroups, each comprising the majority of strains clustering below 5 distance levels, indicating a high similarity between the isolates. Our results indicate that these isolates are clonally-related and the methods used afforded a valuable contribution to the epidemiology, prevention and control of the infections caused by this pathogen.

  4. Characterization of lysozyme films produced by matrix assisted pulsed laser evaporation (MAPLE)

    DEFF Research Database (Denmark)

    Purice, Andreea; Schou, Jørgen; Kingshott, Peter

    2007-01-01

    Thin lysozyme films of thickness up to more than 100 nm have been produced in a dry environment by MAPLE (matrix assisted pulsed laser evaporation) from a water ice matrix. Analysis of the films demonstrates that a significant part of the lysozyme molecules is transferred to the substrate without...

  5. Enhanced resolution of DNA restriction fragments: A procedure by two-dimensional electrophoresis and double-labeling

    International Nuclear Information System (INIS)

    Yi, M.; Au, L.C.; Ichikawa, N.; Ts'o, P.O.

    1990-01-01

    A probe-free method was developed to detect DNA rearrangement in bacteria based on the electrophoretic separation of twice-digested restriction fragments of genomic DNA into a two-dimensional (2-D) pattern. The first restriction enzyme digestion was done in solution, followed by electrophoresis of the restriction fragments in one dimension. A second restriction enzyme digestion was carried out in situ in the gel, followed by electrophoresis in a second dimension perpendicular to the first electrophoresis. The 2-D pattern provides for the resolution of 300-400 spots, which are defined and indexed by an x,y coordinate system with size markers. This approach has greatly increased the resolution power over conventional one-dimensional (1-D) electrophoresis. To study DNA rearrangement, a 2-D pattern from a test strain was compared with the 2-D pattern from a reference strain. After the first digestion, genomic DNA fragments from the test strain were labeled with 35S, while those from the reference strain were labeled with 32P. This was done to utilize the difference in the energy emission of 35S and 32P isotopes for autoradiography when two x-ray films were exposed simultaneously on top of the gel after the 2-D electrophoresis. The irradiation from the decay of 35S exposed only the lower film, whereas the irradiation from the decay of 32P exposed both the lower and upper films. Different DNA fragments existed in the test DNA compared with the reference DNA can be identified unambiguously by the differential two 2-D patterns produced on two films upon exposure to the 35S and 32P fragments in the same gel. An appropriate photographic procedure further simplified the process, allowing only the difference in DNA fragments between these two patterns to be shown in the map

  6. Studies on lectins. XXXII. Application of affinity electrophoresis to the study of the interaction of lectins and their derivatives with sugars.

    Science.gov (United States)

    Horejsí, V; Tichá, M; Kocourek, J

    1977-09-29

    Affinity electrophoresis was used to study the sugar binding heterogeneity of lectins or their derivatives. Commercial and demetallized preparations of concanavalin A could be resolved by affinity electrophoresis into three components with different affinity to immobilized sugar. Similarly the Vicia cracca lectin obtained by affinity chromatography behaved on affinity gels as a mixture of active and inactive molecular species. Affinity electrophoresis has shown that the nonhemagglutinating acetylated lentil lectin and photo-oxidized or sulfenylated pea lectin retain their sugar binding properties; dissociation constants of saccharide complexes of these derivatives are similar to those of native lectins. The presence of specific immobilized sugar in the affinity gel improved the resolution of isolectins from Dolichos biflorus and Ricinus communis seeds.

  7. Ionization techniques in capillary electrophoresis-mass spectrometry: principles, design, and application.

    Science.gov (United States)

    Hommerson, Paul; Khan, Amjad M; de Jong, Gerhardus J; Somsen, Govert W

    2011-01-01

    A major step forward in the development and application of capillary electrophoresis (CE) was its coupling to ESI-MS, first reported in 1987. More than two decades later, ESI has remained the principal ionization technique in CE-MS, but a number of other ionization techniques have also been implemented. In this review the state-of-the-art in the employment of soft ionization techniques for CE-MS is presented. First the fundamentals and general challenges of hyphenating conventional CE and microchip electrophoresis with MS are outlined. After elaborating on the characteristics and role of ESI, emphasis is put on alternative ionization techniques including sonic spray ionization (SSI), thermospray ionization (TSI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), matrix-assisted laser desorption ionization (MALDI) and continuous-flow fast atom bombardment (CF-FAB). The principle of each ionization technique is outlined and the experimental set-ups of the CE-MS couplings are described. The strengths and limitations of each ionization technique with respect to CE-MS are discussed and the applicability of the various systems is illustrated by a number of typical examples. Copyright © 2011 Wiley Periodicals, Inc.

  8. Heat stress-induced loss of eukaryotic initiation factor 5A (eIF-5A) in a human pancreatic cancer cell line, MIA PaCa-2, analyzed by two-dimensional gel electrophoresis.

    Science.gov (United States)

    Takeuchi, Kana; Nakamura, Kazuyuki; Fujimoto, Masanori; Kaino, Seiji; Kondoh, Satoshi; Okita, Kiwamu

    2002-02-01

    Alterations of intracellular proteins during the process of heat stress-induced cell death of a human pancreatic cancer cell line, MIA PaCa-2, were investigated using two-dimensional gel electrophoresis (2-DE), agarose gel electrophoresis, and cell biology techniques. Incubation of MIA PaCa-2 at 45 degrees C for 30 min decreased the cell growth rate and cell viability without causing chromosomal DNA fragmentation. Incubation at 51 degrees C for 30 min suppressed cell growth and again led to death without DNA fragmentation. The cell death was associated with the loss of an intracellular protein of M(r) 17,500 and pI 5.2 on 2-DE gel. This protein was determined to be eukaryotic initiation factor SA (eIF-5A) by microsequencing of the N-terminal region of peptide fragments obtained by cyanogen bromide treatment of the protein blotted onto a polyvinylidene difluoride (PVDF) membrane. The sequences detected were QXSALRKNGFVVLKGRP and STSKTGXHGHAKVHLVGID, which were homologous with the sequence of eIF-5A from Gln 20 to Pro 36 and from Ser 43 to Asp 61, respectively. Furthermore, the result of sequencing suggested that the protein was an active form of hypusinated eIF-5A, because Lys 46 could be detected but not Lys 49, which is the site for hypusination. These results suggest that loss of the active form of eIF-5A is an important factor in the irreversible process of heat stress-induced death of MIA PaCa-2 cells.

