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Sample records for 2-d gel electrophoresis

  1. How to use 2D gel electrophoresis in plant proteomics.

    Rabilloud, Thierry

    2014-01-01

    International audience Two-dimensional electrophoresis has nurtured the birth of proteomics. It is however no longer the exclusive setup used in proteomics, with the development of shotgun proteomics techniques that appear more fancy and fashionable nowadays.Nevertheless, 2D gel-based proteomics still has valuable features, and sometimes unique ones, which make it often an attractive choice when a proteomics strategy must be selected. These features are detailed in this chapter, as is the ...

  2. Gel2DE - A software tool for correlation analysis of 2D gel electrophoresis data

    Øye, Ola Kristoffer; Jørgensen, Katarina Mariann; Hjelle, Sigrun Margrethe; Sulen, André; Ulvang, Dag Magne; Gjertsen, Bjørn Tore

    2013-01-01

    Background: Two-dimensional gel electrophoresis (2DE) is a powerful technique for studying protein isoforms and their modifications. Existing commercial 2D image analysis tools rely on spot detection that limits analysis of complex protein profiles, e.g. spot appearance/disappearance or overlapping spots. Pixel-by-pixel correlation analysis, an analysis technique for identifying relations between protein patterns in gel images and external variables, can overcome such limitations ...

  3. Two dimensional (2-D) electrophoresis and silver staining of proteins in SDS gels

    Two dimensional electrophoresis is a powerful way to analyze protein samples. It separates molecules by charge, then by size in a polyacrylamide or agarose gel. Combining these two techniques allows visualization of up to 1000 peptides on a single gel. Silver staining of proteins provides a rapid and ultra-sensitive method for the detection of nanogram amounts of proteins. The procedures for two dimensional electrophoresis and silver staining of proteins in gels are described

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

    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.

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

    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…

  6. Identification of Methanococcus Jannaschii Proteins in 2-D Gel Electrophoresis Patterns by Mass Spectrometry

    Liang, X.

    1998-06-10

    The genome of Methanococcus jannaschii has been sequenced completely and has been found to contain approximately 1,770 predicted protein-coding regions. When these coding regions are expressed and how their expression is regulated, however, remain open questions. In this work, mass spectrometry was combined with two-dimensional gel electrophoresis to identify which proteins the genes produce under different growth conditions, and thus investigate the regulation of genes responsible for functions characteristic of this thermophilic representative of the methanogenic Archaea.

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

    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.

  8. Quality Control and Stability Studies with the Monoclonal Antibody, Trastuzumab: Application of 1D- vs. 2D-Gel Electrophoresis

    Dashnor Nebija

    2014-04-01

    Full Text Available Recombinant monoclonal antibodies (rmAbs are medicinal products obtained by rDNA technology. Consequently, like other biopharmaceuticals, they require the extensive and rigorous characterization of the quality attributes, such as identity, structural integrity, purity and stability. The aim of this work was to study the suitability of gel electrophoresis for the assessment of charge heterogeneity, post-translational modifications and the stability of the therapeutic, recombinant monoclonal antibody, trastuzumab. One-dimensional, SDS-PAGE, under reducing and non-reducing conditions, and two-dimensional gel electrophoresis were used for the determination of molecular mass (Mr, the isoelectric point (pI, charge-related isoform patterns and the stability of trastuzumab, subjected to stressed degradation and long-term conditions. For the assessment of the influence of glycosylation in the charge heterogeneity pattern of trastuzumab, an enzymatic deglycosylation study has been performed using N-glycosidase F and sialidase, whereas carboxypeptidase B was used for the lysine truncation study. Experimental data documented that 1D and 2D gel electrophoresis represent fast and easy methods to evaluate the quality of biological medicinal products. Important stability parameters, such as the protein aggregation, can be assessed, as well.

  9. Polyacrylamide gel plugs enabling 2-D microfluidic protein separations via isoelectric focusing and multiplexed sodium dodecyl sulfate gel electrophoresis.

    Liu, Jikun; Yang, Shuang; Lee, Cheng S; DeVoe, Don L

    2008-06-01

    In situ photopolymerized polyacrylamide (PAAm) gel plugs are used as hydrodynamic flow control elements in a multidimensional microfluidic system combining IEF and parallel SDS gel electrophoresis for protein separations. The PAAm gel plugs offer a simple method to reduce undesirable bulk flow and limit reagent/sample crosstalk without placing unwanted constraints on the selection of separation media, and without hindering electrokinetic ion migration in the complex microchannel network. In addition to improving separation reproducibility, the discrete gel plugs integrated into critical regions of the chip enable the use of a simple pressure-driven sample injection method which avoids electrokinetic injection bias. The gel plugs also serve to greatly simplify operation of the spatially multiplexed system by eliminating the need for complex external fluidic interfaces. Using an FITC-labeled Escherichia coli cell lysate as a model system, the use of gel plugs is shown to significantly enhance separation reproducibility in a chip containing five parallel CGE channels, with an average variance in peak elution time of only 4.1%. PMID:18449857

  10. Analysis of differentially expressed mitochondrial proteins in chromophobe renal cell carcinomas and renal oncocytomas by 2-D gel electrophoresis

    Maria V. Yusenko, Thomas Ruppert, Gyula Kovacs

    2010-01-01

    Full Text Available Renal oncocytomas (RO and chromophobe renal cell carcinomas (RCC display morphological and functional alterations of the mitochondria. Previous studies showed that accumulation of mitochondria in ROs is associated with somatic mutations of mitochondrial DNA (mtDNA resulting in decreased activity of the respiratory chain complex I, whereas in chromophobe RCC only heteroplasmic mtDNA mutations were found. To identify proteins associated with these changes, for the first time we have compared the mitochondrial proteomes of mitochondria isolated from ROs and chromophobe RCCs as well as from normal kidney tissues by two-dimensional polyacrylamide gel electrophoresis. The proteome profiles were reproducible within the same group of tissues in subsequent experiments. The expression patterns within each group of samples were compared and 81 in-gel digested spots were subjected to nanoLC-MS/MS-based identification of proteins. Although the list of mitochondrial proteins identified in this study is incomplete, we identified the downregulation of NDUFS3 from complex I of the respiratory chain and upregulation of COX5A, COX5B, and ATP5H from complex IV and V in ROs. In chromophobe RCCs downregulation of ATP5A1, the alpha subunit of complex V, has been observed, but no changes in expression of other complexes of the respiratory chain were detected. To confirm the role of respiratory chain complex alterations in the morphological and/or functional changes in chromophobe RCCs and ROs, further studies will be necessary.

  11. 2-D GEL ELECTROPHORESIS MAP OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS TREATED WITH QUERCUS INFECTORIA GALL EXTRACT

    Dayang Fredalina Basri

    2013-01-01

    Full Text Available The widespread outbreak of Methicillin-Resistant Staphylococcus Aureus (MRSA has caused clinical and epidemiological concern in hospital environment. The emergence of Vancomycin-Intermediate S. Aureus (VISA and, more recently, Vancomycin-Resistant S. Aureus (VRSA has further alarmed clinician and scientist worlwide. The objective of this study is to determine the optimum concentration of sample protein from MRSA after treatment with acetone extract from Quercus infectoria gall. Comparison of the Protein Expression Profile (PEP between the treated MRSA and untreated strain as control was obtained using 2-dimensional gel electrophoresis. The Minimum Inhibitory Concentration (MIC value of acetone extract of galls from Q. infectoria against two strains of MRSA; ATCC 33591 and PPUKM clinical isolate was determined by broth microdilution method. The MIC value of acetone extracts against both strains of MRSA was 0.3125 mg mL-1 compared to vancomycin (0.00195 mg mL-1. The optimum concentration of MRSA protein that produced the best resolution was 100 µg. Manifold technique was observed to produce a gel with better resolution and greater number of spot compared with the strip holder technique. This study showed that there were 7 protein spots that represented the increased in the protein expression of more than 2-fold in the MRSA treated with acetone extract of galls Q. infectoria compared to the untreated group. This preliminary study on the PEP of Q. infectoria galls extract-treated MRSA may provide an insight of its antimicrobial mechanism which could lead to the identification of target protein in the future development of a new effective regimen for the treatment of MRSA infections.

  12. Denaturing gradient gel electrophoresis

    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

  13. Comparison of two label-free global quantitation methods, APEX and 2D gel electrophoresis, applied to the Shigella dysenteriae proteome

    Fleischmann Robert D

    2009-06-01

    Full Text Available Abstract The in vitro stationary phase proteome of the human pathogen Shigella dysenteriae serotype 1 (SD1 was quantitatively analyzed in Coomassie Blue G250 (CBB-stained 2D gels. More than four hundred and fifty proteins, of which 271 were associated with distinct gel spots, were identified. In parallel, we employed 2D-LC-MS/MS followed by the label-free computationally modified spectral counting method APEX for absolute protein expression measurements. Of the 4502 genome-predicted SD1 proteins, 1148 proteins were identified with a false positive discovery rate of 5% and quantitated using 2D-LC-MS/MS and APEX. The dynamic range of the APEX method was approximately one order of magnitude higher than that of CBB-stained spot intensity quantitation. A squared Pearson correlation analysis revealed a reasonably good correlation (R2 = 0.67 for protein quantities surveyed by both methods. The correlation was decreased for protein subsets with specific physicochemical properties, such as low Mr values and high hydropathy scores. Stoichiometric ratios of subunits of protein complexes characterized in E. coli were compared with APEX quantitative ratios of orthologous SD1 protein complexes. A high correlation was observed for subunits of soluble cellular protein complexes in several cases, demonstrating versatile applications of the APEX method in quantitative proteomics.

  14. Research on pre-staining gel electrophoresis

    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)

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

    Singh, Pramod Kumar; Shrivastava, Nidhi; Chaturvedi, Krishna; Sharma, Bechan; Bhagyawant, Sameer S.

    2016-01-01

    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 ...

  16. Preprocessing of 1D gel electrophoresis image

    Hlavatý, Matej

    2015-01-01

    Gel electrophoresis is an important method used for separation and analysis of macromolecules with electric potential. This well-established method has a big potential in medicine and science, however, the output images often suffer from distortion. This work describes several known sources of distortion and elaborates on existing methods of the track detection in gel electrophoresis. The core of this thesis introduces a new method to detect individual tracks and image pre-processing based on...

  17. Conducting Polymer Electrodes for Gel Electrophoresis

    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 ...

  18. DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.

    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.

  19. The SWISS-2DPAGE database of two-dimensional polyacrylamide gel electrophoresis.

    Appel, Ron David; Sanchez, Jean-Charles; Bairoch, Amos Marc; Golaz, Olivier Georges; Ravier, Florence; Pasquali, Christian; Hughes, Graham; Hochstrasser, Denis

    1994-01-01

    SWISS-2DPAGE is a database of proteins identified on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), created and maintained at the University Hospital of Geneva in collaboration with the Department of Medical Biochemistry of Geneva University. The proteins have been identified on various 2-D PAGE reference maps by microsequencing, immunoblotting, gel comparison and amino acid composition.

  20. The SWISS-2DPAGE database of two-dimensional polyacrylamide gel electrophoresis.

    Appel, R D; Sanchez, J C; Bairoch, A; Golaz, O; Ravier, F; Pasquali, C; Hughes, G J; Hochstrasser, D F

    1994-01-01

    SWISS-2DPAGE is a database of proteins identified on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), created and maintained at the University Hospital of Geneva in collaboration with the Department of Medical Biochemistry of Geneva University. The proteins have been identified on various 2-D PAGE reference maps by microsequencing, immunoblotting, gel comparison and amino acid composition. Images PMID:7937063

  1. Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

    Summer, Heike; Grämer, René; Dröge, Peter

    2009-01-01

    Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecula...

  2. Polyacrylamide gel electrophoresis of soybean seed proteins

    W. Lassocińsk; J. S. Knypl

    2015-01-01

    Four major and 14 minor protein bands were detected when total salt soluble proteins of soybean (Glycine max cultivar Warszawska) seed were subjected to polyacrylamide gel electrophoresis under nondissociating conditions, and 16 protein bands were detected under dissociating conditions. Molecular weights of three major protein fractions in PAGE SDS were determined for around 18 500, 36 000 and 80 000 daltons.

  3. The repton model of gel electrophoresis

    Barkema, G. T.; Newman, M. E. J.

    1996-01-01

    We discuss the repton model of agarose gel electrophoresis of DNA. We review previous results, both analytic and numerical, as well as presenting a new numerical algorithm for the efficient simulation of the model, and suggesting a new approach to the model's analytic solution.

  4. Matching Two-dimensional Gel Electrophoresis' Spots

    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...

  5. Pulsed field gel electrophoresis a practical guide

    Birren, Bruce

    2014-01-01

    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

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

    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.

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

    Singh, Pramod Kumar; Shrivastava, Nidhi; Chaturvedi, Krishna; Sharma, Bechan; Bhagyawant, Sameer S

    2016-01-01

    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. PMID:27144024

  8. Optimizing Human Bile Preparation for Two-Dimensional Gel Electrophoresis

    Hao-Tsai Cheng

    2016-01-01

    Full Text Available Aims. Bile is an important body fluid which assists in the digestion of fat and excretion of endogenous and exogenous compounds. In the present study, an improved sample preparation for human bile was established. Methods and Material. The method involved acetone precipitation followed by protein extraction using commercially available 2D Clean-Up kit. The effectiveness was evaluated by 2-dimensional electrophoresis (2DE profiling quality, including number of protein spots and spot distribution. Results. The total protein of bile fluid in benign biliary disorders was 0.797 ± 0.465 μg/μL. The sample preparation method using acetone precipitation first followed by 2D Clean-Up kit protein extraction resulted in better quality of 2DE gel images in terms of resolution as compared with other sample preparation methods. Using this protocol, we obtained approximately 558 protein spots on the gel images and with better protein spots presentation of haptoglobin, serum albumin, serotransferrin, and transthyretin. Conclusions. Protein samples of bile prepared using acetone precipitation followed by 2D Clean-Up kit exhibited high protein resolution and significant protein profile. This optimized protein preparation protocol can effectively concentrate bile proteins, remove abundant proteins and debris, and yield clear presentation of nonabundant proteins and its isoforms on 2-dimensional electrophoresis gel images.

  9. Characterization of schistosome antigens by SDS-polyacrylamide gel electrophoresis

    Separation of polypeptides by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is based upon the relationship between the electrophoretic mobility of SDS-protein complexes and their molecular weights. Tegumental proteins extracted from Schistosoma mansoni have been analyzed by SDS-PAGE using slab gels by a number of investigators. Valuable information has also been obtained using tube gels to analyze radiolabeled proteins. The procedures for electrophoresis using tube gels and electrophoresis using gradient slab gels are described

  10. Comparative proteomics and difference gel electrophoresis.

    Minden, Jonathan

    2007-12-01

    The goal of comparative proteomics is to analyze proteome changes in response to development, disease, or environment. This is a two-step process in which proteins within cellular extracts are first fractionated to reduce sample complexity, and then the proteins are identified by mass spectrometry. Two-dimensional electrophoresis (2DE) is the long-time standard for protein separation, but it has suffered from poor reproducibility and limited sensitivity. Difference gel electrophoresis (DIGE), in which two protein samples are separately labeled with different fluorescent dyes and then co-electrophoresed on the same 2DE gel, was developed to overcome the reproducibility and sensitivity limitations. In this essay, I discuss the principles of comparative proteomics and the development of DIGE. PMID:18251249

  11. RegStatGel: proteomic software for identifying differentially expressed proteins based on 2D gel images

    Li, Feng; Seillier-Moiseiwitsch, Françoise

    2011-01-01

    Image analysis of two-dimensional gel electrophoresis is a key step in proteomic workflow for identifying proteins that change under different experimental conditions. Since there are usually large amount of proteins and variations shown in the gel images, the use of software for analysis of 2D gel images is inevitable. We developed open-source software with graphical user interface for differential analysis of 2D gel images. The user-friendly software, RegStatGel, contains fully automated as well as interactive procedures. It was developed and has been tested under Matlab 7.01. Availability The database is available for free at http://www.mediafire.com/FengLi/2DGelsoftware PMID:21904427

  12. Gel Electrophoresis of Gold-DNA Nanoconjugates

    T. Pellegrino

    2007-01-01

    Full Text Available Gold-DNA conjugates were investigated in detail by a comprehensive gel electrophoresis study based on 1200 gels. A controlled number of single-stranded DNA of different length was attached specifically via thiol-Au bonds to phosphine-stabilized colloidal gold nanoparticles. Alternatively, the surface of the gold particles was saturated with single stranded DNA of different length either specifically via thiol-Au bonds or by nonspecific adsorption. From the experimentally determined electrophoretic mobilities, estimates for the effective diameters of the gold-DNA conjugates were derived by applying two different data treatment approaches. The first method is based on making a calibration curve for the relation between effective diameters and mobilities with gold nanoparticles of known diameter. The second method is based on Ferguson analysis which uses gold nanoparticles of known diameter as reference database. Our study shows that effective diameters derived from gel electrophoresis measurements are affected with a high error bar as the determined values strongly depend on the method of evaluation, though relative changes in size upon binding of molecules can be detected with high precision. Furthermore, in this study, the specific attachment of DNA via gold-thiol bonds to Au nanoparticles is compared to nonspecific adsorption of DNA. Also, the maximum number of DNA molecules that can be bound per particle was determined.

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

    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)

  14. 2-D gel electrophoresis-based proteomic analysis reveals that ormeloxifen induces G0-G1 growth arrest and ERK-mediated apoptosis in chronic myeloid leukemia cells K562.

    Pal, Pooja; Kanaujiya, Jitendra K; Lochab, Savita; Tripathi, Shashi B; Bhatt, Madan L B; Singh, Pradhyumna K; Sanyal, Sabyasachi; Trivedi, Arun K

    2011-04-01

    Ormeloxifen is a nonsteroidal selective estrogen receptor modulator (SERM) and has been shown to possess anticancer activities in breast and uterine cancer. Here, we show that ormeloxifen induces apoptosis in dose-dependent manner in a variety of leukemia cells, more strikingly in K562. 2-DE-gel electrophoresis of K562 cells induced with ormeloxifen showed that 57 and 30% of proteins belong to apoptosis and cell-cycle pathways, respectively. Our data demonstrate that ormeloxifen-induced apoptosis in K562 cells involves activation of extracellular signal-regulated kinases (ERKs) and subsequent cytochrome c release, leading to mitochondria-mediated caspase-3 activation. Ormeloxifen-induced apoptosis via ERK activation was drastically inhibited by prior treatment of K562 cells with ERK inhibitor PD98059. Ormeloxifen also inhibits proliferation of K562 cells by blocking them in G0-G1 phase by inhibiting c-myc promoter via ormeloxifen-induced MBP-1 (c-myc promoter-binding protein) and upregulation of p21 expression. We further show that ormeloxifen-induced apoptosis in K562 is translatable to mononuclear cells isolated from chronic myeloid leukemia (CML) patients. Thus, ormeloxifen induces apoptosis in K562 cells via phosphorylation of ERK and arrests them in G0-G1 phase by reciprocal regulation of p21 and c-myc. Therefore, inclusion of ormeloxifen in the therapy of chronic myeloid leukemia can be of potential utility. PMID:21360677

  15. Functional annotation of 2D protein maps: The GelMap portal

    Hans-Peter eBraun

    2012-05-01

    Full Text Available In classical proteome analyses, final experimental data are (a images of 2D protein separations obtained by gel electrophoresis and (b corresponding lists of proteins which were identified by mass spectrometry (MS. For data annotation, software tools were developed which allow to link protein identity data directly to 2D gels (clickable gels. GelMap is a new online software tool to annotate 2D protein maps. It allows (i functional annotation of all identified proteins according to biological categories defined by the user, e.g. subcellular localization, metabolic pathway, or assignment to a protein complex and (ii annotation of several proteins per analyzed protein spot according to MS primary data. Options to differentially display proteins of functional categories offer new opportunities for data evaluation. For instance, if used for the annotation of 2D Blue native / SDS gels, GelMap allows identifying protein complexes of low abundance. A web portal has been established for presentation and evaluation of protein identity data related to 2D gels and is freely accessible at http://www.gelmap.de/.

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

    Roghaye Khodadadi; Azim Khajeali; Alireza Farajollahi; Parisa Hajalioghli; Noorallah Raeisi

    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 t...

  17. Micro-size polyacrylamide gel electrophoresis system

    Hinson, W. G.; Pipkin, J. L.; Anson, J. F.; Casciano, D. A.; Burns, E. R.

    1987-09-01

    The development and characterization of a micro-size two-dimensional polyacrylamide gel electrophoresis system is described. Some of the techniques which have evolved with use of the system are also discussed. This apparatus has unique features which provide advantages over other small scale units. Up to ten first- and second-dimension gels can be processed simultaneously with excellent resolution of protein regions. Consistent reproducibility is possible from protein samples as small as 400 ng and individual protein regions as small as 1 pg can be visualized by silver staining of the two-dimensional gels. Similar sensitivities are achieved in autoradiographs of 3H-labeled proteins extracted from the nuclei of cultured cells. The application of this system in conjunction with flow cytometric examination of nuclear DNA and electrostatic cell sorting of specific cell nuclei to provide homogeneous sample populations, allows subtle variations in isotope incorporation in proteins to be detected; whereas many times in generalized tissue samples these changes are masked. Also, these techniques elucidate the effects of external stimuli (chemicals, drugs, or environment) on protein synthesis and phosphorylation for analyses and comparison. Fabrication drawings are available upon request.

  18. Inexpensive and Safe DNA Gel Electrophoresis Using Household Materials

    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…

  19. Purification of radiolabeled RNA products using denaturing gel electrophoresis

    Adachi, Hironori; Yu, Yi-Tao

    2014-01-01

    This unit discusses a basic method for purification of radiolabeled RNAs using denaturing polyacrylamide gel electrophoresis. The method consists of a number of experimental procedures, including total RNA preparation from yeast cells, isolation of a specific RNA from total yeast RNA, RNA 3' terminal labeling using nucleotide (5’[32P]pCp) addition (via ligation), denaturing (8 M urea) polyacrylamide gel electrophoresis, and RNA extraction from the gel slice. Key points for achieving good elec...

  20. Guidelines for reporting the use of gel electrophoresis in proteomics.

    Gibson, Frank; Anderson, Leigh; Babnigg, Gyorgy; Baker, Mark; Berth, Matthias; Binz, Pierre-Alain; Borthwick, Andy; Cash, Phil; Day, Billy W.; Friedman, David B; Garland, Donita; Gutstein, Howard B.; Hoogland, Christine; Jones, Neil A.; Khan, Alamgir

    2008-01-01

    the MIAPE Gel Electrophoresis (MIAPE-GE) guidelines specify the minimum information that should be provided when reporting the use of n-dimensional gel electrophoresis in a proteomics experiment. Developed through a joint effort between the gel-based analysis working group of the Human Proteome Organisation's Proteomics Standards Initiative (HUPO-PSI; http://www.psidev.info/) and the wider proteomics community, they constitute one part of the overall Minimum Information about a Proteomics Exp...

  1. Agarose Gel Electrophoresis for the Separation of DNA Fragments

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

    2012-01-01

    Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. 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....

  2. A systematic study of field inversion gel electrophoresis.

    Heller, C.; Pohl, F M

    1989-01-01

    The mobilities of oligomers of phage lambda DNA and of yeast chromosomes in agarose gels during field inversion gel electrophoresis (FIGE) were measured at different pulse times and electric fields. Also the ratios between forward and backward pulse times and/or field gradients were varied. The problem of 'band inversion' during FIGE, leading to an ambiguity in the mobility of large DNA fragments, was solved by using two dimensional gel electrophoresis with different parameters in the first a...

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

    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

  4. The SWISS-2DPAGE database of two-dimensional polyacrylamide gel electrophoresis, its status in 1995.

    Appel, Ron David; Sanchez, Jean-Charles; Bairoch, Amos Marc; Golaz, Olivier Georges; Ravier, Florence; Pasquali, Christian; Hughes, Graham; Hochstrasser, Denis

    1996-01-01

    SWISS-2DPAGE is a database of proteins identified on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The current release contains 343 entries of human, yeast (Saccharomyces cerevisiae) and Escherichia coli origin, as well as virtual entries for each of the protein sequences in the SWISS-PROT database.

  5. The SWISS-2DPAGE database of two-dimensional polyacrylamide gel electrophoresis, its status in 1995.

    Appel, R D; Sanchez, J C; Bairoch, A; Golaz, O; Ravier, F; Pasquali, C; Hughes, G J; Hochstrasser, D F

    1996-01-01

    SWISS-2DPAGE is a database of proteins identified on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The current release contains 343 entries of human, yeast (Saccharomyces cerevisiae) and Escherichia coli origin, as well as virtual entries for each of the protein sequences in the SWISS-PROT database. PMID:8594575

  6. Analysis of soybean embryonic axis proteins by two-dimensional gel electrophoresis and mass spectrometry

    A proteomic approach based on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for protein separation and subsequent mass spectrometry (MS) for protein identification was applied to establish a proteomic reference map for the soybean embryonic axis. Proteins were extracted from dissecte...

  7. The Gel Electrophoresis Markup Language (GelML) from the Proteomics Standards Initiative

    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.

    2010-01-01

    The Human Proteome Organisation’s Proteomics Standards Initiative (HUPO-PSI) has developed the GelML 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 f...

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

    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.

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

    Mandal Nakul

    2009-12-01

    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.

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

    Mandal Nakul; Heegaard Steffen; Prause Jan; Honoré Bent; Vorum Henrik

    2009-01-01

    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 differe...

  11. 1-D and 2-D electrophoresis protein profiles of the scorpion venom from Brotheas amazonicus

    Higa, A.M.; Noronha, M.D.N. [Universidade do Estado do Amazonas (UEA), Manaus, AM (Brazil). Rede Proteomica do Amazonas (Proteam). Lab. de Genomica e Proteomica; Rocha-Oliveira, F.; Lopez-Lozano, J.L.L. [Universidade Federal do Amazonas (UFAM), Manaus, AM (Brazil). Pos-Graduacao em Biotecnologia

    2008-07-01

    Full text: Introduction: Scorpions venoms show specific neurotoxins to insect or mammals. These toxins are very important molecular tools to development of news drugs or bioinsecticides. Brotheas amazonicus scorpion is an endemic specie in Amazonian Rain Forest, but your venom do not show toxicity in humans. Information about biological specific activity on insect of this venom is not known yet. Objectives: Molecular protein toxins profiles of the venom from Brotheas amazonicus scorpion by 1-D and 2-D electrophoresis methods to detected toxins with potential biotech applications. Results: Several spots 'families' with {approx} 60, 70 and 80 kDa were detected in gel acidic region with pI {approx} 4,5 - 6 range, in the same region 1-D zimography showed proteolytic activity on gelatin and fibrinogen and proteolytic activity was inhibited by PMSF, suggesting scorpion serine proteinases. 50 kDa proteins were detected with pI {approx} 6,5 - 7 range. In 23 - 50 kDa gel acid region were observed some proteins. In 23 - 14 kDa gel acidic region were detected proteins with pI 4 - 7 range. 1-D Tris-tricine gel showed proteins with {approx} 7 kDa, suggesting scorpion neurotoxins. In gel basic region only 14 kDa proteins were observed with pI {approx} 9 - 10 range. Conclusion: Molecular profile of the scorpion venom from B. amazonicus showed proteins with high and low molecular masses, mainly with acidic pI. Proteolytic activity suggest serine proteinases with high molecular masses and 7 kDa proteins in B. amazonicus venom suggest scorpion neurotoxins. Purification and molecular characterization of these toxins are in course.

  12. 1-D and 2-D electrophoresis protein profiles of the scorpion venom from Brotheas amazonicus

    Full text: Introduction: Scorpions venoms show specific neurotoxins to insect or mammals. These toxins are very important molecular tools to development of news drugs or bioinsecticides. Brotheas amazonicus scorpion is an endemic specie in Amazonian Rain Forest, but your venom do not show toxicity in humans. Information about biological specific activity on insect of this venom is not known yet. Objectives: Molecular protein toxins profiles of the venom from Brotheas amazonicus scorpion by 1-D and 2-D electrophoresis methods to detected toxins with potential biotech applications. Results: Several spots 'families' with ∼ 60, 70 and 80 kDa were detected in gel acidic region with pI ∼ 4,5 - 6 range, in the same region 1-D zimography showed proteolytic activity on gelatin and fibrinogen and proteolytic activity was inhibited by PMSF, suggesting scorpion serine proteinases. 50 kDa proteins were detected with pI ∼ 6,5 - 7 range. In 23 - 50 kDa gel acid region were observed some proteins. In 23 - 14 kDa gel acidic region were detected proteins with pI 4 - 7 range. 1-D Tris-tricine gel showed proteins with ∼ 7 kDa, suggesting scorpion neurotoxins. In gel basic region only 14 kDa proteins were observed with pI ∼ 9 - 10 range. Conclusion: Molecular profile of the scorpion venom from B. amazonicus showed proteins with high and low molecular masses, mainly with acidic pI. Proteolytic activity suggest serine proteinases with high molecular masses and 7 kDa proteins in B. amazonicus venom suggest scorpion neurotoxins. Purification and molecular characterization of these toxins are in course

  13. Gel Electrophoresis on a Budget to Dye for

    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…

  14. Field inversion gel electrophoresis with different pulse time ramps.

    Heller, C.(Fakultät für Physik, Ludwig-Maximilians-Universität München, München, Germany); Pohl, F M

    1990-01-01

    The influence of different pulse time ramps on the separation of yeast chromosomes with field inversion gel electrophoresis (FIGE) was investigated by the means of two dimensional gel electrophoresis. The problem of band inversion, which makes it difficult to distinguish DNA molecules of different size, has been solved by using double randomized pulse times. A major disadvantage of the field inversion technique is thereby overcome, making this system comparable to other pulsed field techniques.

  15. Pulsed field gel electrophoresis on frozen tumour tissue sections.

    Boultwood, J. (Jacqueline); Kaklamanis, L.; Gatter, K. C.; Wainscoat, J S

    1992-01-01

    The application of pulsed field gel electrophoresis (PFGE) to the molecular genetic analysis of solid tumours has been restricted by the requirement for whole single cells as a DNA source. A simple technique which allows for the direct analysis of histologically characterised solid tumour material by pulsed field gel electrophoresis was developed. Single frozen tissue sections obtained from colonic carcinoma specimens were embedded without further manipulation in molten, low melting temperatu...

  16. Further characterization of filarial antigens by SDS polyacrylamide gel electrophoresis

    Dissanayake, S.; Galahitiyawa, S. C.; Ismail, M. M.

    1983-01-01

    SDS (sodium dodecyl sulfate)-polyacrylamide gel electrophoresis of an antigen isolated from sera of Wuchereria bancrofti-infected patients and Setaria digitata antigen SD2-4 is reported. Both antigens showed carbohydrate (glycoprotein) staining. The W. bancrofti antigen had an apparent relative molecular mass of 35 000 while the S. digitata antigen SD2-4 migrated at the marker dye position on SDS-polyacrylamide gel electrophoresis. SDS treatment of these antigens did not abolish the precipita...

  17. Strategic analysis for a novel gel electrophoresis technology

    Yu, Jeff

    2007-01-01

    A novel gel electrophoresis technology for separating protein and nucleic acid is under development by a research team at Simon Fraser University (SFU. Compared with the conventional gel electrophoresis technology, the new technology is easier to use, has higher throughput, and lower use cost. The purpose of this project is to help the researchers to commercialize the new technology. This report begins with introduction of the project, followed by an industry analysis. Then it develops and di...

  18. Use of capillary electrophoresis as a method development tool for classical gel electrophoresis

    Sutton, Richard M. C.; Gratz, Samuel R.; Stalcup, Apryll M.

    1998-01-01

    Capillary electrophoresis (CE) was used to optimize the buffer pH, ionic strength and sulfated cyclodextrin concentrations for enantiomeric separation of piperoxan. These enantioseparation conditions were then applied to a classical gel electrophoresis system. Binding constants of the sulfated beta-cyclodextrin–piperoxan couple were approximated using CE and the effects of organic solvents on the system were also investigated.

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

    Andersen, H; Birkelund, Svend; Christiansen, Gunna; Freundt, A

    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...

  20. Separation of Protein Oligomers by Blue Native Gel Electrophoresis

    Braz, Valerie A.; Howard, Kathryn J.

    2009-01-01

    Native gel electrophoresis is used as a tool to assess structural differences in proteins. This note presents an application to separate oligomeric forms of proteins, such as HIV-1 reverse transcriptase monomers and homodimers. Technical difficulties encountered with various native gel techniques and ways to circumvent them are described.

  1. Gel Electrophoresis--The Easy Way for Students

    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…

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

    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 and...... arabinose and other sugars as enzyme inducers. After electrophoresis the gels were separated into several slabs by a gel cutter. Each slab was stained for a particular enzyme. Among numerous enzyme systems tested we found useful variation in esterases (EC 3.1.1.1, EC 3.1.1.2), 3-hydroxybutyrate...

  3. Electron beam sterilization of the agarose gel used for electrophoresis

    The results obtained by electron beam (EB) sterilization of the plates with agarose gel used for human serum protein electrophoresis are presented. Also, the results obtained by human serum protein electrophoresis performed with agarose gel plates irradiated at different EB doses, from 4 kGy to 20 kGy, are presented. The microbiological results demonstrate that above 5 kGy the irradiated agarose plates are sterile. The EB irradiation of the agarose gel plates in the dose range of 7-9 kGy gives the best results for both, sterilization and protein fraction separation processes. (author)

  4. Gel Electrophoresis of Gold-DNA Nano-Conjugates

    Pellegrino, T.; Sperling, R.A.; Alivisatos, A.P.; Parak, W.J.

    2006-01-10

    Single stranded DNA of different lengths and different amounts was attached to colloidal phosphine stabilized Au nanoparticles. The resulting conjugates were investigated in detail by a gel electrophoresis study based on 1200 gels. We demonstrate how these experiments help to understand the binding of DNA to Au particles. In particular we compare specific attachment of DNA via gold-thiol bonds with nonspecific adsorption of DNA. The maximum number of DNA molecules that can be bound per particle was determined. We also compare several methods to used gel electrophoresis for investigating the effective diameter of DNA-Au conjugates, such as using a calibration curve of particles with known diameters and Ferguson plots.

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

    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.

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

    Shaw, AC; Rossel 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...

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

    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...

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

    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 polyacrylami...

  9. Nanoparticle gel electrophoresis: bare charged spheres in polyelectrolyte hydrogels.

    Li, Fei; Hill, Reghan J

    2013-03-15

    Nanoparticle gel electrophoresis has recently emerged as an attractive means of separating and characterizing nanoparticles. Consequently, a theory that accounts for electroosmotic flow in the gel, and coupling of the nanoparticle and hydrogel electrostatics and hydrodynamics, is required, particularly for gels in which the mesh size is comparable to or smaller than the particle radii. Here, we present an electrokinetic model for charged, spherical colloidal particles undergoing electrophoresis in charged (polyelectrolyte) hydrogels: the gel-electrophoresis analogue of Henry's theory for electrophoresis in Newtonian electrolytes. We compare numerically exact solutions of the model with several independent asymptotic approximations, identifying regions in the parameter space where these approximations are accurate or break down. As previously assumed in the literature, Henry's formula, modified by the addition of a constant electroosmotic flow mobility, is accurate only for nanoparticles that are small compared to the hydrogel mesh size. We derived an exact analytical solution of the full model by judiciously modifying the theory of Allison et al. for uncharged gels, drawing on the superposition methodology of Doane et al. to account for hydrogel charge. This furnishes accurate and economical mobility predictions for the entire parameter space. The present model suggests that nanoparticle size separations (with diameters ≲40 nm) are optimal at low ionic strength, with a gel mesh size that is selected according to the particle charging mechanism. For weakly charged particles, optimal size separation is achieved when the Brinkman screening length is matched to the mean particle size. PMID:23153681

  10. Scrambling of bands in gel electrophoresis of DNA.

    Lalande, M; Noolandi, J; Turmel, C; Brousseau, R.; Rousseau, J.; Slater, G W

    1988-01-01

    Under certain conditions of agarose gel electrophoresis, larger DNA molecules migrate faster than smaller ones. This anomalous mobility of DNA, which can lead to serious errors in the measurement of DNA fragment lengths, is related to near-zero velocity conformations which can trap DNA chains during electrophoresis. Intermittent electric fields can be used to alter the chain conformations so as to restore the monotonic mobility-size relationship which is necessary for a correct interpretation...

  11. Purification of coated vesicles by agarose gel electrophoresis

    1981-01-01

    We have applied agarose gel electrophoresis as a novel step in the purification of clathrin-coated vesicles. Preparations of coated vesicles obtained by sedimentation velocity and isopycnic centrifugation are resolved into two distinct fractions upon electrophoresis. The slower migrating fraction contains smooth vesicles, whereas the faster contains only coated vesicles and empty clathrin coats. The faster mobility of the coated vesicles is primarily caused by the acidic nature of clathrin. C...

  12. Biochemical Identification of the Two Races of Radopholus similis by Polyacrylamide Gel Electrophoresis

    Huettel, R. N.; Dickson, D. W.; Kaplan, D. T.

    1983-01-01

    Analysis of proteins of the banana and citrus race of Radopholus similis was carried out by several different types of polyacrylamide gel electrophoresis. These included standard slab gel, SDS slab gel, gradient slab gel, and two-ditnensional slab gel electrophoresis. A major band difference was detected between the two races by slab gel electrophoresis. However, several other poorly resolved but consistent hands of high molecular weight proteins near the gel origin also were considered as di...

  13. Basics and recent advances of two dimensional- polyacrylamide gel electrophoresis

    Magdeldin, Sameh; Enany, Shymaa; Yoshida, Yutaka; Xu, Bo; Zhang, Ying; Zureena, Zam; Lokamani, Ilambarthi; Yaoita, Eishin; Yamamoto, Tadashi

    2014-01-01

    Gel- based proteomics is one of the most versatile methods for fractionating protein complexes. Among these methods, two dimensional- polyacrylamide gel electrophoresis (2-DE) represents a mainstay orthogonal approach, which is popularly used to simultaneously fractionate, identify, and quantify proteins when coupled with mass spectrometric identification or other immunological tests. Although 2-DE was first introduced more than three decades ago, several challenges and limitations to its uti...

  14. Analysis of photoaffinity-labeled aryl hydrocarbon receptor heterogeneity by two-dimensional gel electrophoresis

    The level of charge heterogeneity in the aryl hydrocarbon receptor (AhR) was examined by high-resolution denaturing two-dimensional (2D) gel electrophoresis. Hepa 1c1c7 cell cytosolic fraction was photoaffinity-labeled with 2-azido-3-[125I]-iodo-7,8-dibromodibenzo-p-dioxin and applied to isoelectric focusing (IEF) tube gels. After optimization of focusing conditions a broad peak of radioactivity was detected in the apparent pI range of 5.2-5.7. IEF tube gels were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by visualization of the radiolabeled AhR by autoradiography; three distinct isoforms were detected. The same 2D electrophoretic isoform pattern was obtained when the AhR from Hepa 1c1c7 was photoaffinity-labeled in cell culture. BPrCl cells, a mutant line derived from Hepa 1c1c7 cells, contain an AhR that is unable to bind to DNA. Photoaffinity-labeled BPrCl cytosolic fractions were subjected to 2D gel electrophoretic analysis resulting in essentially the same molecular weight and isoform pattern as seen in Hepa 1c1c7 cytosol. This result would suggest that if a mutation is present in the BPrCl AhR it has not caused a significant change in its IEF pattern, although a small shift in the pI values was observed. Two-dimensional gel electrophoresis of photoaffinity-labeled cytosolic fractions from HeLa cells, the rat liver tumor cell line McA-RH777, and buffalo rat thymus revealed three isoforms, essentially the same isoform pattern as in Hepa 1c1c7 cells. This would indicate that despite the considerable molecular weight polymorphism between species the level of charge heterogeneity is high conserved

  15. Explorative data analysis of two-dimensional electrophoresis gels

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

    2004-01-01

    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 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...... 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...

  16. Applications of single cell gel electrophoresis assay in radiation research

    The single cell gel electrophoresis (SCGE), also called comet assay, is a sensitive and rapid method for DNA damage detection in individual mammalian cell. Its use has increased significantly in the past few years. Applications in the field of radiation medicine are reviewed and possible future directions of the technique are briefly explored

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

    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 and...

  18. Field inversion gel electrophoresis in denaturing polyacrylamide gels.

    Heller, C.; Beck, S

    1992-01-01

    The velocities of single stranded DNA molecules in denaturing polyacrylamide gels during symmetric and asymmetric field inversion were measured at different pulse times and gel concentrations. Under the conditions chosen in our study, pulse times as short as a few milliseconds lead to a retardation of DNA molecules larger than 400 bases. We found that a field inversion with an electric field in the forward direction of about double the strength of that applied in the backward direction is a g...

  19. Optimisation of the two-dimensional gel electrophoresis protocol using the Taguchi approach

    Blow J Julian

    2004-09-01

    Full Text Available Abstract Background Quantitative proteomic analyses have traditionally used two-dimensional gel electrophoresis (2DE for separation and characterisation of complex protein mixtures. Among the difficulties associated with this approach is the solubilisation of protein mixtures for isoelectric focusing (IEF. To find the optimal formulation of the multi-component IEF rehydration buffer (RB we applied the Taguchi method, a widely used approach for the robust optimisation of complex industrial processes, to determine optimal concentrations for the detergents, carrier ampholytes and reducing agents in RB for 2DE using commercially supplied immobilised pH gradient (IPG gel strips. Results Our optimisation resulted in increased protein solubility, improved resolution and reproducibility of 2D gels, using a wide variety of samples. With the updated protocol we routinely detected approximately 4-fold more polypeptides on samples containing complex protein mixtures resolved on small format 2D gels. In addition the pI and size ranges over which proteins could be resolved was substantially improved. Moreover, with improved sample loading and resolution, analysis of individual spots by immunoblotting and mass spectrometry revealed previously uncharacterised posttranscriptional modifications in a variety of chromatin proteins. Conclusions While the optimised RB (oRB is specific to the gels and analysis approach we use, our use of the Taguchi method should be generally applicable to a broad range of electrophoresis and analysis systems.

  20. Manual for starch gel electrophoresis: A method for the detection of genetic variation

    Aebersold, Paul B.; Winans, Gary A.; David J Teel; Milner, George B.; Utter, Fred M

    1987-01-01

    The procedure to conduct horizontal starch gel electrophoresis on enzymes is described in detail. Areas covered are (I) collection and storage of specimens, (2) preparation of tissues, (3) preparation of a starch gel, (4) application of enzyme extracts to a gel, (5) setting up a gel for electrophoresis, (6) slicing a gel, and (7) staining a gel. Recipes are also included for 47 enzyme stains and 3 selected gel buffers. (PDF file contains 26 pages.)

  1. The electrophoresis of transferrins in urea/polyacrylamide gels

    Evans, RW; Williams, J.

    1980-01-01

    The denaturation of transferrin by urea has been studied by (a) electrophoresis in polyacrylamide gels incorporating a urea gradient, (b) measurements of the loss in iron-binding capacity and (c) u.v. difference spectrometry. In human serum transferrin and hen ovotransferrin the N-terminal and C-terminal domains of the iron-free protein were found to denature at different urea concentrations.

  2. Polyacrylamide gel electrophoresis of isoenzymes from Entamoeba species.

    Mathews, H M; Moss, D M; Healy, G R; Visvesvara, G S

    1983-01-01

    In this preliminary report, we describe a polyacrylamide gel electrophoresis technique for the resolution of isoenzyme patterns of four isolates of Entamoeba histolytica and one isolate of Entamoeba coli. Our findings were similar to previous findings for three enzyme systems: maleic enzyme (malate dehydrogenase [EC 1.1.1.40]), hexokinase (EC 2.7.1.1), and phosphoglucomutase (EC 2.7.5.1). We found preliminary evidence that glucosephosphate isomerase (EC 5.3.1.9) may also differentiate invasiv...

  3. Small angle neutron scattering from DNA molecules during gel electrophoresis

    We have performed small angle neutron scattering experiments on agarose-DNA gels undergoing electrophoresis. Two kinds of DNA (5 and 50 kilobase pairs) were used with applied fields up to 5 V/cm. The SANS patterns obtained do not show evidence of any anisotropic scattering. This result is discussed in the context of current theories of DNA fragments migrating through a polysaccharide network. (orig.)

  4. Single Cell Gel Electrophoresis in DNA Damage Detection (Comet Assay)

    Aysen Durmaz; Nurten Dikmen; Cumhur Gunduz

    2010-01-01

    “Single cell gel electrophoresis (SCGE)”, also called “Comet Assay”, is a sensitive, reliable and rapid technique for quantifying and analyzing DNA damage in individual cells. The comet assay is widely used in living cells, researches and the applications on comet assay is becoming broader day by day. To date, the comet assay has been used for a variety of applications, including genotoxic and cytotoxic agent analyses, environmental toxicology, cancer research, and radiati...

  5. Protein Mobility Shifts Contribute to Gel Electrophoresis Liquid Chromatography Analysis

    Carruthers, Nicholas J.; Parker, Graham C.; Gratsch, Theresa; Caruso, Joseph A.; Stemmer, Paul M.

    2015-01-01

    Profiling of cellular and subcellular proteomes by liquid chromatography with tandem mass spectrometry (MS) after fractionation by SDS-PAGE is referred to as GeLC (gel electrophoresis liquid chromatography)-MS. The GeLC approach decreases complexity within individual MS analyses by size fractionation with SDS-PAGE. SDS-PAGE is considered an excellent fractionation technique for intact proteins because of good resolution for proteins of all sizes, isoelectric points, and hydrophobicities. Addi...

  6. Insight of Saffron Proteome by Gel-Electrophoresis

    Gianluca Paredi; Samanta Raboni; Francesco Marchesani; ORDOUDI, Stella A; TSIMIDOU, Maria Z; Andrea Mozzarelli

    2016-01-01

    Saffron is a spice comprised of the dried stigmas and styles of Crocus sativus L. flowers and, since it is very expensive, it is frequently adulterated. So far, proteomic tools have never been applied to characterize the proteome of saffron or identify possible cases of fraud. In this study, 1D-Gel Electrophoresis was carried out to characterize the protein profile of (i) fresh stigmas and styles of the plant; (ii) dried stigmas and styles from different geographical origins (Spanish, Italian...

  7. Statistical Analyses of Complex Denaturing Gradient Gel Electrophoresis Profiles

    Gafan, Gavin P.; Lucas, Victoria S.; Roberts, Graham J.; Petrie, Aviva; Wilson, Michael; David A. Spratt

    2005-01-01

    Studies using molecular techniques have demonstrated that a culture-based approach can severely underestimate the bacterial diversity in most environments. One of the molecular techniques that has been applied in microbial ecology is denaturing gradient gel electrophoresis (DGGE). The purpose of this study was to investigate differences in the microbiota of plaque, using a number of analysis techniques, from children without gingivitis (n = 30) and from those with gingivitis (n = 30). Extract...

  8. Statistical analyses of complex denaturing gradient gel electrophoresis profiles

    Gafan, G. P.; Lucas, V S; Roberts, G J; Petrie, A; Wilson, M; Spratt, D. A.

    2005-01-01

    Studies using molecular techniques have demonstrated that a culture-based approach can severely underestimate the bacterial diversity in most environments. One of the molecular techniques that has been applied in microbial ecology is denaturing gradient gel electrophoresis (DGGE). The purpose of this study was to investigate differences in the microbiota of plaque, using a number of analysis techniques, from children without gingivitis (n = 30) and from those with gingivitis (n = 30). Extract...

  9. Optimizing Human Bile Preparation for Two-Dimensional Gel Electrophoresis

    Hao-Tsai Cheng; Sen-Yung Hsieh; Chang-Mu Sung; Betty Chien-Jung Pai; Nai-Jen Liu; Carl PC Chen

    2016-01-01

    Aims. Bile is an important body fluid which assists in the digestion of fat and excretion of endogenous and exogenous compounds. In the present study, an improved sample preparation for human bile was established. Methods and Material. The method involved acetone precipitation followed by protein extraction using commercially available 2D Clean-Up kit. The effectiveness was evaluated by 2-dimensional electrophoresis (2DE) profiling quality, including number of protein spots and spot distribut...

  10. Determination of microbial genome sizes by two-dimensional denaturing gradient gel electrophoresis.

    Poddar, S K; Maniloff, J

    1989-01-01

    In two-dimensional denaturing gradient gel electrophoresis, DNA is digested with a restriction endonuclease and the resulting DNA fragments are separated as a function of size by conventional agarose gel electrophoresis. Following this first dimension electrophoresis, the fragment distribution is placed at the top of a denaturing gradient slab gel and electrophoresis is carried out parallel to the gradient direction. This second dimension separation is a complex function of the base sequence ...

  11. Classification of Clostridium butyricum based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and pulsed-field gel electrophoresis.

    Yokota, K; Fujinaga, Y; Inoue, K; Shimazaki, S; Seo, G; Takeshi, K; Nagamachi, E; Oguma, K

    1998-08-01

    Eleven strains of Clostridium butyricum collected from different sources were analysed by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and pulsed-field gel electrophoresis (PFGE). The strains could be classified into four groups based on their banding profiles of the proteins extracted from the cells on SDS-PAGE. Group I consisted of seven strains, and these strains were further divided into five subgroups by PFGE. The strains belonging to groups II, III, and IV on SDS-PAGE were also classified into the same II to IV groups by PFGE. These data indicate that grouping of the strains of C. butyricum can be performed by employing both SDS-PAGE and PFGE. PMID:16887639

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

    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

  13. Two-dimensional polyacrylamide gel electrophoresis of intracellular proteins

    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

  14. Topological complexity of different populations of pBR322 as visualized by two-dimensional agarose gel electrophoresis

    Martin-Parras, Luis; Lucas, Isabelle A.; Martínez-Robles, María Luisa; Hernandez, Pablo; Krimer, Dora B.; Hyrien, Olivier; Schvartzman, Jorge Bernardo

    1998-01-01

    Neutral/neutral two-dimensional (2D) agarose gel electrophoresis was used to investigate populations of the different topological conformations that pBR322 can adopt in vivo in bacterial cells as well as in Xenopus egg extracts. To help in interpretation and identification of all the different signals, undigested as well as DNA samples pretreated with DNase I, topoisomerase I and topoisomerase II were analyzed. The second dimension of the 2D gel system was run with or without ethidium bromide...

  15. The migration behaviour of DNA replicative intermediates containing an internal bubble analyzed by two-dimensional agarose gel electrophoresis

    Schvartzman, Jorge Bernardo; Martínez-Robles, María Luisa; Hernandez, Pablo

    1993-01-01

    Initiation of DNA replication in higher eukaryotes is still a matter of controversy. Some evidence suggests it occurs at specific sites. Data obtained using two-dimensional (2D) agarose gel electrophoresis, however, led to the notion that it may occur at random in broad zones. This hypothesis is primarily based on the observation that several contiguous DNA fragments generate a mixture of the so-called 'bubble' and 'simple Y' patterns in Neutral/neutral 2D gels. The interpretation that this m...

  16. Band broadening of DNA fragments isolated by polyacrylamide gel electrophoresis in capillary electrophoresis.

    Kaneta, Takashi; Ogura, Takehito; Yamato, Shuhei; Imasaka, Totaro

    2012-02-01

    Polyacrylamide gel electrophoresis (PAGE) is used frequently for isolation and purification of DNA fragments. In the present study, DNA fragments extracted from polyacrylamide gels showed significant band broadening in capillary electrophoresis (CE). A pHY300PLK (a shuttle vector functioning in Escherichia coli and Bacillus subtilis) marker, which contained nine fragments ranging from 80 to 4870 bp, was separated by PAGE, and each fragment was isolated by phenol/chloroform extraction and ethanol precipitation. After extraction from the polyacrylamide gel, the peaks of the isolated DNA fragments exhibited band broadening in CE, where a linear poly(ethylene oxide) was used as a sieving matrix. The theoretical plate numbers of the DNA fragments contained in the pHY300PLK marker were >10(6) for all the fragments before extraction. However, the DNA fragments extracted from the polyacrylamide gel showed decreased theoretical plate numbers (5-20 times smaller). The degradation of the theoretical plate number was significant for middle sizes of the DNA fragments ranging from 489 to 1360 bp, whereas the largest and smallest fragments (80 and 4870 bp) had no obvious influence. The band broadening was attributed to contamination of the DNA fragments by polyacrylamide fibers during the separation and extraction process. PMID:22258810

  17. Comparative proteomics of E. coli O157:H7: two-dimensional gel electrophoresis vs. two-dimensional liquid chromatography separation

    The accepted method for comparing bacterial proteomes has traditionally been two-dimensional gel electrophoresis (2-D GE). However, in recent years, new procedures for protein separation have been introduced. One of these new procedures utilizes column-based liquid chromatography (2-D LC) separati...