  9. Comparison of lipoprotein electrophoresis and apolipoprotein e genotyping in investigating dysbetalipoproteinemia

    International Nuclear Information System (INIS)

    Ahmed, F.; Kadiki, A.E.

    2017-01-01

    Dysbetalipoproteinemia is often associated with apolipoprotein E2E2 homozygosity; however, lipoprotein electrophoresis may also be used to assist in the diagnosis. The aim of this study was to compare apolipoprotein E (apo E) genotyping and lipoprotein electrophoresis in investigating dysbetalipoproteinemia. Data were collected over a three-year period from a lipid clinic in a tertiary referral centre and reviewed for apo E genotyping and lipoprotein electrophoresis. Sixty-two patients had both apo E genotyping and lipoprotein electrophoresis. Of these, 16 patients showed broad beta band on electrophoresis. However, only 3 of them had apo E2E2 homozygosity on genotyping. Lipoprotein electrophoresis and apo E genotyping results showed poor concordance. This was primarily due to visual interpretation error of lipoprotein electrophoresis which may over diagnose dysbetalipoproteinemia. (author)

  10. Comparison of Lipoprotein Electrophoresis and Apolipoprotein E Genotyping in Investigating Dysbetalipoproteinemia.

    Science.gov (United States)

    Ahmed, Farhan; El-Kadiki, Alia; Gibbons, Stephen

    2017-06-01

    Dysbetalipoproteinemia is often associated with apolipoprotein E2E2 homozygosity; however, lipoprotein electrophoresis may also be used to assist in the diagnosis. The aim of this study was to compare apolipoprotein E (apo E) genotyping and lipoprotein electrophoresis in investigating dysbetalipoproteinemia. Data were collected over a three-year period from a lipid clinic in a tertiary referral centre and reviewed for apo E genotyping and lipoprotein electrophoresis. Sixty-two patients had both apo E genotyping and lipoprotein electrophoresis. Of these, 16 patients showed broad beta band on electrophoresis. However, only 3 of them had apo E2E2 homozygosity on genotyping. Lipoprotein electrophoresis and apo E genotyping results showed poor concordance. This was primarily due to visual interpretation error of lipoprotein electrophoresis which may over diagnose dysbetalipoproteinemia.

  11. Studies on the proteinaceous gel secretion from the skin of the ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-12-15

    Dec 15, 2009 ... and SDS-PAGE analysis of the proteinaceous gel of catfish, A. ... SDS–PAGE. One dimensional sodium dodecyl sulphate (SDS) poly acrylamide gel electrophoresis (PAGE) was carried out to estimate the mole- cular weight of the ... 0.2% Triton X-100, was defined as percent hemolysis (Park et al.,. 1997).

  12. Matrix molecularly imprinted mesoporous sol-gel sorbent for efficient solid-phase extraction of chloramphenicol from milk.

    Science.gov (United States)

    Samanidou, Victoria; Kehagia, Maria; Kabir, Abuzar; Furton, Kenneth G

    2016-03-31

    Highly selective and efficient chloramphenicol imprinted sol-gel silica based inorganic polymeric sorbent (sol-gel MIP) was synthesized via matrix imprinting approach for the extraction of chloramphenicol in milk. Chloramphenicol was used as the template molecule, 3-aminopropyltriethoxysilane (3-APTES) and triethoxyphenylsilane (TEPS) as the functional precursors, tetramethyl orthosilicate (TMOS) as the cross-linker, isopropanol as the solvent/porogen, and HCl as the sol-gel catalyst. Non-imprinted sol-gel polymer (sol-gel NIP) was synthesized under identical conditions in absence of template molecules for comparison purpose. Both synthesized materials were characterized by Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FT-IR) and nitrogen adsorption porosimetry, which unambiguously confirmed their significant structural and morphological differences. The synthesized MIP and NIP materials were evaluated as sorbents for molecularly imprinted solid phase extraction (MISPE) of chloramphenicol in milk. The effect of critical extraction parameters (flow rate, elution solvent, sample and eluent volume, selectivity coefficient, retention capacity) was studied in terms of retention and desorption of chloramphenicol. Competition and cross reactivity tests have proved that sol-gel MIP sorbent possesses significantly higher specific retention and enrichment capacity for chloramphenicol compared to its non-imprinted analogue. The maximum imprinting factor (IF) was found as 9.7, whereas the highest adsorption capacity of chloramphenicol by sol-gel MIP was 23 mg/g. The sol-gel MIP was found to be adequately selective towards chloramphenicol to provide the necessary minimum required performance limit (MRPL) of 0.3 μg/kg set forth by European Commission after analysis by LC-MS even without requiring time consuming solvent evaporation and sample reconstitution step, often considered as an integral part in solid phase extraction work-flow. Intra and

  13. Investigations into ultraviolet matrix-assisted laser desorption

    Energy Technology Data Exchange (ETDEWEB)

    Heise, Theodore W. [Iowa State Univ., Ames, IA (United States)