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

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

  19. System for loading slab-gel holders for electrophoresis separation

    Anderson, Norman G.; Anderson, Norman L.

    1979-01-01

    A slab-gel loading system includes a prismatic chamber for filling a plurality of slab-gel holders simultaneously. Each slab-gel holder comprises a pair of spaced apart plates defining an intermediate volume for gel containment. The holders are vertically positioned in the chamber with their major surfaces parallel to the chamber end walls. A liquid inlet is provided at the corner between the bottom and a side wall of the chamber for distributing a polymerizable monomer solution or a coagulable colloidal solution into each of the holders. The chamber is rotatably supported so that filling can begin with the corner having the liquid inlet directed downwardly such that the solution is gently funneled upwardly, without mixing, along the diverging side and bottom surfaces. As filling proceeds, the chamber is gradually rotated to position the bottom wall in a horizontal mode. The liquid filling means includes a plastic envelope with a septum dividing it into two compartments for intermixing two solutions of different density and thereby providing a liquid flow having a density gradient. The resulting gels have a density gradient between opposite edges for subsequent use in electrophoresis separations.

  20. Electrophoresis and orientation of F-actin in agarose gels.

    Borejdo, J; Ortega, H.

    1989-01-01

    F-Actin was electrophoresed on agarose gels. In the presence of 2 mM MgCl2 and above pH 8.5 F-actin entered 1% agarose; when the electric field was 2.1 V/cm and the pH was 8.8, F-actin migrated through a gel as a single band at a rate of 2.5 mm/h. Labeling of actin with fluorophores did not affect its rate of migration, but an increase in ionic strength slowed it down. After the electrophoresis actin was able to bind phalloidin and heavy meromyosin (HMM) and it activated Mg2+-dependent ATPase...

  1. Gel Electrophoresis as Quality Control Method of the Radiolabeled Monoclonal Antibodies

    Kocurová, Veronika

    Rijeka : InTech, 2012 - (Magdeldin, S.), s. 447-462 ISBN 978-953-51-0457-5 R&D Projects: GA AV ČR IBS1048301 Institutional research plan: CEZ:AV0Z10480505 Keywords : monoclonal antibodies * gel electrophoresis * radiodiagnostic Subject RIV: FR - Pharmacology ; Medidal Chemistry http://www.intechopen.com/books/ gel - electrophoresis -advanced-techniques/ gel - electrophoresis -as-quality-control-method-of-the-radiolabeled-monoclonal-antibodies

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

    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). PMID:27251892

  3. Novel display of knotted DNA molecules by two-dimensional gel electrophoresis

    Trigueros, Sonia; Arsuaga, Javier; Vázquez, María E.; Sumners, De Witt; Roca, Joaquim

    2001-01-01

    We describe a two-dimensional agarose gel electrophoresis procedure that improves the resolution of knotted DNA molecules. The first gel dimension is run at low voltage, and DNA knots migrate according to their compactness. The second gel dimension is run at high voltage, and DNA knots migrate according to other physical parameters such as shape and flexibility. In comparison with one-dimensional gel electrophoresis, this procedure segregates the knotted DNA molecules ...

  4. The Application of Pulsed Field Gel Electrophoresis in Clinical Studies.

    Parizad, Elaheh Gholami; Parizad, Eskandar Gholami; Valizadeh, Azar

    2016-01-01

    Pulsed-field gel electrophoresis is a method applied in separating large segments of deoxyribonucleotide using an alternating and cross field. In a uniform magnetic field, components larger than 50kb pass a route through the gel and since the movement of DNA (Deoxyribonucleic acid) molecules are in a Zigzag form, separation of DNAs as bands carried out better via gel. PFGE in microbiology is a standard method which is used for typing of bacteria. It is also a very useful tool in epidemiological studies and gene mapping in microbes and mammalian cell, also motivated development of large-insert cloning system such as bacterial and yeast artifical chromosomes. In this method, close and similar species in terms of genetic patterns show alike profiles regarding DNA separation, and those ones which don't have similarity or are less similar, reveal different separation profiles. So this feature can be used to determine the common species as the prevalence agent of a disease. PFGE can be utilized for monitoring and evaluating different micro-organisms in clinical samples and existing ones in soil and water. This method can also be a reliable and standard method in vaccine preparation. In recent decades, PFGE is highly regarded as a powerful tool in control, prevention and monitoring diseases in different populations. PMID:26894068

  5. [Detection of picobirnaviruses by electrophoresis of RNA in polyacrylamide gel].

    Novikova, N A; Epifanova, N V; Fedorova, O F; Golitsyna, L N; Kupriianova, N V

    2003-01-01

    Double-segment profiles typical of picobirnavirus (PBV) were detected (during 1994-2001) in nucleic acids extracted from feces of children (3 cases) and calf (1 case) with diarrhea by using the method of electrophoresis. The human genomic PBV segments migrated in the polyacrylamide gel (PAAG) as dsRNA segments sized 1.7 and 2.4 kbp for small and large segments, respectively; the similar calf sizes were 1.5 and 2.6 kbp. PBVs were detected in various places of the Nizhny Novgorod Region and at different time periods. It was for the first time that the PBV circulation was proven to be present in Russia's territory. However, their association with diarrhea was not reliably established, and the pathogenic PBV potential needs further investigations. PMID:14708231

  6. Analysis of Two-Dimensional Electrophoresis Gel Images

    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 and...... methods based on image analysis techniques in order to significantly accelerate this key technology. The methods described and developed fall into three categories: image segmentation, point pattern matching, and a unified approach simultaneously segmentation the image and matching the spots. The main...... pharmaceutical applications, e.g., drug development. The technique results in an image, where the proteins appear as dark spots on a bright background. However, the analysis of these images is very time consuming and requires a large amount of manual work so there is a great need for fast, objective, and robust...

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

    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

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

    Full Text Available ta contents Detailed information and images of gel electrophoresis of each marker. Data file File name: rgp_caps_electrophoresis_imag...aps/LATEST/rgp_caps_electrophoresis_image.zip File size: 28.7 MB Simple search URL - Data acquisition method

  9. Electron beam sterilization of the Agaroze gel used for electrophoresis

    Electron beam sterilization is based on its ability to kill pathogenic microorganisms. It is applied to a broad range of disposable medical products such as syringes, needles, surgical sutures, transplant kits, inhalation and dialysis equipment, blood-handling equipment, wound and burn dressings, gloves, masks, gowns, Petri dishes and pipettes. By this work we intend to extend the EB irradiation to the sterilization of the agarose gel put on plastic plates. Electrophoresis of serum proteins on agarose gel is an important investigation method of variety disproteinemia types, used in clinical laboratory. By this method are separated six proteic fractions: Albumin; Alpha 1 globulin consisting of α1 lipoproteins and α1 antitrypsin; Alpha 2 globulin consisting of α2 macroglobulin and haptoglobin; Beta 1 globulin consisting of transferrin and β lipoprotein; Beta 2 globulin consisting of complement C3; Gamma globulin consisting of immunoglobulins and fibrogen. Agarose is a natural colloid extracted from seaweed. It is very fragile and easily destroyed by handling. Agarose gel can be processed faster than polyacrylamide gels, but the agarose gel is a favorable medium for the microorganisms' development, which is the cause for the degradation of the gel and of the results. Thus, the sterilization of the plates with agarose gels becomes important. A disadvantage is that ionizing radiation degrades some plastic gels above a certain absorbed dose level. In view of this important aspects the sterilization of the agarose gel, which is put on plastic plates, by electron beam irradiation has been studied. Also, the effects of the electron beam irradiation upon the agarose gel and on the process of the proteic fraction separation have been investigated. In the experimental studies are used the plastic plates with 1% agarose gel in Tris-barbital tampon, supplied by DDS DIAGNOSTIC-Romania. The used migration solution is Tris-barbital tampon pH 8.6. In order to establish the

  10. Analysis of strains of Campylobacter fetus by pulsed-field gel electrophoresis.

    Fujita, M; Fujimoto, S.; Morooka, T; Amako, K.

    1995-01-01

    Campylobacter fetus chromosomal DNA from 21 strains was analyzed by pulsed-field gel electrophoresis. The fingerprint patterns generated with SmaI and SalI were distinctive. Using the profiles obtained by pulsed-field gel electrophoresis, we established the phylogenetic dendrogram of C. fetus to identify the genetic relationship of the strains.

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

    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...... in a buffer without propan-2-ol but containing sodium dodecyl sulfate....

  12. Rifaximin-mediated changes to the epithelial cell proteome: 2-D gel analysis.

    Caroline Schrodt

    Full Text Available Rifaximin is a semi-synthetic rifamycin derivative that is used to treat different conditions including bacterial diarrhea and hepatic encephalopathy. Rifaximin is of particular interest because it is poorly adsorbed in the intestines and has minimal effect on colonic microflora. We previously demonstrated that rifaximin affected epithelial cell physiology by altering infectivity by enteric pathogens and baseline inflammation suggesting that rifaximin conferred cytoprotection against colonization and infection. Effects of rifaximin on epithelial cells were further examined by comparing the protein expression profile of cells pretreated with rifaximin, rifampin (control antibiotic, or media (untreated. Two-dimensional (2-D gel electrophoresis identified 36 protein spots that were up- or down-regulated by over 1.7-fold in rifaximin treated cells compared to controls. 15 of these spots were down-regulated, including annexin A5, intestinal-type alkaline phosphatase, histone H4, and histone-binding protein RbbP4. 21 spots were up-regulated, including heat shock protein (HSP 90α and fascin. Many of the identified proteins are associated with cell structure and cytoskeleton, transcription and translation, and cellular metabolism. These data suggested that in addition to its antimicrobial properties, rifaximin may alter host cell physiology that provides cytoprotective effects against bacterial pathogens.

  13. Two-dimensional gel electrophoresis in platelet proteomics research.

    García, Angel

    2007-01-01

    Proteomics technology allows a comprehensive and efficient analysis of the proteome and has become an indispensable tool in biomedical research. Since the late 80s, advances on mass spectrometry (MS) instrumentation and techniques have revolutionized the way proteins can be analyzed. Such analysis would only be possible with a proper sample preparation and separation ahead of the MS step. Different gel and nongel-based methods are available for protein separation. This chapter will focus on the use of two-dimensional gel electrophoresis (2-DE) in proteomics and its application to platelet research. 2-DE separates proteins according to their isoelectric point (pI) and size (molecular weight) and allows the detection of thousands of proteins at a time. Platelets are enucleated cells that play a critical function in the control of bleeding and wound healing. As platelets do not have a nucleus, proteomics offers a powerful alternative approach to provide data on protein expression in these cells, helping to address their biology. This chapter presents a protocol for an efficient sample preparation, protein separation by 2-DE, and protein digestion ahead of the MS analysis. The experimental approach, already successfully applied to the study of the platelet proteome, includes recommendations for an efficient platelet preparation for proteomics studies. PMID:18287684

  14. Stability measurements of antisense oligonucleotides by capillary gel electrophoresis.

    Bruin, G J; Börnsen, K O; Hüsken, D; Gassmann, E; Widmer, H M; Paulus, A

    1995-08-11

    The approach of using antisense oligonucleotides as potential drugs is based on hybridization of a short chemically-modified oligonucleotide with complementary cellular DNA or RNA sequences. A critical question is the stability of chemically modified antisense oligonucleotides in cellular environments. In a model system, resistance against various nucleases was evaluated by capillary gel electrophoresis (CGE). For some of the samples, matrix assisted laser desorption and ionization mass spectrometry (MALDI-MS) was used as an additional analytical tool to perform stability measurements. Using CGE, the enzymatic degradation of single nucleotides from the oligomer can be followed after different incubation times. 10% T polyacrylamide gels give baseline resolution for oligonucleotides ranging between 5 and 30 bases in length. The kinetic influence of a specific nuclease concentration and the antisense oligonucleotide structure on the cleavage reaction are discussed. Also, a simple desalting method to improve the injection efficiency and sensitivity of the method are described. Examples of measurements of chemically modified antisense 19-mers are presented. PMID:7581844

  15. Two previously undetected variants of glutamic-pyruvic transaminase found by acidic polyacrylamide gel electrophoresis.

    McLellan, T

    1982-01-01

    Two new electrophoretic variants of glutamic-pyruvic transaminase (GPT) have been found by polyacrylamide gel electrophoresis at acidic pH. They appeared to represent a single allele, GPT 2, by the standard method of starch gel electrophoresis. Studies in families show that they are inherited as codominant alleles at the GPT locus. Population frequencies are about the same as those of other rare GPT variants. Their behavior on gels is consistent with both of them having substitutions of histi...

  16. Identification of the multiple beta-thalassemia mutations by denaturing gradient gel electrophoresis.

    Cai, S P; Kan, Y W

    1990-01-01

    We used denaturing gradient gel electrophoresis to detect the beta-thalassemia mutations in the Chinese population. By amplifying the beta-globin gene in four separate fragments and electrophoresing the amplified DNA in two gels, we were able to distinguish all the 12 known mutations on the basis of the mobility of the homoduplexes and heteroduplexes. We conclude that denaturing gradient gel electrophoresis offers a nonradioactive means of detecting multiple mutations in genetic disorders.

  17. Molecular sequestration stabilizes CAP-DNA complexes during polyacrylamide gel electrophoresis.

    Fried, M G; G.Liu

    1994-01-01

    The gel electrophoresis mobility shift assay is widely used for qualitative and quantitative characterization of protein complexes with nucleic acids. Often it is found that complexes that are short-lived in free solution (t1/2 of the order of minutes) persist for hours under the conditions of gel electrophoresis. We have investigated the influence of polyacrylamide gels on the pseudo first-order dissociation kinetics of complexes containing the E.coli cyclic AMP receptor protein (CAP) and la...

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

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

    2014-01-01

    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 electrop...

  19. Adaptation of a 2D in-gel kinase assay to trace phosphotransferase activities in the human pathogen Leishmania donovani.

    Schmidt-Arras, Dirk; Leclercq, Olivier; Gherardini, Pier Federico; Helmer-Citterich, Manuela; Faigle, Wolfgang; Loew, Damarys; Späth, Gerald F

    2011-08-24

    The protozoan parasite Leishmania donovani undergoes various developmental transitions during its infectious cycle that are triggered by environmental signals encountered inside insect and vertebrate hosts. Intracellular differentiation of the pathogenic amastigote stage is induced by pH and temperature shifts that affect protein kinase activities and downstream protein phosphorylation. Identification of parasite proteins with phosphotransferase activity during intracellular infection may reveal new targets for pharmacological intervention. Here we describe an improved protocol to trace this activity in L. donovani extracts at high resolution combining in-gel kinase assay and two-dimensional gel electrophoresis. This 2D procedure allowed us to identify proteins that are associated with amastigote ATP-binding, ATPase, and phosphotransferase activities. The 2D in-gel kinase assay, in combination with recombinant phospho-protein substrates previously identified by phospho-proteomics analyses, provides a novel tool to establish specific protein kinase-substrate relationships thus improving our understanding of Leishmania signal transduction with relevance for future drug development. PMID:21443974

  20. Determination of phosphorus and metals in human brain proteins after isolation by gel electrophoresis by laser ablation inductively coupled plasma source mass spectrometry

    Becker, J. S.; M. Zoriy; Becker, J. Su.; Pickhardt, C.; Przybylski, M.

    2004-01-01

    Phosphorus, sulfur, silicon and metal concentrations (Al, Cu and Zn) were determined in human brain, proteins by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) after separation of protein mixtures by two dimensional (2-D) gel electrophoresis. The analysis of phosphorus, silicon and metals in single protein spots in the gel was' performed with an optimized microanalytical method using a double-focusing sector field inductively coupled plasma mass spectrometer coupled t...

  1. Parameters of field inversion gel electrophoresis for the analysis of pox virus genomes.

    Bostock, C J

    1988-01-01

    The effects of variation in the lengths of forward and reverse pulses, voltage gradient, gel concentration and gel temperature on the mobility of DNA molecules in agarose gels during field inversion gel electrophoresis (FIGE) have been determined. A curve, which best fits the empirical data, is presented and allows the choice of pulse conditions and voltage gradient most suitable for the resolution of molecules of chosen size. The use of FIGE in the analysis and direct mapping of large virus ...

  2. Constant denaturant gel electrophoresis as a rapid screening technique for p53 mutations.

    Børresen, A.L.; Hovig, E; Smith-Sørensen, B; Malkin, D; Lystad, S; Andersen, T.I.; Nesland, J. M.; Isselbacher, K J; Friend, S H

    1991-01-01

    At present, mutation of the p53 gene appears to be the most common genetic alteration found in human cancers. These mutations can occur within many different regions of the gene. We have developed a modification of denaturing gradient gel electrophoresis termed "constant denaturant gel electrophoresis" (CDGE), which provides a rapid and sensitive method to screen the four conserved regions within the p53 gene where the majority of p53 mutations have been reported. The sensitivity of CDGE was ...

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

    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. Trapping of megabase-sized DNA molecules during agarose gel electrophoresis

    Gurrieri, Sergio; Smith, Steven B.; Bustamante, Carlos

    1999-01-01

    Megabase DNA molecules become trapped in agarose gels during electrophoresis if the electric field exceeds a few volts per cm. Fluorescence microscopy reveals that these molecules invariably arrest in U-shaped conformations. The field-vs.-size dependence for trapping indicates that a critical molecular tension is required for trapping. The size of unligated λ-ladders, sheared during gel electrophoresis at a given field, coincides with the size of molecules trapped at that field, suggesting th...

  5. Use of agarose gel electrophoresis of plasmid deoxyribonucleic acid to fingerprint gram-negative bacilli.

    Schaberg, D.R.; Tompkins, L S; Falkow, S

    1981-01-01

    Agarose gel electrophoresis of the plasmid deoxyribonucleic acids from 60 gram-negative bacilli recovered during investigations of nosocomial epidemics was used to fingerprint the strains. This method was as specific at differentiating bacterial strains as more conventional phenotyping methods. In all cases, plasmid band fingerprints of epidermic strains isolates were identical whereas coisolate plasmid deoxyribonucleic acid patterns were different. Agarose gel electrophoresis of plasmid deox...

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

    José Carlos Pelielo De Mattos; Ellen Serri da Motta; Márcia Betania Nunes de Oliveira; Flávio José da Silva Dantas; Adriano Caldeira de Araujo

    2008-01-01

    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 ...

  7. Insight of Saffron Proteome by Gel-Electrophoresis.

    Paredi, Gianluca; Raboni, Samanta; Marchesani, Francesco; Ordoudi, Stella A; Tsimidou, Maria Z; Mozzarelli, Andrea

    2016-01-01

    Saffron is a spice comprised of the dried stigmas and styles of Crocus sativus L. flowers and, since it is very expensive, it is frequently adulterated. So far, proteomic tools have never been applied to characterize the proteome of saffron or identify possible cases of fraud. In this study, 1D-Gel Electrophoresis was carried out to characterize the protein profile of (i) fresh stigmas and styles of the plant; (ii) dried stigmas and styles from different geographical origins (Spanish, Italian, Greek and Iranian) that had been stored for various periods of time after their processing; and (iii) two common plant adulterants, dried petals of Carthamus tinctorius L. and dried fruits of Gardenia jasminoides Ellis. A selective protein extraction protocol was applied to avoid interference from colored saffron metabolites, such as crocins, during electrophoretic analyses of saffron. We succeeded in separating and assigning the molecular weights to more than 20 proteins. In spite of the unavailability of the genome of saffron, we were able to identify five proteins by Peptide Mass Fingerprinting: phosphoenolpyruvate carboxylase 3, heat shock cognate 70 KDa protein, crocetin glucosyltransferase 2, α-1,4-glucan-protein synthase and glyceraldehydes-3-phosphate dehydrogenase-2. Our findings indicate that (i) few bands are present in all saffron samples independently of origin and storage time, with amounts that significantly vary among samples and (ii) aging during saffron storage is associated with a reduction in the number of detectable bands, suggesting that proteases are still active. The protein pattern of saffron was quite distinct from those of two common adulterants, such as the dried petals of Carthamus tinctorius and the dried fruits of Gardenia jasminoides indicating that proteomic analyses could be exploited for detecting possible frauds. PMID:26840283

  8. Insight of Saffron Proteome by Gel-Electrophoresis

    Gianluca Paredi

    2016-01-01

    Full Text Available Saffron is a spice comprised of the dried stigmas and styles of Crocus sativus L. flowers and, since it is very expensive, it is frequently adulterated. So far, proteomic tools have never been applied to characterize the proteome of saffron or identify possible cases of fraud. In this study, 1D-Gel Electrophoresis was carried out to characterize the protein profile of (i fresh stigmas and styles of the plant; (ii dried stigmas and styles from different geographical origins (Spanish, Italian, Greek and Iranian that had been stored for various periods of time after their processing; and (iii two common plant adulterants, dried petals of Carthamus tinctorius L. and dried fruits of Gardenia jasminoides Ellis. A selective protein extraction protocol was applied to avoid interference from colored saffron metabolites, such as crocins, during electrophoretic analyses of saffron. We succeeded in separating and assigning the molecular weights to more than 20 proteins. In spite of the unavailability of the genome of saffron, we were able to identify five proteins by Peptide Mass Fingerprinting: phosphoenolpyruvate carboxylase 3, heat shock cognate 70 KDa protein, crocetin glucosyltransferase 2, α-1,4-glucan-protein synthase and glyceraldehydes-3-phosphate dehydrogenase-2. Our findings indicate that (i few bands are present in all saffron samples independently of origin and storage time, with amounts that significantly vary among samples and (ii aging during saffron storage is associated with a reduction in the number of detectable bands, suggesting that proteases are still active. The protein pattern of saffron was quite distinct from those of two common adulterants, such as the dried petals of Carthamus tinctorius and the dried fruits of Gardenia jasminoides indicating that proteomic analyses could be exploited for detecting possible frauds.

  9. In-gel DNA radiolabelling and two-dimensional pulsed field gel electrophoresis procedures suitable for fingerprinting and mapping small eukaryotic genomes

    Brugère, Jean-François; Cornillot, Emmanuel; Méténier, Guy; Vivarès, Christian P.

    2000-01-01

    A simple method for complete genome radiolabelling is described, involving long-wave UV exposure of agarose-embedded chromosomal DNA and [α-32P]dCTP incorporation mediated by the Klenow fragment. Experiments on the budding yeast genome show that the labelling procedure can be coupled with two new two-dimensional pulsed field gel electrophoresis (2D-PFGE) protocols of genome analysis: (i) the KARD (karyotype and restriction display)-PFGE which provides a complete view of the fragments resultin...

  10. Assessing CMT cell line stability by two dimensional polyacrylamide gel electrophoresis and mass spectrometry based proteome analysis

    Zhang, Kelan; Wrzesinski, Krzysztof; Fey, Stephen J;

    2008-01-01

    Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) followed by mass spectrometric identification of the proteins in the protein spots has become a central tool in proteomics. CMT167(H), CMT64(M) and CMT170(L) cell lines, selected from a spontaneous mouse lung adenocarcinoma, with high...... useful tool for assessing differences in cell line stability. This approach provided a tool to select the best cell line and optimal subculture period for studies of cancer related phenomena and for testing the effect of potential anticancer drugs....

  11. 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

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

    2001-01-01

    varieties. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was applied to three pairs of wheat varieties, which can not be classified correctly by ANN. By 2-D PAGE the varieties in the three pairs can be discriminated and these six wheat varieties can be separated from each other, which could...

  12. Polyvinylpyrrolidone-Agarose Gel Electrophoresis Purification of Polymerase Chain Reaction-Amplifiable DNA from Soils

    Young, Charles C.; Burghoff, Robert L.; Keim, Lois G.; Minak-Bernero, Vera; Lute, James R.; Hinton, Stephen M.

    1993-01-01

    This communication describes a modification of agarose gel electrophoresis to provide a rapid and simple method for the purification of polymerase chain reaction-amplifiable DNA from soil. This modification is to add polyvinylpyrrolidone to the agarose gel. The polyvinylpyrrolidone addition retards the electrophoretic mobility of denaturing phenolic compounds so that they do not comigrate with nucleic acids.

  13. Investigation of the repair of single-strand breaks in human DNA using alkaline gel electrophoresis

    Unstimulated lymphocytes from eight healthy persons were exposed to 10-, 30-, and 100-Gy doses of 60Co gamma radiation. The repair of damaged DNA was measured by (1) alkaline gel electrophoresis (extracted DNA loaded on 0.25% agarose gel, run at 1 V/cm for 39-44 h) at 0, 1, and 2 h after exposure and (2) incorporation of [3H]thymidine into unstimulated lymphocytes in the presence of 2 mM hydroxyurea 1 and 2 h after exposure. Both methods--alkaline gel electrophoresis and thymidine incorporation--showed that repair was completed within 2 h

  14. High-sensitivity detection of proteins using gel electrophoresis and atomic force microscopy

    We have developed a method to detect specific proteins with a high sensitivity using a gel electrophoresis method and force measurement of atomic force microscopy (AFM). Biotinylated proteins were separated by electrophoresis and fixed with cross-linking chemicals on the gel, followed by direct force measurement between the biotinylated proteins on the gel and a streptavidin-modified tip of an AFM cantilever. We were able to achieve a high enough sensitivity to detect the picogram order of the biotinylated proteins by evaluating the frequency of the interaction force larger than 100 pN in the force profile, which corresponds to the rupture force of interaction between streptavidin and biotin.

  15. In-gel staining of proteins in native poly acryl amide gel electrophoresis using tetrakis(4-sulfonato phenyl)porphyrin.