    1993-07-01

    Matrix-assisted laser desorption (MALD) is a technique for converting large biomolecules into gas phase ions. Some characteristics of the commonly used uv matrices are determined. Solubilities in methanol range from 0.1 to 0.5 M. Solid phase absorption spectra are found to be similar to solution, but slightly red-shifted. Acoustic and quartz crystal microbalance signals are investigated as possible means of uv-MALD quantitation. Evidence for the existence of desorption thresholds is presented. Threshold values are determined to be in the range of 2 to 3 MW/cm2. A transient imaging technique based on laser-excited fluorescence for monitoring MALD plumes is described. Sensitivity is well within the levels required for studying matrix-assisted laser desorption, where analyte concentrations are significantly lower than those in conventional laser desorption. Results showing the effect of film morphology, particularly film thickness, on plume dynamics are presented. In particular, MALD plumes from thicker films tend to exhibit higher axial velocities. Fluorescent labeling of protein and of DNA is used to allow imaging of their uv-MALD generated plumes. Integrated concentrations are available with respect to time, making it possible to assess the rate of fragmentation. The spatial and temporal distributions are important for the design of secondary ionization schemes to enhance ion yields and for the optimization of ion collection in time-of-flight MS instruments to maximize resolution. Such information could also provide insight into whether ionization is closely associated with the desorption step or whether it is a result of subsequent collisions with the matrix gas (e.g., proton transfer). Although the present study involves plumes in a normal atmosphere, adaptation to measurements in vacuum (e.g., inside a mass spectrometer) should be straightforward.

  14. Dynamic computer simulations of electrophoresis: three decades of active research.

    Science.gov (United States)

    Thormann, Wolfgang; Caslavska, Jitka; Breadmore, Michael C; Mosher, Richard A

    2009-06-01

    Dynamic models for electrophoresis are based upon model equations derived from the transport concepts in solution together with user-inputted conditions. They are able to predict theoretically the movement of ions and are as such the most versatile tool to explore the fundamentals of electrokinetic separations. Since its inception three decades ago, the state of dynamic computer simulation software and its use has progressed significantly and Electrophoresis played a pivotal role in that endeavor as a large proportion of the fundamental and application papers were published in this periodical. Software is available that simulates all basic electrophoretic systems, including moving boundary electrophoresis, zone electrophoresis, ITP, IEF and EKC, and their combinations under almost exactly the same conditions used in the laboratory. This has been employed to show the detailed mechanisms of many of the fundamental phenomena that occur in electrophoretic separations. Dynamic electrophoretic simulations are relevant for separations on any scale and instrumental format, including free-fluid preparative, gel, capillary and chip electrophoresis. This review includes a historical overview, a survey of current simulators, simulation examples and a discussion of the applications and achievements of dynamic simulation.

  15. Mapping of Chlamydia trachomatis proteins by immobiline-polyacrylamide two-dimensional electrophoresis: spot identification by N-terminal sequencing and immunoblotting

    DEFF Research Database (Denmark)

    Bini, L; Sanchez-Campillo, M; Santucci, A

    1996-01-01

    Proteins from purified elementary bodies of Chlamydia trachomatis were separated by two-dimensional gel electrophoresis on nonlinear wide-range immobilized pH gradients in the first dimension and polyacrylamide gradient gels in the second dimension. The maps obtained with this system are highly...

  16. Calcium alginate gels as stem cell matrix-making paracrine stem cell activity available for enhanced healing after surgery.

    Directory of Open Access Journals (Sweden)

    Andreas Schmitt

    Full Text Available Regeneration after surgery can be improved by the administration of anabolic growth factors. However, to locally maintain these factors at the site of regeneration is problematic. The aim of this study was to develop a matrix system containing human mesenchymal stem cells (MSCs which can be applied to the surgical site and allows the secretion of endogenous healing factors from the cells. Calcium alginate gels were prepared by a combination of internal and external gelation. The gelling behaviour, mechanical stability, surface adhesive properties and injectability of the gels were investigated. The permeability of the gels for growth factors was analysed using bovine serum albumin and lysozyme as model proteins. Human MSCs were isolated, cultivated and seeded into the alginate gels. Cell viability was determined by AlamarBlue assay and fluorescence microscopy. The release of human VEGF and bFGF from the cells was determined using an enzyme-linked immunoassay. Gels with sufficient mechanical properties were prepared which remained injectable through a syringe and solidified in a sufficient time frame after application. Surface adhesion was improved by the addition of polyethylene glycol 300,000 and hyaluronic acid. Humans MSCs remained viable for the duration of 6 weeks within the gels. Human VEGF and bFGF was found in quantifiable concentrations in cell culture supernatants of gels loaded with MSCs and incubated for a period of 6 weeks. This work shows that calcium alginate gels can function as immobilization matrices for human MSCs.

  17. Study of the mechanisms of matrix assisted laser desorption / ionization

    International Nuclear Information System (INIS)

    Manuelli, Pascal

    1995-01-01

    This research thesis aims at a better knowledge of some aspects of a complex mechanism: the matrix-assisted laser desorption/ionization (MALDI). The author first proposes a comparative analysis of results obtained by time-of-flight (TOF) mass spectrometry and by Fourier transform mass spectrometry. He reports the study of the matrix role (notably a polymeric matrix) as a matter submitted to laser desorption. In this respect, the influence of the incident wavelength has been studied. The author also reports a comparative of ions produced by matrix laser desorption (study performed by Fourier transform mass spectrometry) and of neutral molecules (study performed by flash pyrolysis coupled with gas chromatography and with mass spectrometry). Finally, results obtained on derivatives and complexes based on beta-cyclodextrins highlight benefits as well as limitations of this technique [fr

  18. Comparative analysis of inflamed and non-inflamed colon biopsies reveals strong proteomic inflammation profile in patients with ulcerative colitis

    DEFF Research Database (Denmark)

    Poulsen, Nina Aagaard; Andersen, Vibeke; Moller, Jens Christian

    2012-01-01

    colonic biopsies were characterized using 2D-gel electrophoresis, and peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied for identification of differently expressed protein spots. Results: A total of 597 spots were...... mucosa with acute UC is strong. Totally, 43 individual protein spots were identified, including proteins involved in energy metabolism (triosephosphate isomerase, glycerol-3-phosphate-dehydrogenase, alpha enolase and L-lactate dehydrogenase B-chain) and in oxidative stress (superoxide dismutase...

  19. Efficacy of Pulsed-Field Gel Electrophoresis and Repetitive Element Sequence-Based PCR in Typing of Salmonella Isolates from Assam, India.

    Science.gov (United States)

    Gogoi, Purnima; Borah, Probodh; Hussain, Iftikar; Das, Leena; Hazarika, Girin; Tamuly, Shantanu; Barkalita, Luit Moni

    2018-05-01

    A total of 12 Salmonella isolates belonging to different serovars, viz , Salmonella enterica serovar Enteritidis ( n = 4), Salmonella enterica serovar Weltevreden ( n = 4), Salmonella enterica serovar Newport ( n = 1), Salmonella enterica serovar Litchifield ( n = 1), and untypeable strains ( n = 2) were isolated from 332 diarrheic fecal samples collected from animals, birds, and humans. Of the two molecular typing methods applied, viz , repetitive element sequence-based PCR (REP-PCR) and pulsed-field gel electrophoresis (PFGE), PFGE could clearly differentiate the strains belonging to different serovars as well as differentiate between strains of the same serovar with respect to their source of isolation, whereas REP-PCR could not differentiate between strains of the same serovar. Thus, it can be suggested that PFGE is more useful and appropriate for molecular typing of Salmonella isolates during epidemiological investigations than REP-PCR. Copyright © 2018 American Society for Microbiology.

  20. Single-cell gel electrophoresis applied to the analysis of UV-C damage and its repair in human cells

    International Nuclear Information System (INIS)

    Gedik, C.M.; Collins, A.R.; Ewen, S.W.B.

    1992-01-01

    The authors have adapted procedure of single cell gel electrophoresis (SCGE) for studying DNA damage and repair induced by UV-C-radiation, using HeLa cells. UV-C itself does not induce DNA breakage, and though cellular repair of UV-C damage produces DNA breaks as intermediates, these are too short-lived to be detected by SCGE. Incubation of UV-C-irradiated cells with the DNA synthesis inhibitor aphidicolin causes accumulation of incomplete repair sites to a level readily detected by SCGE even after doses as low as 0.5 J m -2 and incubation for as little as 5 min. The authors also studied UV-C-dependent incision, repair synthesis and ligation in permeable cells. Finally, key incubated permeable cells, after UV-C-irradiation, with exogenous UV endonuclease, examined consequent breaks both by SCGE and by alkaline unwinding to express results of the electrophoretic method in terms of DNA break frequencies. The sensitivity of the SCGE technique can thus be estimated; as few as 0.1 DNA breaks per 10 9 daltons are detected. (Author)

  1. Genotyping of Yersinia enterocolitica biotype 1A strains from clinical and nonclinical origins by pulsed-field gel electrophoresis.

    Science.gov (United States)

    Campioni, Fábio; Falcão, Juliana P

    2014-06-01

    Yersinia enterocolitica biotype 1A (B1A) strains are considered mainly nonpathogenic. However, some studies considered strains of this biotype to be the causal agents of infections in humans and animals. In South America, there are no studies that have compared clinical and nonclinical strains of B1A typed by pulsed-field gel electrophoresis (PFGE) and none that have compared the capability of different enzymes on typing these strains. This study typed 51 Y. enterocolitica B1A strains isolated in Brazil and Chile by PFGE, testing the enzymes XbaI, NotI, and XhoI. The resulting dendrograms discriminated the strains in 47, 40, and 49 pulsotypes generated by the cleavage with the enzymes XbaI, NotI, and XhoI, respectively. The majority of the strains were grouped independently of their clinical or nonclinical origins. The high discriminatory power of PFGE confirmed the heterogeneity of B1A strains but could not divide the strains studied into clusters that differed in the frequency of some virulence genes as observed in studies using other methodologies.

  2. Assessment of DNA damage and Chromosome aberration in human lymphocyte exposed to low dose radiation detected by FISH(Fluorescence In Situ Hybridization) and SCGE(Single Cell Gel Electrophoresis)

    International Nuclear Information System (INIS)

    Chung, Hai Won; Kim, Su Young; Kim, Byung Mo; Kim, Sun Jin; Ha, Sung Whan; Kim, Tae Hwan; Cho, Chul Koo

    2000-01-01

    Comparative study was performed for the assessment of DNA damage and Chromosomal aberration in human lymphocyte exposed to low dose radiation using Fluorescence In Situ Hybridization(FISH) and Single Cell Gel Electrophoresis(SCGE). Chromosomal aberrations in human lymphocyte exposed to radiation at doses of 5, 10, 30 and 50cGy were analysed with whole chromosome-specific probes by human chromosome 1, 2 and 4 according to PAINT system. FISH with chromosome-specific probe has been used to be a valid and rapid method for detection of chromosome rearrangements induced by low dose radiation. The frequencies of stable translocation per cell equivalents were 0.0116, 0.0375, 0.0407, 0.0727 and 0.0814 for 0, 5, 10, 30 and 50cGy, respectively, and those of dicentric were 0.00, 0.0125, 0.174, 0.0291 and 0.0407 respectively. Radiation induced DNA damage in human lymphocyte in a dose-dependent manner at low doses from 5cGy to 50cGy, which were analysed by single Cell Gel Electrophoresis(SCGE). From above results, FISH seemed to be useful for radiation biodosimetry by which the frequencies of stable aberrations in human lymphocyte can be observed more easily than by conventional method and SCGE also seemed to be sensitive method for detecting DNA damage by low dose radiation exposure, so that those methods will improve our technique to perform meaningful biodosimetry for radiation at low doses