    Divakar, Kalivarathan; Sujatha, Vijayan; Barath, Sridhar; Srinath, Krishnamurthy; Gautam, Pennathur

    2011-01-01

    Protein identification in polyacrylamide gel electrophoresis (PAGE) requires post-electrophoretic steps like fixing, staining and destaining of the gel, which are time-consuming and cumbersome. We have developed a method for direct visualization of protein bands in PAGE using tetrakis(4-sulfonato phenyl)porphyrin (TPPS) as a dye without the need for any post electrophoretic steps, where separation and recovery of enzymes become much easier for further analysis. Activity staining was done to prove that the biochemical activity of the enzymes was preserved after electrophoresis. PMID:21233569

  16. In-gel staining of proteins in native polyacrylamide gel electrophoresis using meso-tetrakis(4-sulfonatophenyl) porphyrin.

    Divakar, K; Devi, G Nandhini; Gautam, Pennathur

    2012-01-01

    Protein identification in polyacrylamide gel electrophoresis (PAGE) requires post-electrophoretic steps like fixing, staining, and destaining of the gel, which are time-consuming and cumbersome. A new method for direct visualization of protein bands in PAGE has been developed using meso-tetrakis(4-sulfonatophenyl)porphyrin (TPPS) as a dye without the need for any post-electrophoretic steps; thus, separation and recovery of enzymes become much easier for further analysis. Activity staining was carried out to show that the biochemical activity of the enzymes was preserved after electrophoresis. PMID:22585523

  17. Resolving mitochondrial protein complexes using non-gradient blue native polyacrylamide gel electrophoresis

    Yan, Liang-Jun; Forster, Michael J.

    2009-01-01

    Blue native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful technique for separation and proteomic analysis of high molecular weight protein complexes. It is often performed on gradient gels and is widely used for studying mitochondrial membrane complexes involved in electron transportation and oxidative phosphorylation. In this paper, we present an alternative BN-PAGE method that uses highly porous, non-gradient polyacrylamide gels for separation of rat brain mitochondrial protein...

  18. Functional Characterization of Reductive Dehalogenases by Using Blue Native Polyacrylamide Gel Electrophoresis

    Tang, S; Chan, W. W. M.; Fletcher, K. E.; Seifert, J.; Liang, X.; Loffler, F. E.; Edwards, E A; Adrian, L.

    2013-01-01

    Dehalococcoides mccartyi strains are obligate organohalide-respiring bacteria harboring multiple distinct reductive dehalogenase (RDase) genes within their genomes. A major challenge is to identify substrates for the enzymes encoded by these RDase genes. We demonstrate an approach that involves blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by enzyme activity assays with gel slices and subsequent identification of proteins in gel slices using liquid chromatography coupled t...

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

    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 pr...

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

    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.

  1. Generating high peak capacity 2-D maps of complex proteomes using PMMA microchip electrophoresis.

    Osiri, John K; Shadpour, Hamed; Park, Sunjung; Snowden, Brandy C; Chen, Zhi-Yuan; Soper, Steven A

    2008-12-01

    A high peak capacity 2-D protein separation system combining SDS micro-CGE (SDS micro-CGE) with microchip MEKC (micro-MEKC) using a PMMA microfluidic is reported. The utility of the 2-D microchip was demonstrated by generating a 2-D map from a complex biological sample containing a large number of constituent proteins using fetal calf serum (FCS) as the model system. The proteins were labeled with a thiol-reactive AlexaFluor 633 fluorophore (excitation/emission: 633/652 nm) to allow for ultra-sensitive on-chip detection using LIF following the 2-D separation. The high-resolution separation of the proteins was accomplished based on their size in the SDS micro-CGE dimension and their interaction with micelles in the micro-MEKC dimension. A comprehensive 2-D SDS micro-CGE x micro-MEKC separation of the FCS proteins was completed in less than <30 min using this 2-D microchip format, which consisted of 60 mm and 50 mm effective separation lengths for the first and second separation dimensions, respectively. Results obtained from the microchip separation were compared with protein maps acquired using conventional 2-D IEF and SDS-PAGE of a similar FCS sample. The microchip 2-D separation was found to be approximately 60x faster and yielded an average peak capacity of 2600 (+/- 149), nearly three times larger than that obtained using conventional IEF/SDS-PAGE. PMID:19130578

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

    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. (author)

  3. Measurement of body fluid proteins by polyacrylamide gel electrophoresis

    Johnson, MH; Thompson, EJ

    1982-01-01

    A development of the disc polyacrylamide gel electrophoretic system of Ornstein and Davis, which has been applied to the analysis of unconcentrated cerebrospinal fluid, is described. Modifications to the scanning densitometer have improved the signal:noise ratio of the gel scanning system. Using this technique, we have studied the dye-binding properties of albumin, and of beta- and gamma-globulins, and have shown that reproducible quantification of proteins can be achieved. The advantages of ...

  4. Characterization of DNA metabolizing enzymes in situ following polyacrylamide gel electrophoresis

    The authors have detected the in situ activities of DNA glycosylase, endonuclease, exonuclease, DNA polymerase, and DNA ligase using a novel polyacrylamide activity gel electrophoresis procedure. DNA metabolizing enzymes were resolved through either native or SDS-polyacrylamide gels containing defined 32P-labeled oligonucleotides annealed to M13 DNA. After electrophoresis, these enzymes catalyzed in situ reactions and their [32P]DNA products were resolved from the gel by a second dimension of electrophoresis through a denaturing DNA sequencing gel. Detection of modified (degraded or elongated) oligonucleotide chains was used to locate various enzyme activities. The catalytic and physical properties of Novikoff hepatoma DNA polymerase β were found to be similar under both in vitro and in situ conditions. With 3'-terminally matched and mismatched [32P]DNA substrates in the same activity gel, DNA polymerase and/or 3' to 5' exonuclease activities of Escherichia coli DNA polymerase I (large fragment), DNA polymerase III (holoenzyme), and exonuclease III were detected and characterized. Several restriction endonucleases and the tripeptide (Lys-Try-Lys), which acts as an apurinic/apyrimidinic endonuclease, were able to diffuse into gels and modify DNA. This ability to create intermediate substrates within activity gels could prove extremely useful in delineating the steps of DNA replication and repair pathways

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

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

    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 and...... 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....

  6. Phenols content and 2-D electrophoresis protein pattern: a promising tool to monitor Posidonia meadows health state

    Randazzo Davide

    2007-07-01

    Full Text Available Abstract Background The endemic seagrass Posidonia oceanica (L. Delile colonizes soft bottoms producing highly productive meadows that play a crucial role in coastal ecosystems dynamics. Human activities and natural events are responsible for a widespread meadows regression; to date the identification of "diagnostic" tools to monitor conservation status is a critical issue. In this study the feasibility of a novel tool to evaluate ecological impacts on Posidonia meadows has been tested. Quantification of a putative stress indicator, i.e. phenols content, has been coupled to 2-D electrophoretic protein analysis of rhizome samples. Results The overall expression pattern from Posidonia rhizome was determined using a preliminary proteomic approach, 437 protein spots were characterized by pI and molecular weight. We found that protein expression differs in samples belonging to sites with high or low phenols: 22 unique protein spots are peculiar of "low phenols" and 27 other spots characterize "high phenols" samples. Conclusion Posidonia showed phenols variations within the meadow, that probably reflect the heterogeneity of environmental pressures. In addition, comparison of the 2-D electrophoresis patterns allowed to highlight qualitative protein expression differences in response to these pressures. These differences may account for changes in metabolic/physiological pathways as adaptation to stress. A combined approach, based on phenols content determination and 2-D electrophoresis protein pattern, seems a promising tool to monitor Posidonia meadows health state.

  7. [Characterization of Acholeplasma Strains by Horizontal Polyacrylamide Flat Gel Electrophoresis (author's transl)].

    Boden, K; Kirchhoff, H

    1977-01-01

    Proteins extracted with phenol-acetic acid-water (2:1:0.5, w/v/v) from Acholeplasma laidlawii (PG 8), A. granularum (BTS-39), A. oculi (19L), A. modicum (PG 49), A. axanthum (S743) and the Acholeplasma strains C1 and C112 (which were isolated from aborted horse foetuses) were compared by electrophoresis in horizontal acidic polyacylamide flat gel using the electrophoresis equipment LKB Multiphor 2117. In this system the gels are not prepared in the electrophoresis chamber but between glas plates. For electrophoresis they are applied onto a special cooling plate. This makes it possible to produce a number of identical gels (from the same gel mixture and polymerized under the same conditions) what can be important for comparing investigations. The gels can be stored for more than 4 weeks in the refrigerator at +4 degrees C. Marked differences were observed between the electrophoretic patterns of each of the established species and the horse strains C1 and C112. The results are in agreement with those obtained in serological investigations in which the strains C1 and C112 were different from the established Acholeplasma species. PMID:848216

  8. Nitroproteins in Human Astrocytomas Discovered by Gel Electrophoresis and Tandem Mass Spectrometry

    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

  9. Rapid Pulsed-Field Gel Electrophoresis Method for Group B Streptococcus Isolates

    Benson, Jeffrey A.; Ferrieri, Patricia

    2001-01-01

    We developed a rapid pulsed-field gel electrophoresis (PFGE) method that required 3 days to complete, an improvement over the standard method that required as many as 8 days. The accuracy and reproducibility of the rapid method were verified by analysis of DNA band sizes of our control group B streptococcus isolate. The rapid method was superior to the standard method, providing more precise molecular sizing and gels of higher image quality. The reproducibility of rapid PFGE substantiated its...

  10. Difference in the homology of two nuclear nonhistone protein fractions as compared by polyacrylamide gel electrophoresis.

    Tsutsui, Ken; Tsutsui, Kimiko; Aoyama, Koji; Oda,Takuzo

    1985-01-01

    The extent of homology between two protein fractions was compared by simple electrophoretic analysis. Nuclear proteins of several rodent cells of different origins were fractionated into acid-soluble and acid-insoluble fractions. The two protein fractions were subjected to polyacrylamide gel electrophoresis in separate gel systems, and protein bands with identical mobilities were sought either in all possible combinational pairs of cell types or in all cell types. The paired and overall homol...

  11. Electron Beam Sterilization of the Plates with Agaroze Gel Used for Electrophoresis

    Ighigeanu, Daniel I.; Martin, Diana I.; Stan, Dana E.; Matei, Constantin I.; Manaila, Elena M.; Craciun, Gabriela D.; Iacob, Nicusor I.; Oproiu, Constantin V.; Ighigeanu, Adelina I.

    2007-04-01

    Electron beam (EB) sterilization applied to the plastic plates with agarose gel used for electrophoresis is presented. The effects of EB irradiation upon the agarose gel and on the process of the proteic fraction separation have been investigated. The investigation were focused on the concentration changes of the six proteic fractions, albumin, alpha 1, alpha 2, beta 1, beta 2 and gamma, versus the dose irradiation as compared with the unirradiated sample.

  12. Polyacrylamide gel electrophoresis and silver staining for detection of rotavirus in stools from diarrheic patients in Thailand.

    Kasempimolporn, S.; Louisirirotchanakul, S; Sinarachatanant, P; Wasi, C

    1988-01-01

    Detection of rotavirus by polyacrylamide gel electrophoresis in combination with silver staining and by enzyme-linked immunosorbent assay showed 96.7% identical results in tests with 1,304 stool specimens from diarrheic patients. The polyacrylamide gel electrophoresis method can be modified to reduce cost and working time. Phenol extraction of stools, however, is essential in maintaining the sensitivity of the method.

  13. The use of SDS-polyacrylamide gel electrophoresis in epidemiological studies of Corynebacterium diphtheriae.

    Hallas, G.

    1988-01-01

    Polyacrylamide gel electrophoresis of cell proteins was investigated as a possible typing method for Corynebacterium diphtheriae. A method was developed using stock strains which were representatives of the five gravis serotypes described by Robinson & Peeney (1936). This technique was then applied to recent isolates sent to our laboratory for identification.

  14. Blue Native Polyacrylamide Gel Electrophoresis and Mass Spectric Identification of Nuclear Protein Complexes

    Novák, Petr; Man, Petr; Nováková, Zora; Havlíček, Vladimír; Bezouška, Karel; Hozák, Pavel

    Florida, 2002, s. 1-2. [ASMS Conference Mass Spectrometry and Allied Topics /50./. Orlando (US), 02.06.2002-06.06.2002] R&D Projects: GA AV ČR 5902 Institutional research plan: CEZ:MSM 113100001 Keywords : polyacrylamide gel * electrophoresis * cid spectra Subject RIV: EE - Microbiology, Virology

  15. Regulation of annexins following infection like tissue damage – investigated by 2-dimensional gel electrophoresis

    Wulff, Tune; Nielsen, Michael Engelbrecht

    , internal- and external control were found using a t-test. To investigate numerous proteins in a single study changes in protein abundance were investigated using 2-dimensional gel electrophoresis. Protein of interest were identified using MALDI MS/MS. The results show that both annexin 4 and 5 are...

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

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

    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...

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

    Møller, Kristian; Jensen, Tim Kåre; Boye, Mette; Leser, T.D.; Ahrens, Peter

    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 to the...

  18. Microbial community fingerprinting by differential display-denaturing gradient gel electrophoresis

    Portillo Guisado, María del Carmen; Villahermosa, Desiré; Corzo Rodríguez, Alfonso; González Grau, Juan Miguel

    2011-01-01

    Complex microbial communities exhibit a large diversity, hampering differentiation by DNA fingerprinting. Herein, differential display-denaturing gradient gel electrophoresis is proposed. By adding a nucleotide to the 3′ ends of PCR primers, 16 primer pairs and fingerprints were generated per community. Complexity reduction in each partial fingerprint facilitates sample comparison.

  19. Disposable pen-shaped capillary gel electrophoresis cartridge for fluorescence detection of bio-molecules

    Amirkhanian, Varoujan; Tsai, Shou-Kuan

    2014-03-01

    We introduce a novel and cost-effective capillary gel electrophoresis (CGE) system utilizing disposable pen-shaped gelcartridges for highly efficient, high speed, high throughput fluorescence detection of bio-molecules. The CGE system has been integrated with dual excitation and emission optical-fibers with micro-ball end design for fluorescence detection of bio-molecules separated and detected in a disposable pen-shaped capillary gel electrophoresis cartridge. The high-performance capillary gel electrophoresis (CGE) analyzer has been optimized for glycoprotein analysis type applications. Using commercially available labeling agent such as ANTS (8-aminonapthalene-1,3,6- trisulfonate) as an indicator, the capillary gel electrophoresis-based glycan analyzer provides high detection sensitivity and high resolving power in 2-5 minutes of separations. The system can hold total of 96 samples, which can be automatically analyzed within 4-5 hours. This affordable fiber optic based fluorescence detection system provides fast run times (4 minutes vs. 20 minutes with other CE systems), provides improved peak resolution, good linear dynamic range and reproducible migration times, that can be used in laboratories for high speed glycan (N-glycan) profiling applications. The CGE-based glycan analyzer will significantly increase the pace at which glycoprotein research is performed in the labs, saving hours of preparation time and assuring accurate, consistent and economical results.

  20. Beverage-Agarose Gel Electrophoresis: An Inquiry-Based Laboratory Exercise with Virtual Adaptation

    Cunningham, Steven C.; McNear, Brad; Pearlman, Rebecca S.; Kern, Scott E.

    2006-01-01

    A wide range of literature and experience has shown that teaching methods that promote active learning, such as inquiry-based approaches, are more effective than those that rely on passive learning. Gel electrophoresis, one of the most common laboratory techniques in molecular biology, has a wide range of applications in the life sciences. As…

  1. Aligning Goals, Assessments, and Activities: An Approach to Teaching PCR and Gel Electrophoresis

    Phillips, Allison R.; Robertson, Amber L.; Batzli, Janet; Harris, Michelle; Miller, Sarah

    2008-01-01

    Polymerase chain reaction (PCR) and gel electrophoresis have become common techniques used in undergraduate molecular and cell biology labs. Although students enjoy learning these techniques, they often cannot fully comprehend and analyze the outcomes of their experiments because of a disconnect between concepts taught in lecture and experiments…

  2. An electrophoretic karyotype for Schizosaccharomyces pombe by pulsed field gel electrophoresis.

    Smith, C.L.; Matsumoto, T.; Niwa, O; Klco, S; Fan, J B; Yanagida, M.; Cantor, C R

    1987-01-01

    The three chromosomal DNAs of S. pombe have been fractionated by pulsed field gel electrophoresis. The resulting molecular karyotype will greatly speed gene mapping in this organism, and it indicates that the separation range of the technique extends to DNA molecules as large as 9,000,000 base pairs.

  3. Separation of long RNA by agarose-formaldehyde gel electrophoresis

    Mansour, Farrah H.; Pestov, Dimitri G.

    2013-01-01

    We describe a method to facilitate electrophoretic separation of high molecular weight RNA species, such as ribosomal RNAs and their precursors, on agarose-formaldehyde gels. Two alternative “pK-matched” buffer systems were substituted for the traditionally used MOPS-based conductive media. The key advantages include shortened run times, a five-fold reduction in formaldehyde concentration, a significantly improved resolution of long RNAs, and consistency in separation. The new procedure has a...

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

    Anjo, Antonio dos; Laurell Blom Møller, Anders; Ersbøll, Bjarne Kjær; Finnie, Christine; Shahbazkia, Hamid R.

    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....... Results: Five sections from different gels are used to test the performance of the presented method concerning the detection of protein spots, and three gel sections are used to test the quantification of sixty protein spots. Comparisons with a state-of-the-art commercial software and an academic state...

  5. Application of polyacrylamide gel electrophoresis/neutron activation analysis for protein quantification

    A combination of two methods, polyacrylamide gel electrophoresis (PAGE) and neutron activation analysis (NAA), has been applied to solutions containing phosphoproteins for the purpose of protein quantification. The proteins were separated by molecular weight using PAGE, and then the whole gel was activated by neutron bombardment. Densitometric measurements of the developed bands from 32P, taken from autoradiographs of the activated gels, resulted in quantification of the phosphorus, and then the related protein. This PAGE/NAA method was applied to several phosphoprotein-containing materials, including commercial milk products and reference materials, i.e., IAEA A-11, milk powder, and SRM 1845, Cholesterol in Egg Powder

  6. Ag-nanoparticle fractionation by low melting point agarose gel electrophoresis

    The separation of surface-enhanced raman scattering (SERS)-active Ag-multi-nanoparticle (NP) assemblies by low melting point agarose gel electrophoresis was accomplished here by controlling surface charge using NP capping agents, and the pore size of agarose gel matrix. Detailed transmission electron microscopy analysis of excised gel fractions showed dimers and small clusters to have the greatest SERS activity and a mobility in between the monomers and large aggregates. This strategy enables one to: (1) stabilize small multispherical Ag clusters against further aggregation during purification; (2) fractionate and recover spherical assemblies by nuclearity; and (3) analyze SERS-enhancements for each fraction to optimize purification conditions.

  7. Ag-nanoparticle fractionation by low melting point agarose gel electrophoresis

    Guarrotxena, Nekane, E-mail: nekane@ictp.csic.es [Consejo Superior de Investigaciones Cientificas (CSIC), Instituto de Ciencia y Tecnologia de Polimeros (ICTP) (Spain); Braun, Gary [University of California, Santa Barbara, Department of Chemistry and Biochemistry (United States)

    2012-10-15

    The separation of surface-enhanced raman scattering (SERS)-active Ag-multi-nanoparticle (NP) assemblies by low melting point agarose gel electrophoresis was accomplished here by controlling surface charge using NP capping agents, and the pore size of agarose gel matrix. Detailed transmission electron microscopy analysis of excised gel fractions showed dimers and small clusters to have the greatest SERS activity and a mobility in between the monomers and large aggregates. This strategy enables one to: (1) stabilize small multispherical Ag clusters against further aggregation during purification; (2) fractionate and recover spherical assemblies by nuclearity; and (3) analyze SERS-enhancements for each fraction to optimize purification conditions.

  8. A comparison of cellulose acetate immunofixation with polyacrylamide gel electrophoresis for the detection of oligoclonal bands in unconcentrated cerebrospinal fluid.

    Goodland, F C; Thompson, E. J.

    1983-01-01

    Two methods of electrophoresis for the detection of oligoclonal bands in unconcentrated CSF were compared. A sample of 98 routine CSFs yielded 18 positives by polyacrylamide gel electrophoresis (PAGE) while cellulose acetate electrophoresis with immunofixation (CAIF) gave 13 positives (72% of the PAGE findings). Despite the loss of sensitivity the cellulose acetate electrophoresis was easier to interpret and more suited to a routine hospital laboratory.

  9. Optimization of electric field strength for DNA sequencing in capillary gel electrophoresis

    Luckey, John A.; Smith, Lloyd M.

    1993-06-01

    Since its development, capillary gel electrophoresis has demonstrated the ability to separate DNA sequencing reactions at speeds roughly 25 times as great as conventional slab gel electrophoresis. These increased speeds are the result of using the more efficient dissipation of Joule heating by capillaries. However, to date there have been no studies which quantitate the advantages of disadvantages in operating these gels at high electric field strength. This work addresses this question by investigating the band-broadening of DNA sequencing reactions as they are separated through a fixed distance of gel at field strengths ranging from 50 V/cm to 400 V/cm. It is found that the bandwidths of DNA fragments do decrease with the higher field strengths due to a reduction in diffusional broadening. However, at sufficiently high electric field strengths, the bands begin to broaden again under the influence of an increasing thermal gradient across the diameter of the capillary. The result is an optimum electric field strength in the intermediate range of 100 - 250 V/cm depending on the length of fragments being separated. The relative importance of diffusion and thermal gradients are discussed and used to generate an equation that models the observed band broadening of DNA in capillary gel electrophoresis (CGE).

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

    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

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

    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.

  12. CHARACTERIZATION OF PATHOGENIC FUNGI GENOMES USING PULSED FIELD GEL ELECTROPHORESIS

    吴绍熙; 郭宁如; 殷正男; 柴建华

    1996-01-01

    Pulsed field gel eleetrophoresis (PFGE) has been firstly introdueed in characterization of the pathogenic fungi Pericillium marneffei and Exophiata dermatitidis genomes. The numbers and sizes of their chromosomes have been detected. Polymorphism was identified on the smallest chromosome of E.derntatitidis. The result shows that PFGE for characterization of large molecular DNA pathogenic fungi is very suitable, it is more simple and more efficacy. The result also shows the diversity of pathogenic fungi is relative common even in rare occurred pathogeafie fungi such as E. dermatitidis.

  13. Identification and suppression of secondary structures formed from deoxy-oligonucleotides during electrophoresis in denaturing polyacrylamide-gels.

    Frank, R; Müller, D.; Wolff, C.

    1981-01-01

    Nature and stability of secondary structures formed from DNA-fragments during electrophoresis in urea containing polyacrylamide-gels were investigated. Duplices and especially hairpin loops were found to be surprisingly stable. Polyacrylamide-gels containing high concentrations of formamide effectively melt these structures. Such gels will improve nucleotide sequence analysis in regions of dyad symmetry that often cause 'band compressions' under standard electrophoresis conditions.

  14. The relationship of agarose gel structure to the sieving of spheres during agarose gel electrophoresis.

    Griess, G A; Guiseley, K B; Serwer, P

    1993-01-01

    To understand the organization of fibers in an agarose gel, digitized electron micrographs are used here to determine the frequency distribution of interfiber distance (2Pc) in thin sections of agarose gels. For a preparation of underivatized agarose, a 1.5% gel has a Pc distribution that is indistinguishable from the Pc distribution of a computer-generated, random-fiber gel; the log of the occurrence frequency (F) decreases linearly as a function of Pc. As the agarose concentration decreases...

  15. PFGE MAPPER and PFGE READER: two tools to aid in the analysis and data input of pulse field gel electrophoresis maps.

    Shifman, M. A.; Nadkarni, P.; Miller, P. L.

    1992-01-01

    Pulse field gel electrophoresis mapping is an important technique for characterizing large segments of DNA. We have developed two tools to aid in the construction of pulse field electrophoresis gel maps: PFGE READER which stores experimental conditions and calculates fragment sizes and PFGE MAPPER which constructs pulse field gel electrophoresis maps.

  16. The Prevalence of ESBL Isolates of Acinetobacter baumannii Using Pulsed-Field Gel Electrophoresis

    Parviz Mohajeri

    2014-12-01

    Full Text Available Background: Antibiotics such as fluoroquinolones are used for treating infections caused by Gram-negative bacteria, including Acinetobacter baumannii strains some time have extended-spectrum β-lactamase (ESBL, but ESBL production is rather rare. Resistance to fluoroquinolones antibiotics is mediated by lactamases and other mechanisms of resistance. The aim of the present study was to investigate of the prevalence of ESBL production and clonal relatedness of A. baumannii in Iran. Materials and Methods: A. baumannii isolates identified from patients at hospitals in Kermanshah, Iran, were studied. The double disk method was used for detection of ESBL production. The susceptibility to different antibiotics was determined by the disk diffusion method (CLSI. Clonal relatedness was determined by pulsed-field gel electrophoresis (PFGE and processed by Bionumerics 7.0 software. Statistical analyses were performed using SPSS-16.0. Results: This study showed high prevalence of resistance to ampicillin and cefpodoxim (98.1 and 92.3%. Fifty-two of the 84 isolates were identified as ESBL producers. Only colistin and tigecycline remained active against all isolates tested. The PFGE identified eight distinct pulsotypes: A (N=9, B (N=10, C (N=2, D (N=5, E (N=9, F (N=15, G (N=1 and H (N=1. The PFGE profiles A, B and F were believed to be endemic (specially clone F that was dominant across different wards of the hospitals and appeared to be endemic in the ICU, emergency, pediatric and infection area throughout the years. Conclusion: Early and timely detection of ESBL-producing A. baumannii clones is useful for preventing their spread within the hospital. PFGE analysis is helpful for detection of common strains in different wards and prevention of further spread of these pulsotypes to other hospital environment.