  3. Visible-light-induced hydrogen production over Pt-Eosin Y catalysts with high surface area silica gel as matrix

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xiaojie [State Key Laboratory for Oxo Synthesis and Selective Oxidation, Lanzhou Institute of Chemical Physics, The Chinese Academy of Sciences, Lanzhou 730000 (China); Graduate University of the Chinese Academy of Sciences, Beijing 100101 (China); Jin, Zhiliang; Li, Shuben; Lu, Gongxuan [State Key Laboratory for Oxo Synthesis and Selective Oxidation, Lanzhou Institute of Chemical Physics, The Chinese Academy of Sciences, Lanzhou 730000 (China); Li, Yuexiang [Department of Chemistry, Nanchang University, Nanjing Road 245, Nanchang, 330047 (China)

    2007-03-30

    A new system for the production of hydrogen, constructed using silica gel as a matrix, Eosin Y as a photosensitizer, and Pt as a cocatalyst, has been reported. It was found that the rate of photosensitized hydrogen evolution in the presence of silica gel is enhanced about 10-fold relative to the homogeneous phase, i.e. in the absence of silica gel. The pH value of the solution and the concentration of Eosin Y have remarkable effects on the rate of hydrogen evolution. The optimal pH and concentration of Eosin Y are 7 and 3.60 x 10{sup -4} mol dm{sup -3} (E/S = 1/3) to 7.24 x 10{sup -4} mol dm{sup -3} (E/S = 1/1), respectively. Triethanolamine (TEOA) as an electron donor, the rate of hydrogen evolution and the apparent quantum efficiency in the silica gel system under visible-light irradiation ({lambda} {>=} 420 nm) can reach about 43 {mu}mol h{sup -1} and 10.4%, respectively. In addition, the roles of silica gel, Pt and TEOA, respectively; and the probable mechanism of photosensitized hydrogen evolution have been discussed. (author)

  4. Visible-light-induced hydrogen production over Pt-Eosin Y catalysts with high surface area silica gel as matrix

    Science.gov (United States)

    Zhang, Xiaojie; Jin, Zhiliang; Li, Yuexiang; Li, Shuben; Lu, Gongxuan

    A new system for the production of hydrogen, constructed using silica gel as a matrix, Eosin Y as a photosensitizer, and Pt as a cocatalyst, has been reported. It was found that the rate of photosensitized hydrogen evolution in the presence of silica gel is enhanced about 10-fold relative to the homogeneous phase, i.e. in the absence of silica gel. The pH value of the solution and the concentration of Eosin Y have remarkable effects on the rate of hydrogen evolution. The optimal pH and concentration of Eosin Y are 7 and 3.60 × 10 -4 mol dm -3 (E/S = 1/3) to 7.24 × 10 -4 mol dm -3 (E/S = 1/1), respectively. Triethanolamine (TEOA) as an electron donor, the rate of hydrogen evolution and the apparent quantum efficiency in the silica gel system under visible-light irradiation (λ ≥ 420 nm) can reach about 43 μmol h -1 and 10.4%, respectively. In addition, the roles of silica gel, Pt and TEOA, respectively; and the probable mechanism of photosensitized hydrogen evolution have been discussed.

  5. DNA double-strand breaks induced by high-energy neon and iron ions in human fibroblasts. I. Pulsed-field gel electrophoresis method

    International Nuclear Information System (INIS)

    Rydberg, B.; Loebrich, M.; Cooper, P.K.

    1994-01-01

    The relative effectiveness of high-energy neon and iron ions for the production of DNA double-strand breaks was measured in one transformed and one nontransformed human fibroblast cell line using pulsed-field gel electrophoresis. The DNA released from the gel plug (fraction of activity released: FAR) as well as the size distribution of the DNA entering the gel were used to compare the effects of the heavy-ion exposure with X-ray exposure. Both methods gave similar results, indicating similar distributions of breaks over megabase-pair distances for the heavy ions and the X rays. The relative biological effectiveness (RBE) compared to 225 kVp X rays of initially induced DNA double-strand breaks was found to be 0.85 for 425 MeV/u neon ions (LET 32 keV/μm) and 0.42-0.55 for 250-600 MeV/u iron ions (LET 190-350 keV/μm). Postirradiation incubation showed less efficient repair of breaks induced by the neon ions and the 600 MeV/u iron ions compared to X rays. Survival experiments demonstrated RBE values larger than one for cell killing by the heavy ions in parallel experiments (neon: RBE = 1.2, iron: RBE = 2.3-3.0, based on D 10 values). It is concluded that either the initial yield of DNA double-strand breaks induced by the high-energy particles is lower than the yield for X rays, or the breaks induced by heavy ions are present in clusters that cannot be resolved with the technique used. These results are confirmed in the accompanying paper. 48 refs., 5 figs., 2 tabs

  6. Development of novel separation techniques for biological samples in capillary electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Huan -Tsung [Iowa State Univ., Ames, IA (United States)

    1994-07-27

    This dissertation includes three different topics: general introduction of capillary electrophoresis (CE); gradient in CE and CE in biological separations; and capillary gel electrophoresis (CGE) for DNA separation. Factors such as temperature, viscosity, pH, and the surface of capillary walls affecting the separation performance are demonstrated. A pH gradient between 3.0 and 5.2 is useful to improve the resolution among eight different organic acids. A flow gradient due to the change in the concentration of surfactant, which is able to coat to the capillary wall to change the flow rate and its direction, is also shown as a good way to improve the resolution for organic compounds. A temperature gradient caused by joule heat is shown by voltage programming to enhance the resolution and shorten the separation time for several phenolic compounds. The author also shows that self-regulating dynamic control of electroosmotic flow in CE by simply running separation in different concentrations of surfactant has less matrix effect on the separation performance. One of the most important demonstrations in this dissertation is that the author proposes on-column reaction which gives several advantages including the use of a small amount of sample, low risk of contamination, and time saving and kinetic features. The author uses this idea with laser induced fluorescence (LIF) as a detection mode to detect an on-column digestion of sub-ng of protein. This technique also is applied to single cell analysis in the group.