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

    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 59Fe. 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)

  18. Two methods that facilitate autoradiography of small 32P-labeled DNA fragments following electrophoresis in agarose gels

    Two methods which permit detection by autoradiography of small 32P-labeled DNA fragments resolved by agarose gel electrophoresis are described. Agarose gel electrophoresis poses problems for autoradiography as (i) the gels are normally too thick to allow autoradiography without being dried first, and (ii) fragments of DNA of 1000 bp or less in length are readily lost during drying. In this study DNA fragments as small as 121 bp have been retained in agarose gels upon drying. This has been achieved by either (i) first fixing the DNA with the cationic detergent cetyltrimethylammonium bromide, or (ii) drying the agarose gels onto Zeta-Probe charge-modified membranes

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

    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

  20. Comparative Analysis of the Endosperm Proteins Separated by 2-D Electrophoresis for Two Cultivars of Hybrid Rice (Oryza sativa L.)

    Pingfang Yang; Shihua Shen; Tingyun Kuang

    2006-01-01

    Liangyoupeijiu is a two-parental-line, and Shanyou63 is a three-parental-line hybrid rice (Oryza sativa L.).Although both belong to the indica subspecies, they have obvious differences with respect to morphology,physiology and grain quality. Variations in endosperm protein compositions were studied by comparing the 2-D electrophoresis (2-DE) maps for these two cultivars of hybrid rice. After matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) analysis, a 21-kDa precursor of 19-kDa globulin was identified as the major storage protein for both cultivars. Some isoforms of peroxiredoxin and seed maturation protein were found to only exist in Shanyou63, whereas aldose reductase and starch granule-bound starch synthase were only detected in Liangyoupeijiu. These data might provide a foundation for further comparative studies of these two cultivars of hybrid rice.

  1. First use of two-dimensional polyacrylamide gel electrophoresis to determine phylogenetic relationships.

    Dopson, Mark; Baker-Austin, Craig; Bond, Philip L

    2004-09-01

    Methods for microbial classification are not always capable of distinguishing between isolates at the species level. We have previously characterised four Ferroplasma isolates that were >98.9% similar at the 16S rDNA level, the isolates showed marked phenotypic differences, and one isolate was borderline on the 70% species boundary from DNA-DNA similarity data. In this study we have used statistical comparisons of two-dimensional polyacylamide gel electrophoresis gels for classification of closely related isolates. From the protein profile similarities an un-rooted tree was constructed that was congruent with a tree derived from DNA-DNA similarities. PMID:15279933

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

    Larsen, Martin; Skottrup, Peter; Enghild, Jan Johannes; Højrup, Peter; Roepstorff, Peter

    . 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...... sensitivity level comparable with state-of-the-art proteomics. Whereas techniques exist for characterization of high abundant glycoproteins, no single method is presently capable of providing information on both site occupancy and glycan structure on a single band or spot excised from an electrophoretic gel...

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

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

    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...... sensitivity level comparable with state-of-the-art proteomics. Whereas techniques exist for characterization of high abundant glycoproteins, no single method is presently capable of providing information on both site occupancy and glycan structure on a single band or spot excised from an electrophoretic gel...

  4. New Microsatellite Markers for Anthyllis vulneraria (Fabaceae, Analyzed with Spreadex Gel Electrophoresis

    Halil Kesselring

    2013-12-01

    Full Text Available Premise of the study: New microsatellite primers were developed for the diploid herb Anthyllis vulneraria. These primers will be used in upcoming studies focusing on random genetic variation, local adaptation, and phenotypic plasticity in alpine plants. Methods and Results: The new primers were adjusted to separate PCR amplicons (70 to 170 bp on precast Spreadex gels using horizontal gel electrophoresis. No capillary sequencer was needed. Three to twelve alleles were found per locus depending on the population studied. Conclusions: Our preliminary results showed that the three studied alpine populations are predominantly outcrossing, but include variable levels of self-fertilization.

  5. Gel Electrophoresis of DNA - New Measurements and the Repton Model at High Fields

    New experimental data are presented on the gel electrophoresis of DNA. Experiment was made for molecules of length 173 kbp, in 1 percent agarose gel, in TAE 1 x buffer and the field intensity between 5 and 9 V/cm. The results are compared with our computer simulations, performed within the repton model of Duke and Rubinstein. The ranges of field and molecule length are determined, where the geometration effect appears. We investigate also the field dependence of the velocity and the diffusion coefficient at the border of the geometration regime. (author)

  6. Detection of connexins in liver cells using sodiumdodecylsulfate polyacrylamide gel electrophoresis and immunoblot analysis

    Willebrords, Joost; Maes, Michaël; Yanguas, Sara Crespo; Cogliati, Bruno; Vinken, Mathieu

    2016-01-01

    Summary Since connexin expression is partly regulated at the protein level, immunoblot analysis represents a frequently addressed technique in the connexin research field. The present chapter describes the set-up of an immunoblot procedure, including protein extraction and quantification from biological samples, gel electrophoresis, protein transfer and immunoblotting, which is optimized for analysis of connexins in liver tissue. In essence, proteins are separated on a polyacrylamide gel using sodiumdodecylsulfate followed by transfer of proteins on a nitrocellulose membrane. The latter allows specific detection of connexins with antibodies combined with revelation through enhanced chemiluminescence. PMID:27207285

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

    Young Chon Choi; Ki Rok, Kwon; Suk Ho, Choi

    2006-01-01

    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...

  8. Polyacrylamide Slab Gel Electrophoresis of Soluble Proteins for Studies of Bacterial Floras

    Moore, W. E. C.; Hash, D E; Holdeman, Lillian V.; Cato, Elizabeth P.

    1980-01-01

    A polyacrylamide slab gel electrophoresis procedure was used to compare cellular proteins from bacterial isolates of gingival crevice floras. Isolates with identical protein patterns consistently were shown to be members of the same species. When used to screen isolates, the procedure reduced total analytical time and expense without sacrificing accuracy, and it provided additional verification of the identity of strains characterized by conventional phenotypic tests.

  9. A rapid 3% polyacrylamide slab gel electrophoresis method for high through put screening of LDL phenotype

    Gupta Ruby; Lakshmy Ramakrishnan; Singh Yogendra; Kranthi Vemparala

    2008-01-01

    Abstract Background Small dense LDL is reported to be associated with increased coronary artery disease risk by various epidemiological studies. The gold standard for separation and identification of LDL subtypes in plasma is ultracentrifugation which is a lengthy procedure and difficult to perform. Various other methods like NMR, HPLC, gradient gel electrophoresis (GGE) have been reported for LDL sub fractionation all of which require specialized equipments and expertise. We report here a hi...

  10. Identification of Frankia Strains by Two-Dimensional Polyacrylamide Gel Electrophoresis

    Benson, David R.; Buchholz, S. E.; Hanna, D. G.

    1984-01-01

    Fifteen Frankia strains from five different plant species were analyzed by two-dimensional polyacryl-amide gel electrophoresis to determine their relatedness by comparing the polypeptide patterns obtained. Three major subgroups (A, C, and D) were found in the Alnus-Comptonia-Myrica cross-inoculation group. An isolate from Purshia tridentata had a unique protein pattern and represents a distinct group of frankiae. Members of group A were isolated from root nodules of Alnus incana subsp. rugosa...

  11. Diversity in the applications of the single cell gel electrophoresis (comet) assay / Cristal Huysamen

    Huysamen, Cristal

    2005-01-01

    The development of the single cell gel electrophoresis assay (Comet assay) as a powerful method for measuring DNA strand breakage and repair, has lead to a broader understanding of the impact of certain internal and external factors on DNA damage. This study describes the establishment of the Comet assay in our laboratory and its application in a diversity of studies. These studies include the monitoring of the effect of exercise on DNA damage and repair with the purpose of ...

  12. Pulsed-Field Gel Electrophoresis (PFGE) Technique and its use in Molecular Biology

    BASIM, Esin (HACIOĞLU)

    2001-01-01

    In recent years, the use of pulsed-field gel electrophoresis (PFGE) in the molecular biology area has been subject to much research. PFGE is a powerful tool for characterizing various strains at the DNA level, obtaining relevant information on genome size and constructing the physical and genetic map of the chromosome of bacteria that are poorly understood at the genetic level as well as in separating chromosomes in microorganisms, and in the long-range mapping of mammalian genes. PFGE also h...

  13. Application of single cell gel electrophoresis assay in radiation biodose estimation and environment monitoring

    Single cell gel electrophoresis(SCGE) or comet assay(CA) is a rapid, simple, sensitive,reliable and inexpensive method for detecting DNA damage in eukaryotic individual cells. The methodology and principles of SCGE are first introduced, such as analytical parameters, automatic analysis of images, advantages of SCGE etc., and then its application is investigated and discussed, including radiation protection and environment monitoring, organism monitoring, biological dose estimation. (authors)

  14. Streptococcus agalactiae pulsed-field gel electrophoresis patterns cross capsular types

    Pillai, P.; Srinivasan, U; ZHANG, L.; BORCHARDT, S.M.; Debusscher, J; Marrs, C F; Foxman, B.

    2009-01-01

    Streptococcus agalactiae is a genetically diverse organism; when typed by pulsed-field gel electrophoresis (PFGE), multiple types appear within a single serotype. We tested whether S. agalactiae PFGE types correspond to a specific serotype within individuals, and different individuals from the same geographic area. A total of 872 S. agalactiae isolates from 152 healthy individuals were classified by PFGE and capsular serotype. Serotype V was the most homogeneous (Simpson’s diversity index 0.5...

  15. Genomic fingerprinting of Borrelia burgdorferi sensu lato by pulsed-field gel electrophoresis.

    1993-01-01

    A total of 46 Borrelia burgdorferi sensu lato isolates that were isolated from patients with Lyme borreliosis and infected animals or were extracted from ticks of the genus Ixodes were analyzed. Large restriction fragment patterns obtained after cleavage of genomic DNAs with MluI were analyzed by pulsed-field gel electrophoresis (PFGE). To eliminate the contribution of plasmid DNA, only fragments greater than 70 kb were used for the analysis. The results indicated that each of the 14 B. burgd...

  16. Plasmid DNA replication and topology as visualized by two-dimensional agarose gel electrophoresis

    Schvartzman, Jorge Bernardo; Martínez-Robles, María Luisa; Krimer, Dora B.

    2010-01-01

    During the last 20 years, two-dimensional agarose gel electrophoresis combined with other techniques such as Polymerase Chain Reaction, helicase assay and electron microscopy, helped to characterize plasmid DNA replication and topology. Here we describe some of the most important findings that were made using this method including the characterization of uni-directional replication, replication origin interference, DNA breakage at the forks, replication fork blockage, replication knotting, re...

  17. Beverage-Agarose Gel Electrophoresis: An Inquiry-based Laboratory Exercise with Virtual Adaptation1

    Cunningham, Steven C.; McNear, Brad; Rebecca S. Pearlman; Kern, Scott E.

    2006-01-01

    A wide range of literature and experience has shown that teaching methods that promote active learning, such as inquiry-based approaches, are more effective than those that rely on passive learning. Gel electrophoresis, one of the most common laboratory techniques in molecular biology, has a wide range of applications in the life sciences. As such, we chose it as a platform to expose high school and undergraduate students to the active process of scientific inquiry in general, while specifica...

  18. Rapid method for epidemiological evaluation of gram-positive cocci by field inversion gel electrophoresis.

    Goering, R V; Winters, M A

    1992-01-01

    We report a rapid method for the isolation of intact chromosomal DNA from gram-positive cocci that is suitable for in situ restriction endonuclease digestion in agarose blocks. When combined with a rapid field inversion gel electrophoresis protocol, this approach allows the preparation and electrophoretic analysis of chromosomal restriction fragments produced by rare-cutting enzymes in a total time period of 2 days from start to finish. The utility of the method is demonstrated in the epidemi...

  19. Genome analysis of Bradyrhizobium japonicum serocluster 123 field isolates by using field inversion gel electrophoresis.

    Sobral, B W; Sadowsky, M. J.; Atherly, A G

    1990-01-01

    The genomes of 11 Bradyrhizobium japonicum serocluster 123 field isolates were analyzed by using field inversion gel electrophoresis. Genomic fingerprints produced by digestion of intact genomic DNA in agarose plugs with the rare-cutting restriction enzymes AseI, DraI, SpeI, and XbaI showed that the isolates were genetically diverse. Few (30 to 50%) isolates exhibited the same fingerprint as the USDA serogroup strain with which they are antigenically related. Southern hybridization with a nif...

  20. Microscopic agglutination and polyacrylamide gel electrophoresis analyses of oral anaerobic spirochetes.

    Tall, B D; Nauman, R K

    1986-01-01

    Microscopic agglutination (MA) analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to determine strain and species similarities and dissimilarities among three species of oral anaerobic spirochetes, Treponema denticola, Treponema pectinovorum, and Treponema vincentii. The MA analysis revealed a diversity of serologic reactivity or sharing of common antigens within each species. However, there was no cross-reactivity or sharing of common antigens among t...

  1. Identification of the Bacterial Microflora in Dairy Products by Temporal Temperature Gradient Gel Electrophoresis

    Ogier, Jean-Claude; Son, Olivier; Gruss, Alexandra; Tailliez, Patrick; Delacroix-Buchet, Agnes

    2002-01-01

    Numerous microorganisms, including bacteria, yeasts, and molds, are present in cheeses, forming a complex ecosystem. Among these organisms, bacteria are responsible for most of the physicochemical and aromatic transformations that are intrinsic to the cheesemaking process. Identification of the bacteria that constitute the cheese ecosystem is essential for understanding their individual contributions to cheese production. We used temporal temperature gradient gel electrophoresis (TTGE) to ide...

  2. Rubella virion polypeptides. Characterization by polyacrylamide gel electrophoresis, isoelectric focusing and peptide mapping

    Ho-Terry, L.; Cohen, A. (University Coll. Hospital Medical School, London (UK))

    1982-01-01

    Four polypeptides with molecular weights of 55K, 47K, 45K, and 33K have been resolved by polyacrylamide gel electrophoresis of immune precipitated rubella virus. The 47K and 45K components have similar peptide maps but different isoelectric points so that the same polypeptide may exist in more than one charged form. The 55K and 45K components have similar isoelectric points but different peptide maps showing that similarity of isoelectric point is not evidence of identity.

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

    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.

  4. Molecular Typing by Pulsed-Field Gel Electrophoresis of Spanish Animal and Human Listeria monocytogenes Isolates

    Vela, A. I.; Fernandez-Garayzabal, J F; Vazquez, J. A.; Latre, M. V.; Blanco, M. M.; Moreno, M. A.; de la Fuente, L.; Marco, J.; C. Franco; Cepeda, A.; Rodriguez Moure, A. A.; Suarez, G; Dominguez, L

    2001-01-01

    A total of 153 strains of Listeria monocytogenes isolated from different sources (72 from sheep, 12 from cattle, 18 from feedstuffs, and 51 from humans) in Spain from 1989 to 2000 were characterized by pulsed-field gel electrophoresis. The strains of L. monocytogenes displayed 55 pulsotypes. The 84 animal, 51 human, and 18 feedstuff strains displayed 31, 29, and 7 different pulsotypes, respectively, indicating a great genetic diversity among the Spanish L. monocytogenes isolates studied. L. m...

  5. Demonstration of a third structurally distinct human Ia beta chain by two-dimensional gel electrophoresis

    1982-01-01

    Previous studies have indicated that HLA-DR homozygous cell lines express two Ia alpha and Ia beta chains that combine to form at least two Ia molecules. This report demonstrates by two-dimensional gel electrophoresis the existence of a third structurally distinct human Ia beta chain on DR2 and DR5 cell lines. This suggests that at least five separate genes control the expression of Ia molecules on HLA-DR homozygous cell lines.

  6. Molecular Analysis of Mycobacterium avium Isolates by Using Pulsed-Field Gel Electrophoresis and PCR

    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 ...

  7. Multilocus Sequence Typing Scheme versus Pulsed-Field Gel Electrophoresis for Typing Mycobacterium abscessus Isolates

    Machado, Gabriel Esquitini; Matsumoto, Cristianne Kayoko; Chimara, Erica; Duarte, Rafael da Silva; Freitas, Denise; Palaci, Moises; Hadad, David Jamil; Lima, Karla Valéria Batista; Lopes, Maria Luiza; Ramos, Jesus Pais; Campos, Carlos Eduardo; Caldas, Paulo César; Heym, Beate; Leão, Sylvia Cardoso

    2014-01-01

    Outbreaks of infections by rapidly growing mycobacteria following invasive procedures, such as ophthalmological, laparoscopic, arthroscopic, plastic, and cardiac surgeries, mesotherapy, and vaccination, have been detected in Brazil since 1998. Members of the Mycobacterium chelonae-Mycobacterium abscessus group have caused most of these outbreaks. As part of an epidemiological investigation, the isolates were typed by pulsed-field gel electrophoresis (PFGE). In this project, we performed a lar...

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

    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

  9. Differential Expression of Proteins in Lung Cancer Using Difference in Gel Electrophoresis (DIGE)

    Beckett, P; Aulak, K. S.; Masri, F.

    2011-01-01

    Background: Lung cancer remains the leading cause of cancer-related mortality worldwide. Early detection of lung cancer is problematic due to the lack of a marker with high diagnosis sensitivity and specificity. The purpose of this study was to develop techniques to identify the differential expression protein profiles between tumor and tumor free of lung cancer tissues. Methods: 2-dimensional differential ingel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of...

  10. High-Yield Separation of Metallic and Semiconducting Single-Wall Carbon Nanotubes by Agarose Gel Electrophoresis

    Tanaka, Takeshi; Jin, Hehua; Miyata, Yasumitsu; Kataura, Hiromichi

    2008-11-01

    We have developed a novel separation method of metallic and semiconducting single-wall carbon nanotubes (SWCNTs) using agarose gel electrophoresis. When the SWCNTs were isolated with sodium dodecyl sulfate (SDS) and embedded in agarose gel, only the metallic SWCNTs separated from the starting gel by an electric field. After 20 min, almost all SWCNTs applied to gel electrophoresis were separated into two fractions, containing ˜95% semiconducting and ˜70% metallic nanotubes. The difference in the response to the electric field between metallic and semiconducting SWCNTs can be explained by the higher affinity of semiconducting SWCNTs to agarose than to SDS.

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

    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...

  12. Local Pixel Value Collection Algorithm for Spot Segmentation in Two-Dimensional Gel Electrophoresis Research

    Peter Peer

    2007-09-01

    Full Text Available Two-dimensional gel-electrophoresis (2-DE images show the expression levels of several hundreds of proteins where each protein is represented as a blob-shaped spot of grey level values. The spot detection, that is, the segmentation process has to be efficient as it is the first step in the gel processing. Such extraction of information is a very complex task. In this paper, we propose a novel spot detector that is basically a morphology-based method with the use of a seeded region growing as a central paradigm and which relies on the spot correlation information. The method is tested on our synthetic as well as on real gels with human samples from SWISS-2DPAGE (two-dimensional polyacrylamide gel electrophoresis database. A comparison of results is done with a method called pixel value collection (PVC. Since our algorithm efficiently uses local spot information, segments the spot by collecting pixel values and its affinity with PVC, we named it local pixel value collection (LPVC. The results show that LPVC achieves similar segmentation results as PVC, but is much faster than PVC.

  13. Local pixel value collection algorithm for spot segmentation in two-dimensional gel electrophoresis research.

    Peer, Peter; Corzo, Luis Galo

    2007-01-01

    Two-dimensional gel-electrophoresis (2-DE) images show the expression levels of several hundreds of proteins where each protein is represented as a blob-shaped spot of grey level values. The spot detection, that is, the segmentation process has to be efficient as it is the first step in the gel processing. Such extraction of information is a very complex task. In this paper, we propose a novel spot detector that is basically a morphology-based method with the use of a seeded region growing as a central paradigm and which relies on the spot correlation information. The method is tested on our synthetic as well as on real gels with human samples from SWISS-2DPAGE (two-dimensional polyacrylamide gel electrophoresis) database. A comparison of results is done with a method called pixel value collection (PVC). Since our algorithm efficiently uses local spot information, segments the spot by collecting pixel values and its affinity with PVC, we named it local pixel value collection (LPVC). The results show that LPVC achieves similar segmentation results as PVC, but is much faster than PVC. PMID:18274608

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

    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

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

    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)

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

    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

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

    Two-dimensional high-resolution gel electrophoresis (2DE) has been applied to the fractionation of 75Se-containing proteins in yeast, grown in 75Se-containing medium, and autoradiography was used for detection of the 75Se-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.)

  18. Proteomic Profiling Of Two-Dimensional Gel Electrophoresis Protein Expression Data

    Ahmad, Norhaiza; Zhang, J.; Brown, P. J.; James, D. C.; Birch, J. R.; Racher, A. J.; Smales, C. M.

    2008-01-01

    We have undertaken two-dimensional gel electrophoresis (2-DE) proteomic profiling on a series of cell lines with different recombinant antibody production rates. Due to the nature of 2-DE proteomic investigations there will always be `process variability' factors in any data set collected in this way. Some of this variation will arise during sample preparation, gel running and staining, while further variation will arise from the gel analysis procedure. Therefore, in order to identify all significant changes in protein expression between biological samples when analysed by 2-DE, the system precision or `error', and how this correlates to protein abundance, must be known. Only then can the system be considered robust and investigators accurately and confidently report all observable statistically significant changes in protein expression. We introduce an expression variability test to identify protein spots whose expression correlates with increased antibody production. The results have highlighted a small number of candidate proteins for further investigation.

  19. Mapping genomic organization by field inversion and two-dimensional gel electrophoresis: application to the murine T-cell receptor gamma gene family

    Woolf, T; Lai, E; Kronenberg, M; Hood, L

    1988-01-01

    A new two-dimensional gel electrophoresis technique has been developed for the mapping of multigene families. Resolution in the first dimension is based on the generation of large size DNA fragments by infrequently-cutting restriction enzymes, and separation of these fragments by field inversion gel (FIG) electrophoresis. A second restriction enzyme digestion is then carried out with the separated DNA fragments in the agarose gel. Standard gel electrophoresis in the second dimension allows on...

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

    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.

  1. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blotting for protein antigen analysis

    Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) has now become a standard tool in most laboratories for protein analysis and purification. Several SDS-PAGE systems have been described but the most widely used one is the discontinuous buffer system introduced for disc gel electrophoresis. Western blotting or the process of transfer of the electrophoretically separated proteins onto immobilizing matrices such as nitrocellulose membrane is an extension of SDS-PAGE system and provides, on the nitrocellulose blot, an identical copy of the electrophoretic separation pattern of the proteins present in the gels. The immobilized proteins can be further reacted with an appropriate probe such as antibody for identification of its corresponding antigen. The protein antigen/antibody complex is then detected by using radioactively labelled or enzyme-linked second antibody probe. The technique is very useful for analysis and characterization of complex protein antigens using immune sera from several sources or vice versa. The protocol given presented here illustrates such a separation by which complex protein antigens of blood stages of Plasmodium vivax obtained from blood of patients with vivax malaria are fractionated by SDS-PAGE and treated with immune sera from patients with acute vivax malaria and the antigen/antibody complex formed are detected by 125I-labelled anti-human immunoglobulins. 5 refs, 2 figs

  2. DNA electrophoresis in tri-block copolymer gels--experiments and Brownian dynamics simulation

    Wei, Ling; van Winkle, David H.

    2015-03-01

    The mobility of double-stranded DNA ladders in Pluronics®P105, P123 and F127, was measured by two-dimensional gel electrophoresis. Pluronics®are triblock copolymers which form gel-like phases of micelles arranged with cubic order at room temperature. A 10 base pair and a 25 base pair DNA ladder were used as samples in gel electrophoresis. The monotonically decreasing mobility with increasing length observed in the agarose separations is not observed in separations in Pluronics®. Rather, a complicated dependence of mobility on DNA length is observed, where mobility vs. length increases for short DNA molecules then decreases for longer molecules. There is also a variation of mobility with length correlated to the micelle diameter. Brownian dynamics simulations of a discrete wormlike chain model were performed to simulate short DNA molecules migrating in free solution and in a face-centered cubic matrix. By incorporating hydrodynamic interactions, the trend of simulated length-dependent mobility qualitatively agrees with experimental measurements.

  3. Genomic DNA detection using cycling probe technology and capillary gel electrophoresis with laser-induced fluorescence.

    Dickinson Laing, Terrina; Mah, David C W; Poirier, Robert T; Bekkaoui, Faouzi; Lee, William E; Bader, Douglas E

    2004-10-01

    Cycling probe technology (CPT) is an isothermal DNA analysis method that has been shown to be useful for identifying genetic markers of antibiotic-resistant bacteria in clinical settings. CPT assays have previously employed several assay methods that include polyacrylamide gel electrophoresis and magnetic beads for separations and radioisotopic and colorimetric detection for detection. Capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) is an alternative separation and detection method that offers attributes such as low sample consumption, short analysis times, no radiation hazards and potential for high throughput. We report on the development of a CGE-LIF CPT assay for genomic DNA from Erwinia herbicola and the comparison of this assay with a conventional 32p radioisotopic PAGE CPT assay. Separation and detection of intact and cleaved fluorescein-labeled CPT probe molecules by CGE-LIF was achieved in under 4 min through a gel-filled capillary (7 cm separation length to detector). Total time, from setup to detection, was about 1 h for the complete assay versus several hours (3-12 h) for the radioisotopic PAGE CPT assay. Similar detection limits of 10(5)-10(6) copies of genomic target DNA were observed with each assay method. This study demonstrated that CGE-LIF CPT is a suitable analysis method for the detection of genomic DNA sequences. PMID:15356906

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

    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

  5. Sodium dodecyl sulfate-capillary gel electrophoresis of proteins using non-cross-linked polyacrylamide.

    Wu, D; Regnier, F E

    1992-09-11

    Proteins with relative molecular masses of 14,000 to 205,000 were separated by sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) using non-cross-linked linear polyacrylamide gels on both coated and uncoated fused-silica capillaries. It was determined that viscosity of the acrylamide solution was a major factor affecting column stability with linear acrylamide gels. When the viscosity of the acrylamide solution reaches 100 cP, electro-osmotically driven displacement of the gels is insignificant. Uncoated capillaries provided better resolution, stability, and reproducibility than surface coated capillaries when the concentration of linear polyacrylamide was greater than 4%. At lower gel concentrations, non-cross-linked polyacrylamide is easily displaced from the columns. A calibration plot of log molecular mass vs. mobility with non-linear polyacrylamide was linear, which indicated that resolution was equivalent to that obtained with cross-linked acrylamide. Separations with model proteins indicated that baseline resolution between protein species that vary 10% in molecular mass can be achieved. PMID:1430034

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

    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.