  7. Mutagenicity assessment of two herbal medicines, Urtan and Carmint in human leukocytes by single cell gel electrophoresis

    International Nuclear Information System (INIS)

    Jalili, M.; Hatami, A.; Kalantari, H.; Kalantar, E.

    2006-01-01

    The use of herbal medicine is an old and still widespread particle, which makes studies their genotoxicity essential. Urtan and Carmint are examples of herbal medicines used in Iran which used for the treatment of hyperplasia, diuretic, urinary diffusion and antispasmodic action, carminative gastrointestinal disfunction respectively. The aim of this study was to evaluate the mutagenicity of these herbal medicines in human leukocytes by Single Cell Gel Electrophoresis (SCGE). Both the herbal medicines were used at four concentrations (0.046, 0.092, 0.138 and 0.184/ 100 ml). The results were compared against positive (Cr VI) and negative (no mutagen) control groups. Fluorescence microscope was used to observe the DNA damage in randomly selected cells, which were stained with ethidium bromide. Microscopic observation of the affected cells due to Urtan and Carmint was encouraging as compared to previous studies using SCGE. Both Urtan and Carmint exhibited considerable DNA damage to the blood cells. For example, Urtan at o.184 g/100ml concentration had almost 23% mutagenic effect and as the concentration increased the mutagenic effect also increased. Similarly Carmint exhibited considerable DNA damage on blood cells. Therefore, both the herbal medicines may have some mutagenic effect. (author)

  8. Recovery of DNA from agarose gel by trap method | Xia | African ...

    African Journals Online (AJOL)

    Recovery of DNA from agarose gel electrophoresis is a basic operation during molecular cloning. Circular or linear DNA fragments which vary from 1.5 to 6.5 kb and correspond to 1 kb marker can be recovered from 0.8 to 1.0% agarose gel smoothly with a simple and rapid trap method. The recovery efficiency could be ...

  9. Review of Fricke gel dosimeters

    International Nuclear Information System (INIS)

    Schreiner, L J

    2004-01-01

    The innovation of adding a gel matrix to the traditional Fricke dosimeter to stabilize geometric information established the field of gel dosimetry for radiation therapy. A discussion of Fricke gels provides an overview of the issues that determine the dose response of all gel dosimeters in general. In this paper we review some of the features of Fricke systems to illustrate these issues and, in addition, to motivate renewed clinical interest in Fricke gels

  10. Electrophoresis for the analysis of heparin purity and quality.

    Science.gov (United States)

    Volpi, Nicola; Maccari, Francesca; Suwan, Jiraporn; Linhardt, Robert J

    2012-06-01

    The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007-2008 produced a global crisis resulting in extensive revisions to the pharmacopeia monographs and prompting the FDA to recommend the development of additional methods for the analysis of heparin purity. As a consequence, a wide variety of innovative analytical approaches have been developed for the quality assurance and purity of unfractionated and low-molecular-weight heparins. This review discusses recent developments in electrophoresis techniques available for the sensitive separation, detection, and partial structural characterization of heparin contaminants. In particular, this review summarizes recent publications on heparin quality and related impurity analysis using electrophoretic separations such as capillary electrophoresis (CE) of intact polysaccharides and hexosamines derived from their acidic hydrolysis, and polyacrylamide gel electrophoresis (PAGE) for the separation of heparin samples without and in the presence of its relatively specific depolymerization process with nitrous acid treatment. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Tetragonal zirconia quantum dots in silica matrix prepared by a modified sol-gel protocol

    Science.gov (United States)

    Verma, Surbhi; Rani, Saruchi; Kumar, Sushil

    2018-05-01

    Tetragonal zirconia quantum dots (t-ZrO2 QDs) in silica matrix with different compositions ( x)ZrO2-(100 - x)SiO2 were fabricated by a modified sol-gel protocol. Acetylacetone was added as a chelating agent to zirconium propoxide to avoid precipitation. The powders as well as thin films were given thermal treatment at 650, 875 and 1100 °C for 4 h. The silica matrix remained amorphous after thermal treatment and acted as an inert support for zirconia quantum dots. The tetragonal zirconia embedded in silica matrix transformed into monoclinic form due to thermal treatment ≥ 1100 °C. The stability of tetragonal phase of zirconia is found to enhance with increase in silica content. A homogenous dispersion of t-ZrO2 QDs in silica matrix was indicated by the mapping of Zr, Si and O elements obtained from scanning electron microscope with energy dispersive X-ray analyser. The transmission electron images confirmed the formation of tetragonal zirconia quantum dots embedded in silica. The optical band gap of zirconia QDs (3.65-5.58 eV) was found to increase with increase in zirconia content in silica. The red shift of PL emission has been exhibited with increase in zirconia content in silica.

  12. Subfractions of enamel matrix derivative differentially influence cytokine secretion from human oral fibroblasts

    Directory of Open Access Journals (Sweden)

    Oscar Villa

    2015-03-01

    Full Text Available Enamel matrix derivative is used to promote periodontal regeneration during the corrective phase of the treatment of periodontal defects. Our main goal was to analyze the bioactivity of different molecular weight fractions of enamel matrix derivative. Enamel matrix derivative, a complex mixture of proteins, was separated into 13 fractions using size-exclusion chromatography and characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and liquid chromatography–electrospray ionization–tandem mass spectrometry. Human periodontal ligament fibroblasts were treated with either enamel matrix derivative or the different fractions. Proliferation and cytokine secretion to the cell culture medium were measured and compared to untreated cells. The liquid chromatography–electrospray ionization–tandem mass spectrometry analyses revealed that the most abundant peptides were amelogenin and leucine-rich amelogenin peptide related. The fractions containing proteins above 20 kDa induced an increase in vascular endothelial growth factor and interleukin-6 secretion, whereas lower molecular weight fractions enhanced proliferation and secretion of interleukin-8 and monocyte chemoattractant protein-1 and reduced interleukin-4 release. The various molecular components in the enamel matrix derivative formulation might contribute to reported effects on tissue regeneration through their influence on vascularization, the immune response, and chemotaxis.