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

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

    2010-03-20

    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.

  8. The latest progress in single cell gel electrophoresis (SCGE) based on DNA damage detection

    DNA damage detection can detect DNA damage caused by the pesticide and irradiation. With the increasing demands of DNA damage detection, the development of a rapid, high throughput and straight forward DNA damage detecting technique has critical biological significance for Single Cell Gel Electrophoresis (SCGE) is a straight and accurate way to detect the DNA damage. In recent years, the throughput and accuracy of the detection SCGE method have been improved significantly by applying new materials and new technologies. This paper reviewed the most recently reported SCGE based DNA damage detection technique-microwell array method and conventional SCGE method, and the prospect were also discussed. (authors)

  9. The single cell gel electrophoresis - a useful technique for DNA damage studies

    Single cell gel electrophoresis assay - SCGE, known also as 'comet assay' was introduced by Oestling and Johanson (1984) and later was developed by Singh (1988) for a rapid and sensitive quantitative analysis of DNA damage and repair in individual eukaryotic cells. The method is specific for detection of single and double DNA strand breaks. The interest in the assay has grown markedly in the last few years. The versatility of applications and the practical features of SCGE render it useful tool for DNA repair studies, genotoxicity testing and large-scale biomonitoring of human populations. (author). 42 refs, 3 figs

  10. The Use of Gel Electrophoresis to Study the Reactions of Activated Amino Acids with Oligonucleotides

    Zieboll, Gerhard; Orgel, Leslie E.

    1994-01-01

    We have used gel electrophoresis to study the primary covalent addition of amino acids to oligonu-cleotides or their analogs and the subsequent addition of further molecules of the amino acids to generate peptides covalently linked to the oligonucleotides. We have surveyed the reactions of a variety of amino acids with the phosphoramidates derived from oligonucleotide 5 inches phosphates and ethylenediamine. We find that arginine and amino acids can interact with oligonucleotidesl through stacking interactions react most efficiently. D- and L-amino acids give indistinguishable families of products.

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

    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.

  12. Sizing of the Haemophilus influenzae Rd genome by pulsed-field agarose gel electrophoresis.

    Lee, J J; Smith, H O

    1988-01-01

    The four restriction enzymes ApaI (5'-GGGCCC), EagI (5'-CGGCCG), NaeI (5'-GCCGGC), and SmaI (5'-CCCGGG) were found to produce distributions of DNA fragment sizes useful for mapping of the Haemophilus influenzae Rd genome by pulsed-field agarose gel electrophoresis. ApaI produced 21 fragments (range, 1.6 to 305 kilobases [kb]), EagI yielded 30 fragments (0.6 to 339 kb), NaeI produced 32 fragments (2.3 to 290 kb), and SmaI yielded 16 fragments (6.0 to 377 kb). Summation of the fragment lengths ...

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

    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

  14. Genome size of human oral Treponema species by pulsed-field gel electrophoresis.

    Correia, F F; Plummer, A R; Paster, B J; Dewhirst, F E

    2004-04-01

    The genome sizes of seven strains of oral treponemes were determined using pulsed-field gel electrophoresis (PFGE). These strains represent members from six of the currently known cultivable oral treponeme groups. The PFGE fragments were digitally recorded and then quantitated using GIMP v 1.2, an image manipulation program. The results show that the six oral treponeme genomes are comparable in size, ranging from approximately 2.2 to 2.5 Mbp. The genome sizes of these strains are 20-25% smaller than Treponema denticola strains, which have genome sizes of approximately 2.8-3.0 Mbp. PMID:14871355

  15. Deciphering metabolic networks by blue native polyacrylamide gel electrophoresis: A functional proteomic exploration

    Christopher Auger

    2015-06-01

    Full Text Available Metabolism is the consortium of reactions within a cell which directs a variety of processes including energy synthesis, signalling and the behaviour of a biological system. Metabolic networks, and more specifically the activity of enzymes within them, provide an accurate status of how cellular information is being executed. The performance of these networks and their ability to siphon metabolites in a number of directions may be the difference between a healthy and diseased state. Blue native polyacrylamide gel electrophoresis (BN-PAGE, owing to its simplicity and wide-ranging applications, permits the inspection of these nodules. The separation of proteins and enzyme complexes in their native format enables the exploration of enzymatic activity in metabolic networks via in-gel assays. These are quick, specific, and amenable to further studies. This electrophoretic technology not only enables the visualization of enzymatic efficacy but reveals the crosstalk among enzymes and their interactions with other organellar partners.

  16. DNA sequencing method using laser-induced fluorescence and capillary gel electrophoresis

    A postelectrophoresis capillary scanning method for increasing the throughput of DNA sequencing has been developed. The method features a spatially and temporally separated arrangement between capillary gel electrophoresis separation of DNA sequencing fragments and the visualization of the separation pattern. Fluorescently labeled DNA sequencing fragments are produced in enzymatic chain-termination reactions and separated by capillary with a transparent polymer coating. The capillary containing all bands of the fragments is then scanned longitudinally with laser beams. The DNA sequence is determined by analyzing the four-color band patterns. The innerwall of the capillary is coated with linear polyacrylamide. The capillary can be used repeatedly by refilling it with crosslinked polyacrylamide gel which can be easily removed out using a HPLC pump. Scanning time of less than 7 s has been achieved and a sequence of about 400 bases can be determined per scan.

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

    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

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

    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 14C-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 14C-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)

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

    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 10B 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)

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

    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.

  1. A tunable isoelectric focusing via moving reaction boundary for two-dimensional gel electrophoresis and proteomics.

    Guo, Chen-Gang; Shang, Zhi; Yan, Jian; Li, Si; Li, Guo-Qing; Liu, Rong-Zhong; Qing, Ying; Fan, Liu-Yin; Xiao, Hua; Cao, Cheng-Xi

    2015-05-01

    Routine native immobilized pH gradient isoelectric focusing (IPG-IEF) and two-dimensional gel electrophoresis (2DE) are still suffering from unfortunate reproducibility, poor resolution (caused by protein precipitation) and instability in characterization of intact protein isoforms and posttranslational modifications. Based on the concept of moving reaction boundary (MRB), we firstly proposed a tunable non-IPG-IEF system to address these issues. By choosing proper pairs of catholyte and anolyte, we could achieve desired cathodic and anodic migrating pH gradients in non-IPG-IEF system, effectively eliminating protein precipitation and uncertainty of quantitation existing in routine IEF and 2DE, and enhancing the resolution and sensitivity of IEF. Then, an adjustable 2DE system was developed by combining non-IPG-IEF with polyacrylamide gel electrophoresis (PAGE). The improved 2DE was evaluated by testing model proteins and colon cancer cell lysates. The experiments revealed that (i) a tunable pH gradient could be designed via MRB; (ii) up to 1.65 fold improvement of resolution was achieved via non-IPG-IEF; (iii) the sensitivity of developed techniques was increased up to 2.7 folds; and (iv) up to about 16.4% more protein spots could be observed via the adjustable 2DE as compared with routine one. The developed techniques might contribute to complex proteome research, especially for screening of biological marker and analysis of extreme acidic/alkaline proteins. PMID:25770625

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

    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)

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

    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

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

    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). PMID:26864499

  5. A control method to inspect the compositional authenticity of Minas Frescal cheese by gel electrophoresis.

    Magenis, Renata B; Prudêncio, Elane S; Molognoni, Luciano; Daguer, Heitor

    2014-08-20

    This study introduces a qualitative method to inspect the compositional authenticity of white nonripened cheeses like Minas Frescal, a typical Brazilian cheese, especially when irregular replacement of milk by whey is suspected. A sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) method, followed by image densitometry, was validated. Cheeses were freeze-dried to electrophoresis, and β-lactoglobulin (β-LG) was chosen as the adulteration marker. In gel trypsin digestion followed by matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry provided its identification. Cheeses with a minimum of 14 mg·g(-1) of β-LG are considered to be adulterated. The method shows satisfactory precision with a detection limit of 7 mg·g(-1). Forty-two commercial samples from inspected establishments were then assessed and subjected to cluster analysis. Compliant and noncompliant groups were set with 24 (57%) authentic samples and 18 (43%) adulterated samples, respectively, showing that proper analytical monitoring is required to inhibit this practice. PMID:25096158

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

    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...... pathogenic strains of R aeruginosa spread between farms and animals either mechanically, or through feed or water from a common source, rather than by random nosocomial infections with strains from the farm environment.......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...... outbreaks of haemorrhagic pneumonia. Isolates from mink were separated into 34 distinct clones by PFGE subtyping. All isolates from mink infected during the same farm outbreak were identical, except in one case where two different strains were isolated from mink obtained from the same farm outbreak. R...

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

    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.

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

    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.

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

    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)

  10. PHProteomicDB: A Module for Two-dimensional Gel Electrophoresis Database Creation on Personal Web Sites

    Pascal Pernet; Arnaud Bruneel; Bruno Baudin; Michel Vaubourdolle

    2006-01-01

    PHProteomicDB is a PHP-written module to help researchers in proteomics to share two-dimensional gel electrophoresis data using personal web sites. No technical or PHP knowledge is necessary except a few basics about web site management. PHProteomicDB has a user-friendly administration interface to enter and update data. It creates web pages on the fly displaying gel characteristics,gel pictures, and numbered gel spots with their related identifications pointing to their reference pages in protein databanks. The module is freely available at http://www.huvec.com/index.php3?rub=Download.

  11. Proteomic Comparison of Two-Dimensional Gel Electrophoresis Pro files from Human Lung Squamous Carcinoma and Normal Bronchial Epithelial Tissues

    Cui Li; Ping Chen; Jingyun Xie; Songping Liang; Xianquan Zhan; Maoyu Li; Xiaoying Wu; Feng Li; Jianling Li; Zhiqiang Xiao; Zhuchu Chen; Xueping Feng

    2003-01-01

    Differential proteome profiles of human lung squamous carcinoma tissue compared to paired tumor-adjacent normal bronchial epithelial tissue were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). The results showed that well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were obtained under the condition of 0.75-ug protein-load. The average deviation of spot position was 0.733+0.101 mm in IEF direction, and 0.925+0.207 mm in SDS-PAGE direction. For tumor tissue, a total of 1241±88 spots were detected, 987±65 spots were matched with an average matching rate of 79.5%. For control, a total of 1190+72 spots were detected, and 875±48 spots were matched with an average matching rate of 73.5%. A total of 864±34 spots were matched between tumors and controls.Forty-three differential proteins were characterized: some proteins were related to oncogenes, and others involved in the regulation of cell cycle and signal transduction. It is suggested that the differential proteomic approach is valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis.These data will be used to establish human lung cancer proteome database to further study human lung squamous carcinoma.

  12. P187-S Differential Proteome Analysis of the Cerebrospinal Fluid of Normal and Equine Protozoal Myeloencephalitis Affected Horses Using Two-Dimensional Fluorescence Difference Gel Electrophoresis and Mass Spectrometry

    Terry, D. E.; Levy, M.; Isaac, I.; Sharma, N

    2007-01-01

    Equine protozoal myeloencephalitis (EPM) is a common and costly neurologic disease of horses caused by Sarcocystis neurona, protozoa that parasitize the nervous system. Confirmation of the clinical diagnosis is a challenge for veterinarians because the current immunologic tests lack specificity. Multiplexing strategy approaches including two-dimensional fluorescence difference gel electrophoresis (2D DIGE) and mass spectrometry were employed to identify differential protein expression associa...

  13. A new method for 2D gel spot alignment: application to the analysis of large sample sets in clinical proteomics

    Granier Claude

    2008-10-01

    Full Text Available Abstract Background In current comparative proteomics studies, the large number of images generated by 2D gels is currently compared using spot matching algorithms. Unfortunately, differences in gel migration and sample variability make efficient spot alignment very difficult to obtain, and, as consequence most of the software alignments return noisy gel matching which needs to be manually adjusted by the user. Results We present Sili2DGel an algorithm for automatic spot alignment that uses data from recursive gel matching and returns meaningful Spot Alignment Positions (SAP for a given set of gels. In the algorithm, the data are represented by a graph and SAP by specific subgraphs. The results are returned under various forms (clickable synthetic gel, text file, etc.. We have applied Sili2DGel to study the variability of the urinary proteome from 20 healthy subjects. Conclusion Sili2DGel performs noiseless automatic spot alignment for variability studies (as well as classical differential expression studies of biological samples. It is very useful for typical clinical proteomic studies with large number of experiments.

  14. Effect of glycerol on the separation of nucleosomes and bent DNA in low ionic strength polyacrylamide gel electrophoresis.

    Pennings, S; Meersseman, G; Bradbury, E M

    1992-01-01

    We report that glycerol changes the separation characteristics of polyacrylamide nucleoprotein gels in which it is included as a stabilizing agent. Polyacrylamide gel electrophoresis fractionates DNA and nucleosomes according to net negative charge, mass and conformation. With glycerol included, fractionation seems to be largely based on particle mass and charge. The conformation factor in separation is progressively lost with increasing glycerol concentrations. Nucleosome positions on the sa...

  15. Hepatitis B surface antigen polypeptides: artifactual bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis caused by aggregation.

    Koistinen, V U

    1980-01-01

    Hepatitis B surface antigen, subtype ad, was purified and studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Two major bands with molecular weights of 23,500 and 27,500 and several weaker bands with higher molecular weights were observed. When the low-molecular-weight bands and the group of high-molecular-weight bands were excised from the gel, eluted, and reelectrophoresed, neither the low-molecular-weight bands nor the high-molecular-weight bands...

  16. Agarose and Polyacrylamide Gel Electrophoresis Methods for Molecular Mass Analysis of 5–500 kDa Hyaluronan

    Bhilocha, Shardul; Amin, Ripal; Pandya, Monika; Yuan, Han; Tank, Mihir; LoBello, Jaclyn; Shytuhina, Anastasia; Wang, Wenlan; Wisniewski, Hans-Georg; De la Motte, Carol; Cowman, Mary K.

    2011-01-01

    Agarose and polyacrylamide gel electrophoresis systems for the molecular mass-dependent separation of hyaluronan (HA) in the size range of approximately 5–500 kDa have been investigated. For agarose-based systems, the suitability of different agarose types, agarose concentrations, and buffers systems were determined. Using chemoenzymatically synthesized HA standards of low polydispersity, the molecular mass range was determined for each gel composition, over which the relationship between HA ...

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

    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.)

  18. Determination of damage and In vivo DNA repairing through the unicellular in gel electrophoresis technique

    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 (H2O2) 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. Epidemiologic study of Taylorella equigenitalis strains by field inversion gel electrophoresis of genomic restriction endonuclease fragments.

    Bleumink-Pluym, N; ter Laak, E A; van der Zeijst, B A

    1990-09-01

    Contagious equine metritis (CEM), a sexually transmitted bacterial disease, was first described in thoroughbred horses. It also occurs in nonthoroughbred horses, in which it produces isolated, apparently unrelated outbreaks. Thirty-two strains of Taylorella equigenitalis, the causative agent of CEM, from all over the world were characterized by field inversion gel electrophoresis of fragments of genomic DNA obtained by digestion with low-cleavage-frequency restriction enzymes. This resulted in a division into five clearly distinct groups. Strains from thoroughbred horses from all continents belonged to one group. Strains from nonthoroughbred horses from various countries were different from strains from thoroughbred horses; four groups could be determined. Two groups contained both streptomycin-resistant and streptomycin-susceptible strains. The data indicate that CEM in nonthoroughbreds did not originate from the thoroughbred population; also, the reverse was not demonstrated. Thus, extensive international transportation directives regarding the testing of nonthoroughbred horses for CEM may need reconsideration. PMID:2172296

  20. Time effectiveness of DNA injury and its repair induced by irradiation using single cell gel electrophoresis

    Objective: To improve the accuracy of estimation of radiation biodosimetry after radiation accident, to explore the time-effect relationship of DNA repair after irradiation, and to describe the curves and plane of DNA repair. Methods: The neutral single cell gel electrophoresis (SCGE) was performed to estimate the repair of DNA double-strand break (DNA-DSB) in human lymphocytes induced by different dose of radiation at 3, 24, 48, 72 h post irradiation. Results: Better goodness of fit was obtained not only in the dose-response curves at different time points, but also in the time-response curves at different doses. A smooth three-dimensional plane model was obtained when combining the two curves. Conclusions: The curves of DNA-DSB repair showed a linear relationship, and repair of radiation-plane model could be used for biological dosimetry. (authors)

  1. Gel Electrophoresis of DNA Knots in Weak and Strong Electric Fields

    Weber, C; Fleurant, M; Rios, P D L; Dietler, G

    2005-01-01

    Gel electrophoresis allows to separate knotted DNA (nicked circular) of equal length according to the knot type. At low electric fields, complex knots being more compact, drift faster than simpler knots. Recent experiments have shown that the drift velocity dependence on the knot type is inverted when changing from low to high electric fields. We present a computer simulation on a lattice of a closed, knotted, charged DNA chain drifting in an external electric field in a topologically restricted medium. Using a simple Monte Carlo algorithm, the dependence of the electrophoretic migration of the DNA molecules on the type of knot and on the electric field intensity was investigated. The results are in qualitative agreement with electrophoretic experiments done under conditions of low and high electric fields: especially the inversion of the behavior from low to high electric field could be reproduced. The knot topology imposes on the problem the constrain of self-avoidance, which is the final cause of the obser...

  2. Gel electrophoresis study of radioprotective effect of hydroxy cinnamic acid derivatives on DNA

    The radioprotective effect exerted by hydroxy cinnamic acid derivatives on the radiation damage of DNA, such as double-strand break (DSB) and intermolecular crosslinking (CL), has been studied by gel electrophoresis. The molecular length distribution of DNA can be measured by this method, and by comparing average molecular length of DNA before and after irradiation, the frequency of DSB and CL can be determined quantitatively. The radioprotective effect of caffeic acid and sinapine on calf thymus DNA in binary system at room temperature have been determined. Both caffeic acid and sinapine reveal apparent protective effects and the former is stronger than the latter. The mechanism of radioprotection at room temperature has been discussed. Based on this finding, combined with the previous ESR study and an analysis of the molecular structure on these two protectors, it is suggested that the target is protected as a result of hydrogen atom transfering from protector to target

  3. Microdisc gel electrophoresis in sodium dodecyl sulfate of organic material from rat otoconial complexes

    Ross, M. D.; Pote, K. G.; Rarey, K. E.; Verma, L. M.

    1981-01-01

    The gravity receptors of all vertebrates utilize a 'test mass' consisting of a complex arrangement of mineral and organic substance that lies over the sensory receptor areas. In most vertebrates, the mineral is a polymorph of calcium carbonate in the form of minute, single crystals called otoconia. An investigation is conducted to determine the number of proteins in otoconial complexes and their molecular weights. The investigation makes use of a microdisk gel electrophoresis method reported by Gainer (1971). The most important finding of the reported research is that analysis of the proteins of the organic material of the otoconial complexes is possible when sensitive microanalytical methods are employed. Further modification of the basic technique employed and the inclusion of other sensitive staining methods should mean that, in the future, protein separation by molecular weight will be possible in sample pools containing only two otoconial masses.

  4. Oxygen effect for DNA double-strand break induction determined by pulsed-field gel electrophoresis

    The induction of DNA double-strand breaks (dsb) following irradiation under oxygenated and hypoxic conditions with and without misonidazole was measured by pulsed-field gel electrophoresis (PFGE) in a human bladder carcinoma cell line. The dose-response curve for DNA dsb detection by PFGE was biphasic with an apparent reduction in rate of dsb induced with dose. Oxygen enhancement ratios (OER) for cell survival (at a surviving fraction of 0.1) and for DNA damage assessed by PFGE (at 80% retained) were 2.0 and 3.0 respectively. Dose-modifying factors for misonidazole (15 mM), of 1.9 (survival) and 2.4 (DNA damage) were found. (author)

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

    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)

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

    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

  7. Non-denaturing gel electrophoresis system for the purification of membrane bound proteins

    A new method is described for the purification of a membrane bound glycoprotein, the kappa opioid receptor from human placental tissue. The method uses preparative slab-gel electrophoresis in the presence of the non-denaturing detergent CHAPS. A linear relationship between log molecular weight and SDS PAGE electrophoretic mobility of known molecular weight markers, in the presence of CHAPS, is observed. Using this method, we were able partially to purify an 3H-etorphine binding glycoprotein, from placental villus tissue, with an apparent molecular weight range of 60-70,000. The iodinated glycoprotein migrates in SDS PAGE with an apparent molecular weight of 63,000. This method may be useful for the isolation of membrane bound proteins, especially when an affinity ligand is not available

  8. Optimization of Pulsed-field Gel Electrophoresis Procedure for Bacillus cereus.

    Zhang, Hui Juan; Pan, Zhuo; Wei, Jian Chun; Zhang, En Min; Cai, Hong; Liang, Xu Dong; Li, Wei

    2016-03-01

    In order to develop a rapid and reliable method for B. cereus genotyping, factors influencing PFGE results, including preparation of bacterial cells embedded in agarose, lysis of embedded cells, enzymatic digestion of intact genomic DNA, and electrophoresis parameters allowing for reproducible and meaningful DNA fragment separation, were controlled. Optimal cellular growth (Luria-Bertani agar plates for 12-18 h) and lysis conditions (4 h incubation with 500 µg/mL lysozyme) produced sharp bands on the gel. Restriction enzyme NotI was chosen as the most suitable. Twenty-two isolates were analyzed by NotI digestion, using three electrophoretic parameters (EPs). The EP-a was optimal for distinguishing between isolates. The optimized protocol could be completed within 40 h which is a significant improvement over the previous methods. PMID:27109136

  9. On-line coupling of capillary gel electrophoresis with electrospray mass spectrometry for oligonucleotide analysis.

    Freudemann, T; von Brocke, A; Bayer, E

    2001-06-01

    Homooligodeoxyribonucleotides differing one nucleotide in length from 12- to 15-mer and from 17- to 20-mer were separated by size with capillary gel electrophoresis (CGE) using an entangled polymer solution in coated capillaries. The resolved components were analyzed by on-line coupling of CGE with electrospray mass spectrometry (ES-MS), denoted as CGE/ES-MS, in the full-scan negative ion detection mode. Baseline separation was achieved for the 12-15-mer oligonucleotide mixtures. Both synthetic phosphodiester oligonucleotide mixtures as well as their phosphorothioate analogues, serving as model compounds for antisense oligonucleotides, could be analyzed by on-line CGE/ES-MS coupling. Terminally phosphorylated and nonphosphorylated synthetic failure sequences could be electrophoretically separated and mass spectrometically characterized as well. This methodology might be a useful tool for synthesis control of phosphodiester oligonucleotides as well as for analysis of phosphorothioate analogues as they are used in antisense drug development. PMID:11403304

  10. Two-dimensional polyacrylamide gel electrophoresis analysis of indomethacin-treated human colon cancer cells

    Yan-Li Cheng; Gui-Ying Zhang; Zhi-Qiang Xiao; Fa-Qing Tang

    2005-01-01

    AIM: To establish the two-dimensional gel electrophoresis (2-DE) profiles of indomethacin (IN)-treated human colon cancer cell line HCT116, and to provide a new way to study its anti-tumor molecular mechanism through analyzing a variety of protein maps.METHODS: Two-DE profiles of HCT116 were established in IN-treated and untreated groups. Total proteins were separated by immobilized pH gradient-based 2-DE. The gels were stained by silver, scanned by ImageScanner,and analyzed with Image Master software.RESULTS: Clear background, well-resolved and reproducible 2-DE patterns of HCT116 cells were acquired in IN-treated and untreated group. The average deviation of spot position was 0.896±0.177 mm in IEF direction and 1.106±0.289 mm in SDS-PAGE direction respectively. In IN-treated group,1 169±36 spots were detected and 1 061±32 spots were matched, the average matching rate was 90.6% in three gels. In untreated group, 1 256±50 spots were detected and 1 168±46 spots were matched, the average matching rate was 93.0% in three gels. Forty-five differential protein spots were displayed between IN-treated and untreated groups. Of which, 34 protein spots decreased and 9showed higher expression in IN-treated group, and only two protein spots showed an expression in untreated cells.CONCLUSION: Two-DE profiles of IN-treated and untreated HCT116 cells were established. Apparent 45 different protein spots were detected in IN-treated and untreated HCT116 cells. The analysis on differential protein spots may serve as a new way to study the molecule mechanism of IN-treated colon cancer.

  11. Comparative Studies on Preparation of Large Plant DNA Fragments by Pulse Field Gel Electrophoresis

    Yang Jiliang(杨继良); Wang Qinghua(王庆华); Deng Daiyong; Yang Dianer; Jin Demin; Weng Manli; Zhang Juren; Wang Bin

    2004-01-01

    The preparation of large plant DNA fragments is extremely important to the construction of large insert DNA libraries (YAC, BAC, PAC and TAC). Although several techniques have been developed in each step of large plant DNA fragments preparation, the whole processing remains complicated and difficult. Based on authors research experience and the recent worldwide development in this field, the following aspects are discussed in this paper: techniques of plant high molecular weight (HMW) DNA purification by pre-electrophoresis, the optimal conditions for the partial digestion of the HMW DNA by HindIII, the isolation effects of of large plant DNA fragments (100~400 kb) with different parameters of pulse field gel electrophoresis (PFGE), and the recovery of large DNA fragments. Through comparative studies, the advantages and disadvantages of each technique are discussed and some recommendations are proposed for preparing high quality large plant DNA fragments. The suggested techniques have been used in preparing the large DNA fragments of maize, rice, moss, laver, sea tangle and peach,and similar results are obtained among all the materials. This paper only reports the results using maize as material.

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

    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.

  13. Rapid pulsed-field gel electrophoresis protocol for Legionella pneumophila typing

    Massimiliano Orsini

    2008-06-01

    Full Text Available

    Background: Genomic DNA patterns generated by pulsed-field gel electrophoresis (PGFE are highly specific for different strains of an organism and have significant value in epidemiologic investigations of infectious disease outbreaks. A disadvantage of PFGE is that the procedure requires up to 6 days to complete.