  13. DNA electrophoresis through microlithographic arrays

    International Nuclear Information System (INIS)

    Sevick, E.M.; Williams, D.R.M.

    1996-01-01

    Electrophoresis is one of the most widely used techniques in biochemistry and genetics for size-separating charged molecular chains such as DNA or synthetic polyelectrolytes. The separation is achieved by driving the chains through a gel with an external electric field. As a result of the field and the obstacles that the medium provides, the chains have different mobilities and are physically separated after a given process time. The macroscopically observed mobility scales inversely with chain size: small molecules move through the medium quickly while larger molecules move more slowly. However, electrophoresis remains a tool that has yet to be optimised for most efficient size separation of polyelectrolytes, particularly large polyelectrolytes, e.g. DNA in excess of 30-50 kbp. Microlithographic arrays etched with an ordered pattern of obstacles provide an attractive alternative to gel media and provide wider avenues for size separation of polyelectrolytes and promote a better understanding of the separation process. Its advantages over gels are (1) the ordered array is durable and can be re-used, (2) the array morphology is ordered and can be standardized for specific separation, and (3) calibration with a marker polyelectrolyte is not required as the array is reproduced to high precision. Most importantly, the array geometry can be graduated along the chip so as to expand the size-dependent regime over larger chain lengths and postpone saturation. In order to predict the effect of obstacles upon the chain-length dependence in mobility and hence, size separation, we study the dynamics of single chains using theory and simulation. We present recent work describing: 1) the release kinetics of a single DNA molecule hooked around a point, frictionless obstacle and in both weak and strong field limits, 2) the mobility of a chain impinging upon point obstacles in an ordered array of obstacles, demonstrating the wide range of interactions possible between the chain and

  14. Oxidizing gel formulation for nuclear decontamination: rheological and acidic properties of the organic matrix and its ozonolysis; Formulation d'un gel oxydant a matrice organique applicable a la decontamination nucleaire: proprietes rheologiques, acido-basiques et ozonolyse de la matrice

    Energy Technology Data Exchange (ETDEWEB)

    Rouy, E

    2003-10-15

    An acidic and oxidizing gel was formulated with a purely organic matrix, xanthan gum, at low concentrations (1 to 2 wt %). This polymer gel was investigated in various media (aqueous, acidic and ceric) by means of rheology: shear thinning behaviour, thixotropy, yield stress... Evidences of unexpected rheological properties in highly concentrated media show that xanthan is quite convenient for industrial projection of this type of gel on metallic walls in nuclear plants, notwithstanding its time-limited resistance to oxidation (about a few hours). Complexation mechanisms between ceric species and polar sites of the polymer led us to characterise acidic properties of our xanthan sample by potentiometric titration and {sup 1}H NMR techniques. The matrix was finally treated by ozonolysis to suppress organic residues, as required to handle nuclear wastes. In acidic medium, ozonolysis of the gel was achieved successfully while in acidic and ceric medium this process showed limited efficiency, needing further investigation to be clarified. (author)

  15. Detection in a Japanese population of a length polymorphism in the 5' flanking region of the human β-globin gene with denaturing gradient gel electrophoresis

    International Nuclear Information System (INIS)

    Takahashi, Noria; Hiyama, Keiko; Kodaira, Mieko; Satoh, Chiyoko

    1992-10-01

    An analysis of the ATTTT repeat polymorphism located approximately 1,400 base pairs upstream from the β-globin structural gene was carried out by denaturing gradient gel electrophoresis (DGGE) of RNA:DNA duplexes. Genomic or cloned DNAs were digested with restriction enzymes and hybridized with 32 P-labeled RNA probes, and resulting RNA:DNA duplexes were examined by DGGE. A difference in the number of repeat units was recognized by differences in duplex mobility on the DGGE gel. In this study of 81 unrelated Japanese from Hiroshima, a sequence heteromorphism was observed at this site. Alleles with 5 and 6 repeats of the ATTTT unit, which had already been reported, were found in polymorphic proportions. In addition, two unreported alleles, one having 7 repeats and the other having an A-to-G nucleotide substitution in the 5th repeat, were detected. Family study data showed that the segregation of these four types of variants is consistent with an autosomal codominant mode of inheritance. This study also demonstrated that DGGE of RNA:DNA duplexes is a sensitive tool for detecting variations in DNA. (author)

  16. Mass spectrometry imaging of illicit drugs in latent fingerprints by matrix-free and matrix-assisted desorption/ionization techniques.

    Science.gov (United States)

    Skriba, Anton; Havlicek, Vladimir

    2018-02-01

    Compared with classical matrix-assisted laser-desorption ionization mass spectrometry (MALDI), the matrix free-based strategies generate a cleaner background, without significant noise or interference coming from an applied matrix, which is beneficial for the analysis of small molecules, such as drugs of abuse. In this work, we probed the detection efficiency of methamphetamine, heroin and cocaine in nanostructure-assisted laser desorption-ionization (NALDI) and desorption electrospray ionization and compared the sensitivity of these two matrix-free tools with a standard MALDI mass spectrometry experiment. In a typical mass spectrometry imaging (MSI) setup, papillary line latent fingerprints were recorded as a mixture a common skin fatty acid or interfering cosmetics with a drug. In a separate experiment, all drugs (1 µL of 1 μM standard solution) were detected by all three ionization techniques on a target. In the case of cocaine and heroin, NALDI mass spectrometry was the most sensitive and revealed signals even from 0.1 μM solution. The drug/drug contaminant (fatty acid or cosmetics) MSI approach could be used by law enforcement personnel to confirm drug abusers of having come into contact with the suspected drug by use of fingerprint scans at time of apprehension which can aid in reducing the work of lab officials.