    Methods: We developed a rapid PFGE protocol for subtyping Legionella pneumophila isolates based on the standardized protocol currently used. Various combinations of reaction conditions (e.g., lysis time and temperature, restriction enzyme concentration and electrophoresis parameters were applied to devise a simple and rapid PFGE protocol that could also be used for frozen bacteria.

    Results: PFGE analysis of Legionella pneumophila isolates can be completed in 26 hours using this protocol compared to 6 days for the conventional one.

    Conclusions: We successfully applied a rapid PFGE protocol for Legionella pneumophila typing and comparison of the patterns obtained from the rapid compared with the conventional method showed that the rapid protocol gave identical and highly reproducible results.

  14. Detection of Helicobacter Colonization of the Murine Lower Bowel by Genus-Specific PCR-Denaturing Gradient Gel Electrophoresis

    Grehan, Martin; Tamotia, Gauri; Robertson, Bronwyn; Mitchell, Hazel

    2002-01-01

    Helicobacter genus-specific PCR and denaturing gradient gel electrophoresis can detect and speciate the helicobacters that colonize the lower bowel of laboratory mice. The method's sensitivity is comparable to that of species-specific PCR and may detect unnamed Helicobacter species. This approach should prove useful for commercial and research murine facilities.

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

    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...

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

    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...

  17. Evaluation of repetitive extragenic palindromic-PCR and denatured gradient gel electrophoresis in identifying Salmonella serotypes isolated from processed turkeys

    Salmonella has been reported as the leading foodborne pathogen in the US. A study was conducted to compare the use of automated repetitive extragenic palindromic (REP-PCR) and denaturing gradient gel electrophoresis (DGGE) as diagnostic tools for identifying Salmonella serotypes. The interspersed ...

  18. Differentiation of Neisseria gonorrhoeae isolates requiring proline, citrulline, and uracil by plasmid content, serotyping, and pulsed-field gel electrophoresis.

    Ng, L K; Carballo, M.; Dillon, J A

    1995-01-01

    A combination of DNA macrorestriction analysis using pulsed-field gel electrophoresis and a serotyping method using three panels of monoclonal antibody was used to discriminate 43 epidemiologically unrelated Neisseria gonorrhoeae isolates requiring proline, citrulline, and uracil (PCU-) into 35 groups. This indicates that PCU- isolates of N. gonorrhoeae are not clonal.

  19. Genetic diversity demonstrated by pulsed field gel electrophoresis of Salmonella enterica isolates obtained from diverse sources in Mexico

    This study was conducted to determine the genetic diversity of Salmonella isolates recovered from a variety of sources using pulsed-field gel electrophoresis (PFGE) to assess their possible relatedness. Salmonella was isolated from ca. 52% of samples from a pepper var. Bell production system. A to...

  20. Analysis of bacterial community in two wastewater treatment plants by denaturing gradient gel electrophoresis and fluorescent in situ hybridization

    A combination of denaturing gradient gel electrophoresis (DGGE) and oligonucleotide probes was used to characterize and follow the dynamic of bacterial community in two wastewater treatment plants with an A2 O system of each plant. To complete this technique, we used Fluorescent in situ Hybridization (FISH) to quantify the main groups in activated sludges samples. (Author)

  1. Characterization of Salmonella isolates from retail foods based on serotyping, pulse field gel electrophoresis, antibiotic resistance and other phenotypic properties

    Sixteen Salmonella strains isolated from a variety of foods during 2000 and 2003, by the Florida State Department of Agriculture, were characterized by various genotypic and phenotypic tests. Among 16 isolates, 15 different serotypes were identified. Pulse-Field Gel Electrophoresis (PFGE) fingerpr...

  2. Improved Pulsed-Field Gel Electrophoresis Procedure for the Analysis of F. columnare Isolates Previously Affected by DNA Degradation

    Flavobacterium columnare is a fresh water bacterium that causes columnaris diseases in over 36 fish species. Intra-species typing of F. columnare can be performed by pulsed-field gel electrophoresis (PFGE). However, this method is hampered by the degradation of chromosomal DNA in about 10% of strain...

  3. Application of multiplex PCR, pulsed-field gel electrophoresis (PFGE), and BOX-PCR for molecular analysis of enterococci

    The objective of the study was to use band-based molecular methods including BOX-PCR (Polymerase Chain Reaction) and Pulsed-Field Gel Electrophoresis (PFGE) to determine if genetically related enterococci were found among different stores, food types, or years. Enterococci were also characterized f...

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

    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 inf...

  5. Comprehensive TP53-denaturing gradient gel electrophoresis mutation detection assay also applicable to archival paraffin-embedded tissue

    Hayes, V M; Bleeker, W; Verlind, E; Timmer, T; Karrenbeld, A; Plukker, J T; Marx, M P; Hofstra, R M; Buys, C H

    1999-01-01

    A comprehensive mutation detection assay is described for the entire coding region and all splice site junctions of TP53. The assay is based on denaturing gradient gel electrophoresis, which follows either multiplex polymerase chain reaction (PCR) applied to DNA extracted from fresh or frozen tissue

  6. Implementation of Salmonella serotype determination using pulsed-field gel electrophoresis in a state public health laboratory.

    Bopp, Dianna J; Baker, Deborah J; Thompson, Lisa; Saylors, Amy; Root, Timothy P; Armstrong, Leeanna; Mitchell, Kara; Dumas, Nellie B; Musser, Kimberlee Arruda

    2016-08-01

    We examined the use of pulsed-field gel electrophoresis (PFGE) to predict serotype for Salmonella isolates. Between 2012 and 2014 we assessed 4481 isolates, resulting in >90% assigned serotypes. PFGE is efficient for determining serotype in the majority of cases and results in expedited serotype determination, as well as cost savings. PMID:27220605

  7. Combination of Multiplex PCR and PCR-Denaturing Gradient Gel Electrophoresis for Monitoring Common Sourdough-Associated Lactobacillus Species

    Settanni, Luca; Valmorri, Sara; van Sinderen, Douwe; Suzzi, Giovanna; Paparella, Antonello; Corsetti, Aldo

    2006-01-01

    A combination of denaturing gradient gel electrophoresis (DGGE) and a previously described multiplex PCR approach was employed to detect sourdough lactobacilli. Primers specific for certain groups of Lactobacillus spp. were used to amplify fragments, which were analyzed by DGGE. DGGE profiles obtained from Lactobacillus type strains acted as standards to analyze lactobacilli from four regional Abruzzo (central Italy) sourdoughs.

  8. Two-dimensional polyacylamide gel electrophoresis of envelope proteins of Escherichia coli.

    Johnson, W C; Silhavy, T J; Boos, W

    1975-03-01

    A method of separating envelope proteins by two-dimensional polyacrylamide gel electrophoresis is described. Escherichia coli envelopes (inner and outer membranes) were prepared by French pressing and washed by repeated centrifugation. Membrane proteins were solubilized with guanidine thiocyanate and were dialyzed against urea prior to two-dimensional electrophoretic analysis. The slab gel apparatus and conditions were similar to the technique developed by Metz and Bogorad (1974) for the separation of ribosomal proteins. This separation occurs in 8 M urea for the first dimension and in 0.2% sodium dodecyl sulfate for the second dimension. The technique separates about 70 different membrane proteins in a highly reproducible fashion according to both intrinsic charge and molecular weight. Some examples of alterations in the membrane protein pattern are demonstrated. These alterations are caused by a mutation affecting a sugar transport system and by growth in the presence of D-fucose, inducer of the transport system. A further example of membrane protein changes introduced by growth at the nonpermissive temperature of a temperature-sensitive cell division mutant is shown. Finally, it is demonstrated that the major outer membrane component of Escherichia coli K-12 contains more than four proteins of similar molecular weight. PMID:803821

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

    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 [125I] 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 24oC 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)

  10. Detection of the irradiation treatment of foods using micro gel electrophoresis of DNA

    Ionizing radiation has profound effects on nucleic acids, reflected by e.g. base damage and strandbreaks. These effects of radiation contribute to the aim of food irradiation: undesirable microorganisms are inactivated, insect infestation eliminated, sprouting inhibited or ripening delayed. It is, therefore, conceivable that methods of detection of a radiation treatment could be based on changes in DNA, either in microbes or insects or in the food itself. A promising method of detecting DNA fragments in irradiated food is the microelectrophoresis of single cells. Advantages of the method are its simplicity and its speed, the electrophoretic separation only requiring 2.5-5 minutes. The principle is based on migration of DNA in an agarose gel exposed to an electric field. Single cells or nuclei are embedded in the agarose and after lysis intact DNA will practically not move upon electrophoresis, due to the gel strains, whereas if DNA fragmentation has occurred, the DNA is able to migrate and ''comets'' following the cells or nuclei become visible after staining. This paper describes our experience obtained by analyzing chicken, beef and pork meat, shrimps and mushrooms. The method therefore has its limitations, although good results both from frozen and fresh meats, fresh fish, onions and potatoes have been reported. Also strawberries have been identified using this technique. Further experience may strengthen the evidence that this simple and cheap technique may have its place among the battery of tests to detect the radiation treatment of foods. (orig./vhe)

  11. A novel multi-scale Hessian based spot enhancement filter for two dimensional gel electrophoresis images.

    Shamekhi, Sina; Miran Baygi, Mohammad Hossein; Azarian, Bahareh; Gooya, Ali

    2015-11-01

    Two dimensional gel electrophoresis (2DGE) is a useful method for studying proteins in a wide variety of applications including identifying post-translation modification (PTM), biomarker discovery, and protein purification. Computerized segmentation and detection of the proteins are the two main processes that are carried out on the scanned image of the gel. Due to the complexities of 2DGE images and the presence of artifacts, the segmentation and detection of protein spots in these images are non-trivial, and involve supervised and time consuming processes. This paper introduces a new spot filter for enhancing, and separating the closely overlapping spots of protein in 2DGE images based on the multi-scale eigenvalue analysis of the image Hessian. Using a Gaussian spot model, we have derived closed form equations to compute the eigen components of the image Hessian of two overlapping spots in a multi-scale fashion. Based on this analysis, we have proposed a novel filter that suppresses the overlapping area and results in a better spot separation. The performance of the proposed filter has been evaluated on the synthetic and real 2DGE images. The comparison with three conventional techniques and a commercial software package reveals the superiority and effectiveness of the proposed filter. PMID:26409228

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

    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.

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

    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

  14. The monitoring of nucleotide diphosphate kinase activity by blue native polyacrylamide gel electrophoresis.

    Mailloux, Ryan J; Darwich, Rami; Lemire, Joseph; Appanna, Vasu

    2008-04-01

    Nucleoside diphosphate kinase (NDPK) has been shown to play a pivotal role in modulating a plethora of cellular processes. In this study, we report on a blue native (BN) PAGE technique which allows the facile assessment of NDPK activity and expression. The in-gel detection of NDPK relies on the precipitation of formazan at the site of immobilized enzyme activity. This is achieved by coupling the formation of ATP, as a consequence of gamma-phosphate transfer from NTP to ADP, to hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PDH), oxidized nicotinamide adenine dinucleotide phosphate (NADP), phenazine methosulfate (PMS), and iodonitrotetrazolium chloride (INT). 2-D denaturing gel analysis confirmed that the activity bands corresponded to NDPK as indicated by subunit composition. Furthermore, the sensitivity and specificity of this readily accessible procedure was assessed by monitoring the in-gel activity of NDPK using different concentrations of GTP and CTP as well as deoxynucleoside triphosphates. This electrophoretic technique allows the quick and easy detection of NDPK, a housekeeping enzyme crucial to cell survival. PMID:18324728

  15. Two-dimensional gel electrophoresis analysis of the proteomes expressed in the human hepatoma cell line BEL-7404 and normal liver cell line L-02

    2000-01-01

    Proteome analysis technology has been used extensively in conducting discovery research of biology and has become one of the most essential technologies in functional genomics. The proteomes of the human hepatoma cell line BEL-7404 and the normal human liver cell line L-02 have been separated by high resolution two-dimensional gel electrophoresis (2-DE) with immobilized pH gradient isoelectric focusing (IPG-IEF) in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension (IPG-DALT). The resulting images have been analyzed using 2-D analysis software. Quantitative analysis reveals that 7 protein spots are detected only in hepatoma BEL-7404 cells, 14 only in L-02 cells, and 78 protein spots show significant fluctuation in quantity in both cell lines (P<0.01).These protein spots have been displayed on a proteome differential expression map. Analysis for the reproducibility of 2-DE indicates that the positional variability in the IEF dimension is 0.73 mm, while the variability in the SDS-PAGE dimension is 0.44 mm, and the quantitative variability is 17.6%-19.2%. These results suggest that the reproducibility of 2-DE has been suitable for the study of differential expression of proteomes. Proteome differential expression maps can be useful tools for disease diagnosis, drug-target validation analysis and biological process elucidation.

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

    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)

  17. Characterization of wheat gliadin proteins by combined two-dimensional gel electrophoresis and tandem mass spectrometry.

    Mamone, Gianfranco; Addeo, Francesco; Chianese, Lina; Di Luccia, Aldo; De Martino, Alessandra; Nappo, Annunziata; Formisano, Annarita; De Vivo, Pasqualina; Ferranti, Pasquale

    2005-07-01

    A proteomics-based approach was used for characterizing wheat gliadins from an Italian common wheat (Triticum aestivum) cultivar. A two-dimensional gel electrophoresis (2-DE) map of roughly 40 spots was obtained by submitting the 70% alcohol-soluble crude protein extract to isoelectric focusing on immobilized pH gradient strips across two pH gradient ranges, i.e., 3-10 or pH 6-11, and to sodium dodecyl sulfate-polyacrylamide electrophoresis in the second dimension. The chymotryptic digest of each spot was characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and nano electrospray ionization-tandem mass spectrometry (MS/MS) analysis, providing a "peptide map" for each digest. The measured masses were subsequently sought in databases for sequences. For accurate identification of the parent protein, it was necessary to determine de novo sequences by MS/MS experiments on the peptides. By partial mass fingerprinting, we identified protein molecules such as alpha/beta-, gamma-, omega-gliadin, and high molecular weight-glutenin. The single spots along the 2-DE map were discriminated on the basis of their amino acid sequence traits. alpha-Gliadin, the most represented wheat protein in databases, was highly conserved as the relative N-terminal sequence of the components from the 2-DE map contained only a few silent amino acid substitutions. The other closely related gliadins were identified by sequencing internal peptide chains. The results gave insight into the complex nature of gliadin heterogeneity. This approach has provided us with sound reference data for differentiating gliadins amongst wheat varieties. PMID:15952231

  18. Proteomic Comparison of Two—Dimensional Gel Electrophoresis Profiles from Human Lung Squamous Carcinoma and Normal Bronchial Epithelial Tissues

    CuiLi; XianquanZhan; MaoyuLi; XiaoyingWu; FengLi; JianlingLi; ZhiqiangXiao; ZhuchuChen; XuepingFeng; PingChen; JingyunXie; SongpingLiang

    2003-01-01

    Differential proteome profiles of human lung squamous carcinoma tissue compared to paired tumor-adjacent normal bronchial epithelial tissue were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis(2-D PAGE)and matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS).The results showed that well-resolved,reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were obtained under the condition of 0.75-mg protein-load.The average deviation of spot position was 0.733±0.101 mm in IEF direction,and 0.925±0.207mm in SDS-PAGE direction.For tumor tissue,a total of 1241±88 spots were detected,987±65 spots were matched with an average matching rate of 79.5%.For control,a total of 1190±72 spots were detected,and 875±48 spots were matched with an average matching rate of 73.5%.A total of 864±34 spots were matched between tumors and controls.Forth-three differential proteins were characterized:some proteins were related to oncogenes,and others involved in the regulation of cell cycle and signal transduction.It is suggested that the differential proteomic approach is valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis.These data will be used to establish human lung cancer proteome database to further study human lung squamous carcinoma.

  19. Two-Dimensional Differential Gel Electrophoresis to Identify Protein Biomarkers in Amniotic Fluid of Edwards Syndrome (Trisomy 18 Pregnancies.

    Te-Yao Hsu

    Full Text Available Edwards syndrome (ES is a severe chromosomal abnormality with a prevalence of about 0.8 in 10,000 infants born alive. The aims of this study were to identify candidate proteins associated with ES pregnancies from amniotic fluid supernatant (AFS using proteomics, and to explore the role of biological networks in the pathophysiology of ES.AFS from six second trimester pregnancies with ES fetuses and six normal cases were included in this study. Fluorescence-based two-dimensional difference gel electrophoresis (2D-DIGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS were used for comparative proteomic analysis. The identified proteins were further validated by Western blotting and the role of biological networks was analyzed.Twelve protein spots were differentially expressed by more than 1.5-fold in the AFS of the ES pregnancies. MALDI-TOF/MS identified one up-regulated protein: apolipoprotein A1 (ApoA1, and four under-regulated proteins: vitamin D binding protein (VDBP, alpha-1-antitrypsin (A1AT, insulin-like growth factor-binding protein 1 (IGFBP-1, and transthyretin (TTR. Western blot and densitometric analysis of ApoA1, A1AT, IGFBP-1, and TTR confirmed the alteration of these proteins in the amniotic fluid samples. Biological network analysis revealed that the proteins of the ES AFS were involved mainly in lipid and hormone metabolism, immune response, and cardiovascular disease.These five proteins may be involved in the pathogenesis of ES. Further studies are needed to explore.

  20. Studies on middle silkgland proteins of cocoon colour sex-limited silkworm (Bombyx mori L.) using two-dimensional polyacrylamide gel electrophoresis

    Yuan-Xiang Jin; Yu-Yin Chen; Meng-Kui Xu; Yong-Huang Jiang

    2004-03-01

    Qualitative and quantitative differences in proteins expressed in the middle silkglands of male and female silkworm larvae that differ in silk colour were investigated by high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), followed by computer assisted image analysis. About 1000 protein spots were resolved in both the sexes and most proteins were shown to be distributed in the area from 15 kDa to 70 kDa and pH 4–8. It was found that some proteins displayed higher expression in yellow cocoon, while two proteins were only expressed in female silkworm silkgland tissue through the comparison and analysis by two-D software. These proteins especially existed in female silkworm middle silkgland tissue of yellow cocoon. Furthermore, these proteins might be involved in the expression of cocoon colour phenotype.

  1. A multi-channel gel electrophoresis and continuous fraction collection apparatus for high throughput protein separation and characterization

    Choi, Megan; Nordmeyer, Robert A.; Cornell, Earl; Dong, Ming; Biggin, Mark D.; Jin, Jian

    2009-10-02

    To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A Counter Free-Flow elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high-resolution separation of a complex protein mixture can be achieved on this system using SDS-PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48-96 fractions over a mass range of 10-150 kDa; sample recovery rates were 50percent or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 L/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 g per channel and reduced resolution.

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

    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 280 +- 40. 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 290 +- 30. Assuming an unwinding angle of 280 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 Mg2+ at 4 to 0.5 mM. The association constant for 4'-hydroxymethyltrioxsalen in 0.5 mM Mg2+ was determined to be 70 M-1

  3. An improved method for detecting genetic variation in DNA using denaturing gradient gel electrophoresis

    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 32P-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)

  4. Microbial Community Structure of Korean Cabbage Kimchi and Ingredients with Denaturing Gradient Gel Electrophoresis.

    Hong, Sung Wook; Choi, Yun-Jeong; Lee, Hae-Won; Yang, Ji-Hee; Lee, Mi-Ai

    2016-06-28

    Kimchi is a traditional Korean fermented vegetable food, the production of which involves brining of Korean cabbage, blending with various other ingredients (red pepper powder, garlic, ginger, salt-pickled seafood, etc.), and fermentation. Recently, kimchi has also become popular in the Western world because of its unique taste and beneficial properties such as antioxidant and antimutagenic activities, which are derived from the various raw materials and secondary metabolites of the fermentative microorganisms used during production. Despite these useful activities, analysis of the microbial community present in kimchi has received relatively little attention. The objective of this study was to evaluate the bacterial community structure from the raw materials, additives, and final kimchi product using the culture-independent method. Specifically, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was used to analyze the 16S rRNA partial sequences of the microflora. One primer set for bacteria, 341F(GC)-518R, reliably produced amplicons from kimchi and its raw materials, and these bands were clearly separated on a 35-65% denaturing gradient gel. Overall, 117 16S rRNA fragments were identified by PCR-DGGE analysis. Pediococcus pentosaceus, Leuconostoc citreum, Leuconostoc gelidum, and Leuconostoc mesenteroides were the dominant bacteria in kimchi. The other strains identified were Tetragenococcus, Pseudomonas, Weissella, and uncultured bacterium. Comprehensive analysis of these microorganisms could provide a more detailed understanding of the biologically active components of kimchi and help improve its quality. PCR-DGGE analysis can be successfully applied to a fermented food to detect unculturable or other species. PMID:26907755

  5. 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

    Garaizar, J.; Lopez-Molina, N.; Laconcha, I.; Baggesen, Dorte Lau; Rementeria, A.; Vivanco, A.; Audicana, A.; Perales, 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 of...... 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...

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

    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

  7. Clinical proteomics in cancer research-promises and limitations of current two-dimensional gel electrophoresis.

    Iwadate, Yasuo

    2008-01-01

    Cancer can be defined as a deviated protein network system toward dysregulated cellular proliferation. Alteration in the content and functional state of the proteins with many linkages may shift the equilibrium state of the protein signaling network to enhance a survival advantage of the affected cells. Searching for such hub proteins is the main purpose of the cancer proteomics. Although the progression in the vanguard proteomic technologies would largely contribute to cancer diagnosis and treatment in the future, the technology most frequently used for the analysis of clinical tissue samples is the two-dimensional gel electrophoresis (2DE) combined with matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Accumulation of 2DE data has generated many candidate biomarkers with potential clinical value. The identified proteins are restricted to a subset of the predicted human proteome, and ubiquitously exist in all normal cells taking important roles in the basic biological functions. Although these proteins can be used as valuable prognostic markers, the low-abundance proteins which is tissue-specific and useful as diagnostic markers could not easily be found by the standard 2DE technology alone. None of the current proteomic technologies can identify the whole proteome by themselves. Adequate combinations of different approaches not only in proteomics but in immunological methods would be necessary for the tissue specific markers. PMID:18855668

  8. Proteomics with two-dimensional gel electrophoresis and mass spectrometry analysis in cardiovascular research.

    You, Sun-Ah; Wang, Qing K

    2006-01-01

    Proteomics is a large-scale, comprehensive study of the proteins of a cell or organism. It is a unique means of characterizing proteins that are expressed in a cell or tissue at any given time-point and of identifying any modifications that they may undergo. Thus, it is a powerful technology that can detect and identify the changes of the structure and function of proteins in response to intra- and extracellular environmental signals or disease states. As proteomics can establish a link for genes and proteins with a disease, it will play an important role in defining the molecular determinants of a disease and in identifying targets for drug discoveries and diagnostics. We have carried out the first proteomics study for coronary artery disease (CAD) and found that the expression of the ferritin light chain was significantly increased in CAD tissues. In this chapter, we use the CAD study as an example to demonstrate the procedures involved in proteomics analysis. The proteome is visualized by two-dimensional gel electrophoresis, a powerful and widely used method for proteomics, and the proteins of interest are then identified by mass spectrometry. This technique should be useful in characterizing cardiovascular diseases and in defining signaling pathways for cardiovascular development and physiology. PMID:17085802

  9. Analysis of yeast and archaeal population dynamics in kimchi using denaturing gradient gel electrophoresis.

    Chang, Ho-Won; Kim, Kyoung-Ho; Nam, Young-Do; Roh, Seong Woon; Kim, Min-Soo; Jeon, Che Ok; Oh, Hee-Mock; Bae, Jin-Woo

    2008-08-15

    Kimchi is a traditional Korean food that is fermented from vegetables such as Chinese cabbage and radish. Many bacteria are involved in kimchi fermentation and lactic acid bacteria are known to perform significant roles. Although kimchi fermentation presents a range of environmental conditions that could support many different archaea and yeasts, their molecular diversity within this process has not been studied. Here, we use PCR-denaturing gradient gel electrophoresis (DGGE) targeting the 16S and 26S rRNA genes, to characterize bacterial, archaeal and yeast dynamics during various types of kimchi fermentation. The DGGE analysis of archaea expressed a change of DGGE banding patterns during kimchi fermentation, however, no significant change was observed in the yeast DGGE banding patterns during kimchi fermentation. No significant difference was indicated in the archaeal DGGE profile among different types of kimchi. In the case of yeasts, the clusters linked to the manufacturing corporation. Haloarchaea such as Halococcus spp., Natronococcus spp., Natrialba spp. and Haloterrigena spp., were detected as the predominant archaea and Lodderomyces spp., Trichosporon spp., Candida spp., Saccharomyces spp., Pichia spp., Sporisorium spp. and Kluyveromyces spp. were the most common yeasts. PMID:18562030

  10. Assessment of DNA damage in radiation workers by using single cell gel electrophoresis

    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)

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

    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, HepG2, 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 HepG2 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 HepG2 and EC-9706 was higher than that of MCF-7. But there was no statistical difference of SF between HepG2 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)

  12. Prokaryotic community composition revealed by denaturing gradient gel electrophoresis in the East Sea

    Jang, Gwang Il; Choi, Dong Han

    2015-12-01

    To understand the temporal and spatial variation of the prokaryotic community in the East Sea, their composition was determined by polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE)-sequencing techniques. The investigations were conducted twice annually in 2007 and 2009 in coastal and offshore stations. Prokaryotic abundance (PA), leucine incorporation rate, and other environmental parameters were also measured. By using the DGGE approach, we obtained 283 bacterial sequences and 160 archaeal sequences. The most frequently detected bacterial phylotypes during the investigations belonged to Alphaproteobacteria, Gammaproteobacteria, and Bacteroidetes. However, their relative compositions differed in time and space. Although Alphaproteobacteria and Bacteroidetes were the dominant groups in the surface water in May 2007 and in May and October 2007, Gammaproteobacteria was dominant in mesopelagic samples. However, Gammaproteobacteria was overwhelmingly dominant in most samples in August 2009. Although Deltaproteobacteria was rarely found as a dominant bacterial group, it occupied the highest fraction in a mesopelagic sample in October 2007. Epsilonproteobacteria also showed a similar trend, although its maximal dominance was found in a mesopelagic sample in August 2009. The archaeal community was dominated overwhelmingly by members of the Euryarchaeota in most of the investigations. However, Nitrosopumilales was dominant in aphotic samples in August 2009. Further, their spatiotemporal composition at the family level changed more dynamically in the East Sea. These temporal and spatial distributions of the prokaryotic community were influenced mainly by seawater temperature and depth in the East Sea.