  17. Use of a commercial agarose gel for analysis of urinary glycosaminoglycans in mucopolysaccharidoses

    Directory of Open Access Journals (Sweden)

    Ana Carolina Breier

    Full Text Available ABSTRACT Mucopolysaccharidoses (MPS are a group of inherited metabolic disorders caused by deficiency of enzymes that degrade glycosaminoglycans (GAGs. Urinary excretion of GAGs is a common feature of MPS, and is considered their major biomarker. We aimed to adapt the GAG electrophoresis method to a commercial agarose gel which would be able to separate urinary GAGs in a simpler way with good sensitivity and reproducibility. Urine samples from patients previously diagnosed with MPS I, IV, and VI were used as electrophoretic standards. Samples from patients on enzyme replacement therapy (ERT were also assessed. Commercial agarose gel electrophoresis was effective, showing proper definition and separation of GAG bands. Detection sensitivity exceeded 0.1 µg and band reproducibility were consistent. GAG bands quantified in urine samples from patients on ERT correlated very strongly (correlation coefficient = 0.98 with total GAG concentrations. This application of gel electrophoresis demonstrates the possibility of monitoring patients with MPS treated with ERT by analyzing separately the GAGs excreted in urine. We suggest this process should be applied to MPS screening as well as to follow-up of patients on treatment.

  18. Structure and dynamics of spin-labeled insulin entrapped in a silica matrix by the sol-gel method.

    Science.gov (United States)

    Vanea, E; Gruian, C; Rickert, C; Steinhoff, H-J; Simon, V

    2013-08-12

    The structure and conformational dynamics of insulin entrapped into a silica matrix was monitored during the sol to maturated-gel transition by electron paramagnetic resonance (EPR) spectroscopy. Insulin was successfully spin-labeled with iodoacetamide and the bifunctional nitroxide reagent HO-1944. Room temperature continuous wave (cw) EPR spectra of insulin were recorded to assess the mobility of the attached spin labels. Insulin conformation and its distribution within the silica matrix were studied using double electron-electron resonance (DEER) and low-temperature cw-EPR. A porous oxide matrix seems to form around insulin molecules with pore diameters in the order of a few nanometers. Secondary structure of the encapsulated insulin investigated by Fourier transform infrared spectroscopy proved a high structural integrity of insulin even in the dried silica matrix. The results show that silica encapsulation can be used as a powerful tool to effectively isolate and functionally preserve biomolecules during preparation, storage, and release.

  19. Application of Pulsed Field Gel Electrophoresis for Study of Genetic Diversity in Mycobacterium tuberculosis Strains Isolated From Tuberculosis Patients.

    Science.gov (United States)

    Khosravi, Azar Dokht; Vatani, Shideh; Feizabadi, Mohammad Mehdi; Abasi Montazeri, Effat; Jolodar, Abbas

    2014-05-01

    Mycobacterium tuberculosis genotyping can effectively improve tuberculosis (TB) control programs by controlling disease transmission. Pulsed field gel electrophoresis (PFGE) is a particularly powerful tool for determination of clonal identity of bacteria providing information for understanding and controlling the spread of disease. The aim of present study was to investigate the genetic diversity of M. tuberculosis strains in Khuzestan province by the PFGE technique. In total, 80 M. tuberculosis positive cultures were obtained from tuberculosis patients. PFGE was performed on 60 PCR-confirmed isolates by using DraI and XbaI restriction enzymes according to standard protocols. Plugs containing digested DNA were then loaded on agarose gels and run using contour-clamped homogenous electric fields. Fifty distinct DNA banding patterns were obtained by digestion of DNA with DraI and 38 DNA banding patterns by digestion with XbaI restriction enzymes. The patterns comprised of 17 different clusters in which cluster I was the major one, containing six strains. Three clusters contained three strains each and the 13 remaining clusters comprised of two strains each. Digestion with DraI yielded 15-20 DNA fragments with 50-485 kb size, while digestion by XbaI produced DNA fragments with a size smaller than 50-242 kb. Despite the ability of PFGE for study of genetic diversity of many mycobacterial species and it being considered as a robust and useful tool, in this study we only found a 15% epidemiological relationship amongst the isolates. Thus, for higher discrimination of genotypic clusters among M. tuberculosis clinical isolates, the application of more sophisticated complementary techniques is required.

  20. Benchmarking sample preparation/digestion protocols reveals tube-gel being a fast and repeatable method for quantitative proteomics.

    Science.gov (United States)

    Muller, Leslie; Fornecker, Luc; Van Dorsselaer, Alain; Cianférani, Sarah; Carapito, Christine

    2016-12-01

    Sample preparation, typically by in-solution or in-gel approaches, has a strong influence on the accuracy and robustness of quantitative proteomics workflows. The major benefit of in-gel procedures is their compatibility with detergents (such as SDS) for protein solubilization. However, SDS-PAGE is a time-consuming approach. Tube-gel (TG) preparation circumvents this drawback as it involves directly trapping the sample in a polyacrylamide gel matrix without electrophoresis. We report here the first global label-free quantitative comparison between TG, stacking gel (SG), and basic liquid digestion (LD). A series of UPS1 standard mixtures (at 0.5, 1, 2.5, 5, 10, and 25 fmol) were spiked in a complex yeast lysate background. TG preparation allowed more yeast proteins to be identified than did the SG and LD approaches, with mean numbers of 1979, 1788, and 1323 proteins identified, respectively. Furthermore, the TG method proved equivalent to SG and superior to LD in terms of the repeatability of the subsequent experiments, with mean CV for yeast protein label-free quantifications of 7, 9, and 10%. Finally, known variant UPS1 proteins were successfully detected in the TG-prepared sample within a complex background with high sensitivity. All the data from this study are accessible on ProteomeXchange (PXD003841). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.