  13. The protective effect of caffeine on DNA photosensitive damage: a gel electrophoresis

    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)

  14. Detection of heavy ion induced DNA double-strand breaks using static-field gel electrophoresis

    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.)

  15. Structure of virioplankton in the Charente Estuary (France): transmission electron microscopy versus pulsed field gel electrophoresis.

    Auguet, J C; Montanié, H; Lebaron, P

    2006-02-01

    Changes in the composition of viral communities were investigated along a salinity gradient and at different times by means of transmission electron microscopy (TEM) and pulsed field gel electrophoresis (PFGE). Samples were collected in fresh (Charente River), estuarine (Charente Estuary), and coastal (Pertuis d'Antioche, French Atlantic coast) waters. Both methods revealed similar patterns in viral community structure with a dominance of small viral particles (capsid and genome size). Viruses with a head size below 65 nm made up 71 +/- 5% of total virus-like particles, and virus-like genomes (VLG) below 100 kb accounted for 89 +/- 9% of total VLG. Despite this apparent stability of virioplankton composition over spatial scale (salinity gradient), the occurrence of large viruses (capsid and genome size) in estuarine and seawater samples indicated the presence of viral populations specific to a geographical location. Temporal changes in the structure (capsid and genome size) of viral communities were more pronounced than those reported at the spatial scale. From January to May 2003, seasonal changes in viral abundance and bacterial production occurred concomitantly with an increase in viral genomic diversity (richness), suggesting that virioplankton composition was strongly linked to changes in microbial activity and/or in the structure of the host communities. Although PFGE and TEM yielded complementary results in the description of virioplankton structures, it seems that the use of PFGE alone should be enough for the monitoring of community changes. PMID:16463133

  16. Molecular Typing of Acinetobacter Baumannii Clinical Strains in Tehran by Pulsed-Field Gel Electrophoresis

    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.  

  17. [Studies on human alpha-2 macroglobulin structure and its complexes with proteases, using polyacrylamide gel electrophoresis].

    Fine, J M; Lambin, P; Steinbuch, M

    1975-09-01

    Pure alpha2M is prepared with fresh plasma as starting material, to prevent the interaction of alpha2M from proteolytic enzymes of plasma such as thrombin, plasmin and kallikrein. During the purification steps, polybrene and aprotin are used as inhibitors and plasminogen is absorbed onto bentonite. When alpha 2M is submitted to polyacrylamide gel electrophoresis (PAA) containing 0.1% SDS, a complete dissociation in two half-molecules of MW 380,000 occurs. When alpha2M is incubated in 1% SDS and 1% beta-mercaptoethanol as reducing agent, only one component of MW 190,000 is observed in PAA-SDS. This experiments show that the alpha2M molecule consist of two symetric halves of same MW (380,000) linked by non covalent bonds. Each two-half-molecules is made of two polypeptides chains MW 190,000 linked by disulfide bonds. Thus alpha2M molecule contains four polypeptides chains having a same MW. The same techniques were applied to the study of alaph2M proteinases complexes. Three different proteinases (plasmin, trypsin and papain) were used in these experiments. Trypsin and papain are commercialy available. Plasminogen was obtained by affinity chromatography and activated into plasmin by insoluble streptokinase fixed on PAB cellulose. PMID:59941

  18. Molecular Analysis of Mycobacterium avium Isolates by Using Pulsed-Field Gel Electrophoresis and PCR

    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

  19. Identification of plant viruses using one-dimensional gel electrophoresis and peptide mass fingerprints.

    Luo, H; Wylie, S J; Jones, M G K

    2010-05-01

    A generic assay to detect and partially characterize unknown viruses from plants was developed. Proteins extracted from virus-infected and uninfected plants were separated in one dimension by SDS polyacrylamide gel electrophoresis. Differentially expressed protein bands were eluted after trypsin digestion and resulting peptide fragments separated according to their mass by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Resulting peptide mass fingerprints (PMF) were compared with those in protein databases. The assay was used to identify three known viruses: the potyviruses Zucchini yellow mosaic virus and Turnip mosaic virus, and an alfamovirus Alfalfa mosaic virus. It was also used to identify a virus that manifested symptoms in wild Cakile maritima plants, tentatively identified as Pelargonium zonate spot virus (PZSV) (genus Anulavirus) by its PMF, and then confirmed by nucleotide sequencing. The detection of PZSV constitutes a first record of this virus in Australia and in this host. It is proposed that this rapid and simple assay is a useful approach for analysis of plant samples known to harbor viruses that could not be identified using antisera or nucleic acid-based assays. PMID:20170682

  20. Indirect fluorometric detection techniques on thin layer chromatography and effect of ultrasound on gel electrophoresis

    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.

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

    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)

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

    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.

  3. Mutations of the Gs α-subunit gene in Albright hereditary osteodystrophy detected by denaturing gradient gel electrophoresis

    Affected members of most kindreds with Albright hereditary osteodystrophy have a partial deficiency of functional Gs, the guanine nucleotide-binding protein that stimulates adenylyl cyclase. By use of the polymerase chain reaction to amplify genomic fragments with the attachment of a high-melting G+C-rich region (GC clamp) and analysis of these fragments by denaturing gradient gel electrophoresis, heterozygous mutations in the Gs α-subunit at the donor splice junction of intron 10 and a coding frameshift created by a single base deletion within exon 10. The findings illustrate the heterogeneity of genetic defects in Albright hereditary osteodystrophy and the usefulness of the polymerase chain reaction-denaturing gradient gel electrophoresis method to search rapidly for mutations in a large candidate gene

  4. GEL-STATE NMR OF BALL-MILLED WHOLE CELL WALLS IN DMSO-d6 USING 2D SOLUTION-STATE NMR SPECTROSCOPY

    Plant cell walls were used for obtaining 2D solution-state NMR spectra without actual solubilization or structural modification. Ball-milled whole cell walls were swelled directly in the NMR tube with DMSO-d6 where they formed a gel. There are relatively few gel-state NMR studies. Most have involved...

  5. A thin-layer multistrip agarose gel electrophoresis apparatus for Ferguson plot analysis at the sub-microgram load level.

    Orbàn, L; Sullivan, J V; Chrambach, A

    1989-07-01

    A method for the simultaneous horizontal agarose gel electrophoresis on thin-layer strips of different gel concentrations was developed for the purpose of generating Ferguson plots at the sub-microgram load level. Seven independent gel strips on a common GelBond support were formed by filling channels created by a comb-shaped spacer (polycarbonate) in a vertical multistrip cassette. Electrophoresis on a horizontal Peltier-cooled surface employed commercial apparatus (E-C Apparatus Corp.) with a modified cover which is airtight and holds anodic and cathodic voltage measurement probes for each strip. The application of the apparatus to Ferguson plot analysis in a single experiment was exemplified on the RNA-containing turnip crinkle virus (TCV) at a load of 50 ng/gel strip, using an optimized silver staining method (a modification of a procedure of FMC Corp. BioProducts) for detection. Within the range of 3.5 to 12.5 V/cm, the plot was found to be independent of field strength. Mobility is also independent of the concentration of detergent (CHAPS) up to 10 mM. PMID:2809063

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

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

    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 b...

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

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

    1986-01-01

    A typing method for Clostridium difficile based on the incorporation of [35S]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 a...

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

    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 pr...

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

    A typing method for Clostridium difficile based on the incorporation of [35S]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

  10. Analysis of cell wall extracts of Candida albicans by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot techniques.

    Ponton, J; J. M. Jones

    1986-01-01

    Cell walls of intact yeast- and mycelial-phase Candida albicans B311 were extracted with different compounds: dithiothreitol, dithiothreitol with protease, dithiothreitol with lyticase, and dithiothreitol with protease followed by beta-glucuronidase with chitinase. Extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot techniques. Dithiothreitol extracts contained the most satisfactory array of components for study. Analysis of these extracts demo...

  11. Detection of a single base exchange in PCR-amplified DNA fragments using agarose gel electrophoresis containing bisbenzimide-PEG.

    Müller, M; Kruse, L; Tabrett, A M; Barbara, D.J.

    1997-01-01

    Using PCR fragments of known sequences derived from isolates of two related fungal species, simple submarine electrophoresis in agarose gels containing a bisbenzimide-PEG conjugate (H.A.-Yellow) has been shown to be capable of distinguishing DNA fragments 567 bp long which differ by as little as a single base change. However, only changes affecting bisbenzimide binding sites (which consist of at least four consecutive A/T bases) alter mobility; other changes are ineffective. A second ligand (...

  12. Determination of the Mutagenicity Potential of Supermint Herbal Medicine by Single Cell Gel Electrophoresis in Rat Hepatocytes

    Zivar Amanpour; Mojtaba Kalantar; Masoud Mahdavinia; Mohsen Rezaei; Heibatullah Kalantari; Golnaz Varnaseri

    2012-01-01

    Purpose: The increasing use of herbal drugs and their easy availability have necessitated the use of mutagenicity test to analyze their toxicity and safety. The aim of this study was to evaluate the genotoxicity of Supermint herbal medicine in DNA breakage of rat hepatocytes in comparison with sodium dichromate by single cell gel electrophoresis technique or comet assay. Methods: Hepatocytes were prepared from male wistar rats and were counted and kept in a bioreactor for 30 minutes....

  13. DNA fragmentation of human infarcted myocardial cells demonstrated by the nick end labeling method and DNA agarose gel electrophoresis.

    Itoh, G; Tamura, J; M. Suzuki; Suzuki, Y.; Ikeda, H; Koike, M; Nomura, M; Jie, T; Ito, K

    1995-01-01

    Myocardial tissue taken from 19 autopsy cases of myocardial infarction were examined both by the nick and labeling method (NELM) and by DNA agarose gel electrophoresis in order to demonstrate the localization of cells with fragmented DNA and to confirm the internucleosomal cleavage of DNA biochemically. The nuclei corresponding to those with the histological features of acute myocardial infarction in hematoxylin and eosin (H&E)-stained sections were stained strongly positive with the nick end...

  14. Characterization of coagulase-negative staphylococci by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses.

    Thomson-Carter, F M; Pennington, T H

    1989-01-01

    Coagulase-negative staphylococci are important nosocomial pathogens. At present, no wholly satisfactory typing scheme exists for these organisms. Therefore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were assessed as characterization methods. A total of 100 type strains and nontyped isolates representing nine species of coagulase-negative staphylococci were analyzed. Each species had a reproducible, characteristic whole-cell banding pattern when an...

  15. Differentiation of Bartonella Species by a Microimmunofluorescence Assay, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, and Western Immunoblotting

    Liang, Zhongxing; Raoult, Didier

    2000-01-01

    Bartonella species can be differentiated by microimmunofluorescence assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting with murine polyclonal antisera to Bartonella henselae, B. quintana, B. elizabethae, and B. bacilliformis. A pairwise comparison on the basis of SDS-PAGE protein profiles demonstrated similarity values for proteins of different Bartonella species ranging from 28.6 to 86.4%. Antigenic relationships revealed by immunoblotting with mu...

  16. Multicolour hybrid nanoprobes of molecular beacon conjugated quantum dots: FRET and gel electrophoresis assisted target DNA detection

    We have developed multicolour hybrid DNA probes employing green, yellow and orange colour quantum dot conjugated molecular beacons with black hole quencher 2. Optical and electrophoretic characterization revealed fluorescent energy transfer that follows the FRET mechanism with single nucleotide discrimination. Target DNA identification was observed to be highly sensitive up to 8 ng in gel electrophoresis. Comparison with the conventional organic dye SYBR Gold(TM) showed that our hybrid nanoprobes exhibit more stable performance with less background signal

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

    Zheng Xiaojuan; Zhang Xin; Zhou Jiyong; Wu Yongping; Jiang Xuetao; Shi Lixue; Yin Wei; Wang Junhua

    2009-01-01

    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 viru...

  18. POLYACRYLAMIDE GEL ELECTROPHORESIS AS A TOOL FOR THE TAXONOMIC IDENTIFICATION OF SNAKES FROM THE ELAPIDAE AND VIPERIDAE FAMILIES

    Soares, A. M.; L. H. ANZALONI PEDROSA; Fontes, M. R. M.; R. J. DA SILVA; Giglio, J. R.

    1998-01-01

    Polyacrylamide gel electrophoresis (PAGE) for basic proteins may be a useful tool for the characterization of whole snake venoms and for the taxonomic classification of snakes of the Elapidae and Viperidae families. However, due to the close proximity of molecular weights among toxins bearing different net charges, sodium dodecyl sulfate (SDS) - PAGE was not able to provide an efficient differentiation. This article reports the electrophoretic analysis of several venoms from the genera Micrur...

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

    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.

  20. Monitoring the Bacterial Population Dynamics in Sourdough Fermentation Processes by Using PCR-Denaturing Gradient Gel Electrophoresis

    Meroth, Christiane B.; Walter, Jens; Hertel, Christian; Brandt, Markus J.; Hammes, Walter P.

    2003-01-01

    Four sourdoughs (A to D) were produced under practical conditions by using a starter mixture of three commercially available sourdough starters and a baker's yeast constitutively containing various species of lactic acid bacteria (LAB). The sourdoughs were continuously propagated until the composition of the LAB flora remained stable. Two LAB-specific PCR-denaturing gradient gel electrophoresis (DGGE) systems were established and used to monitor the development of the microflora. Depending on...

  1. Identification and Population Dynamics of Yeasts in Sourdough Fermentation Processes by PCR-Denaturing Gradient Gel Electrophoresis

    Meroth, Christiane B.; Hammes, Walter P.; Hertel, Christian

    2003-01-01

    Four sourdoughs (A to D) were produced under practical conditions, using a starter obtained from a mixture of three commercially available sourdough starters and baker's yeast. The doughs were continuously propagated until the composition of the microbiota remained stable. A fungi-specific PCR-denaturing gradient gel electrophoresis (DGGE) system was established to monitor the development of the yeast biota. The analysis of the starter mixture revealed the presence of Candida humilis, Debaryo...

  2. Design and Evaluation of PCR Primers for Analysis of Bacterial Populations in Wine by Denaturing Gradient Gel Electrophoresis

    Lopez, Isabel; Ruiz-Larrea, Fernanda; Cocolin, Luca; Orr, Erica; Phister, Trevor; Marshall, Megan; VanderGheynst, Jean; Mills, David A.

    2003-01-01

    Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial p...

  3. Polyacrylamide gel electrophoresis of RNA compared with polyclonal- and monoclonal-antibody-based enzyme immunoassays for rotavirus.

    Pacini, D L; Brady, M T; Budde, C T; Connell, M J; Hamparian, V V; Hughes, J. H.

    1988-01-01

    Polyacrylamide gel electrophoresis (PAGE) of rotaviral RNA, a sensitive and highly specific test for detecting rotavirus in stool, was compared with two commercially available enzyme immunoassays (EIAs), monoclonal (Pathfinder) and polyclonal (Rotazyme II). Stool samples from 204 children with nosocomial diarrhea were tested for rotavirus by both EIAs and by PAGE of RNA extracted from raw stools or 10% stool suspensions. Samples which tested positive by either EIA but were negative by PAGE we...

  4. Reproducibility of an Integrated Quantitation Method Coupling 2D GeLC-MS/MS with the emPAI for Comparative Proteomics

    In 2D gel mapping, most protein spots consist of multiple proteins posing a significant challenge for the proper interpretation of gel-based comparative experiments. Previously we introduced an approach integrating 2-D difference gel electrophoresis and LC-MS/MS analysis with the exponentially modif...

  5. Proteomic analysis of plasma from cows affected with milk fever using two-dimensional differential in-gel electrophoresis and mass spectrometry.

    Xia, C; Zhang, H Y; Wu, L; Xu, C; Zheng, J S; Yan, Y J; Yang, L J; Shu, S

    2012-10-01

    Milk fever is an important metabolic disorder of dairy cows after calving, and is characterized by hypocalcemia, tetany, lateral recumbency, and eventual coma. To date, there have been many reports about the pathogenesis and pathophysiology of milk fever, but the plasma protein profile in milk fever has not been reported. The aim of our study was to investigate novel pathophysiological changes in the plasma proteome of cows affected with milk fever. Plasma samples were collected from eight Holstein cows with milk fever (T), and eight control Holstein cows without milk fever (C), at an intensive Holstein dairy farm in Heilongjiang province, China. Samples were analyzed by fluorescence two-dimensional (2D) differential in-gel electrophoresis (DIGE), followed by in-gel digestion, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) for peptide mass fingerprinting of selected protein spots. Eight of the 23 differential protein spots in the plasma of T and C cows were isolated and identified by 2D-DIGE and MALDI-TOF-MS. The protein spots represented five unique proteins, and had significant alterations in spot volume as determined by DeCyder differential in-gel analysis (DIA) software. The upregulated proteins were identified as serpin peptidase inhibitor (angiotensin), which regulates blood pressure and maintains fluid and electrolyte homeostasis, and endopin 2B which is involved in neural regulation. The downregulated proteins were serum albumin, which acts as a transport protein, fibrinogen beta chain which is involved in blood coagulation, and IgG heavy-chain C-region (IgG-C(H)) which participates in the immune response. In conclusion, we were able to use proteomic technologies to identify several novel plasma proteins in cows affected with milk fever. These findings may reveal new pathophysiological changes that occur in cows with milk fever. PMID:22119234

  6. Phylogenetic compositions of bacterioplankton from two California estuaries compared by denaturing gradient gel electrophoresis of 16S rDNA fragments.

    Murray, A. E.; Hollibaugh, J.T.; Orrego, C.

    1996-01-01

    The phylogenetic compositions of bacterioplankton assemblages from San Francisco Bay and Tomales Bay, Calif., differed substantially when analyzed by PCR-denaturing gradient gel electrophoresis; these differences are consistent with the results of previous studies demonstrating differences in their metabolic capabilities. PCR-denaturing gradient gel electrophoresis analysis of complex microbial assemblages was sensitive and reliable, and the results were reproducible as shown by experiments w...

  7. Random amplified polymorphic DNA assay is less discriminant than pulsed-field gel electrophoresis for typing strains of methicillin-resistant Staphylococcus aureus.

    Saulnier, P; Bourneix, C; Prévost, G; Andremont, A

    1993-01-01

    Twenty-six strains of methicillin-resistant Staphylococcus aureus with different pulsed-field gel electrophoresis fingerprints were tested by random amplified polymorphic DNA assay with three primers, resulting in 15 to 20 different random amplified polymorphic DNA fingerprints. By summing the results for the three primers, the number of different fingerprints increased to 25, but two strains could not be differentiated. We conclude that pulsed-field gel electrophoresis remains the best metho...

  8. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of urinary protein in acute kidney injury.

    Suhail, Sufi M; Woo, K T; Tan, H K; Wong, K S

    2011-07-01

    Recent experimental and clinical studies have shown the importance of urinary proteomics in acute kidney injury (AKI). We analyzed the protein in urine of patients with clinical AKI using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for its diagnostic value, and followed them up for 40 months to evaluate prognosis. Urine from 31 consecutive cases of AKI was analyzed with SDS-PAGE to determine the low, middle and high molecular weight proteins. Fractional excretion of sodium (FENa) was estimated from serum and urine creatinine and sodium (Na). The cases were followed-up for 40 months from the end of the recruitment of study cases. Glomerular protein was higher in the hematuria group when compared with the non-hematuria group (P <0.04) and in the AKI group than in the acute on chronic renal failure (AKI-on-CRF) group (P <0.002). Tubular protein was higher in the AKI-on-CRF group (P <0.003) than in the AKI group. Tubular protein correlated with FENa in groups with diabetes mellitus (DM), AKI-on-CRF, and without hematuria (P <0.03, P <0.02 and P <0.004, respectively). Pattern of protein did not differ between groups with and without DM and clinical acute tubular necrosis (ATN). At the end of 40 months follow-up, category with predominantly glomerular protein progressed to chronic renal failure (CRF) or end-stage renal failure in higher proportion (P <0.05). In clinical AKI, we observed that glomerular protein dominated in cases with glomerular insult, as indicated by hematuria. Tubular protein was common in the study cases with CRF, DM and cases without hematuria. This indicates tubulo-interstitial injury for AKI in these cases. Patients with predominantly glomerular protein had an adverse outcome. PMID:21743220

  9. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE of urinary protein in acute kidney injury

    Sufi M Suhail

    2011-01-01

    Full Text Available Recent experimental and clinical studies have shown the importance of urinary proteomics in acute kidney injury (AKI. We analyzed the protein in urine of patients with clinical AKI using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE for its diagnostic value, and followed them up for 40 months to evaluate prognosis. Urine from 31 consecutive cases of AKI was analyzed with SDS-PAGE to determine the low, middle and high molecular weight proteins. Fractional excretion of sodium (FENa was estimated from serum and urine creatinine and sodium (Na. The cases were followed-up for 40 months from the end of the recruitment of study cases. Glomerular protein was higher in the hematuria group when compared with the non-hematuria group (P <0.04 and in the AKI group than in the acute on chronic renal failure (AKI-on-CRF group (P <0.002. Tubular protein was higher in the AKI-on-CRF group (P <0.003 than in the AKI group. Tubular protein correlated with FENa in groups with diabetes mellitus (DM, AKI-on-CRF, and without hematuria (P <0.03, P <0.02 and P <0.004, respectively. Pattern of protein did not differ between groups with and without DM and clinical acute tubular necrosis (ATN. At the end of 40 months follow-up, category with predominantly glomerular protein progressed to chronic renal failure (CRF or end-stage renal failure in higher proportion (P <0.05. In clinical AKI, we observed that glomerular protein dominated in cases with glomerular insult, as indicated by hematuria. Tubular protein was common in the study cases with CRF, DM and cases without hematuria. This indicates tubulo-interstitial injury for AKI in these cases. Patients with predominantly glomerular protein had an adverse outcome.

  10. Subpopulations of liver coated vesicles resolved by preparative agarose gel electrophoresis

    Rat liver clathrin coated vesicles (CVs) were separated into several distinct subpopulations using non-sieving concentrations of agarose, which allowed the separation of species differing primarily in surface charge. Using preparative agarose electrophoresis, the CVs were recovered and analyzed for differences in morphology, coat protein composition, and stripped vesicle protein composition. Coat proteins from difference populations appeared identical on SDS PAGE, and triskelions stripped from the different populations showed the same mobility on the agarose gel, suggesting that the mobility differences observed in intact CVs were due to differences in the surface charge of underlying vesicles rather than to variations in their clathrin coats. Stripped CVs exhibited considerable heterogeneity when analyzed by Western blotting: the fast-migrating population was enriched in the mannose 6-phosphate receptor, secretory acetyl-choline esterase, and an M/sub r/ 195,000 glycoprotein. The slow-migrating population of CVs was enriched in the asialoglycoprotein receptor, and it appeared to contain all detectable concanavalin A-binding polypeptides as well as the bulk of detectable WGA-binding proteins. When CVs were prepared from 125I-asialoorosomucoid-perfused rat liver, ligand was found in the slow-migrating CVs, suggesting that these were endocytic in origin. Morphological differences were also observed: the fast-migrating population was enriched in smaller CVs, whereas the slow-migrating population exhibited an enrichment in larger CVs. As liver consists largely of hepatocytes, these subpopulations appear to originate from the same cell type and probably represent CVs of different intracellular origin and destination

  11. Molecular analysis of Salmonella enteritidis isolates from the Caribbean by pulsed-field gel electrophoresis

    Abiodun Adesiyun

    2000-11-01

    Full Text Available Using pulsed-field gel electrophoresis (PFGE, between 1987 and 1996 we analyzed Salmonella enteritidis isolates from gastroenteritis cases in four Caribbean countries: Barbados, Saint Kitts and Nevis, Saint Lucia, and Trinidad and Tobago. We also determined the resistance of the isolates to 12 antimicrobial agents. Of the 129 isolates of S. enteritidis available for testing, DNA digested by XbaI revealed 13 distinctive PFGE patterns. The most prevalent XbaI PFGE patterns were group 1 (88 of 129 isolates, 68.2% and group 2 (26 of 129, 20.2%. The patterns found among S. enteritidis isolates correlated with the geographical origin of the isolates. Of the 28 isolates from Barbados, 20 of them (71.4% belonged to XbaI PFGE group 2, and of the 93 isolates from Trinidad and Tobago, 78 of them (83.9% belonged to group 1. SpeI digestion of S. enteritidis genome was not as discriminatory as XbaI. Overall, of the 129 isolates, 67 of them (51.9% exhibited resistance to one or more of the 12 antimicrobial agents that we tested. The prevalence of resistance was 53.8% for the S. enteritidis isolates tested from Trinidad and Tobago, 50.0% for those from Barbados, 28.6% for those from Saint Lucia, and 100.0% for one isolate from the island of Saint Kitts. Resistance was highest to triple sulfur (59 of 129 isolates, 45.7%, followed by furadantoin (10 of 129, 7.8%, ampicillin (7 of 129, 5.4%, and carbamycin (5 of 129, 3.9%.

  12. Multilocus sequence typing scheme versus pulsed-field gel electrophoresis for typing Mycobacterium abscessus isolates.

    Machado, Gabriel Esquitini; Matsumoto, Cristianne Kayoko; Chimara, Erica; Duarte, Rafael da Silva; de Freitas, Denise; Palaci, Moises; Hadad, David Jamil; Lima, Karla Valéria Batista; Lopes, Maria Luiza; Ramos, Jesus Pais; Campos, Carlos Eduardo; Caldas, Paulo César; Heym, Beate; Leão, Sylvia Cardoso

    2014-08-01

    Outbreaks of infections by rapidly growing mycobacteria following invasive procedures, such as ophthalmological, laparoscopic, arthroscopic, plastic, and cardiac surgeries, mesotherapy, and vaccination, have been detected in Brazil since 1998. Members of the Mycobacterium chelonae-Mycobacterium abscessus group have caused most of these outbreaks. As part of an epidemiological investigation, the isolates were typed by pulsed-field gel electrophoresis (PFGE). In this project, we performed a large-scale comparison of PFGE profiles with the results of a recently developed multilocus sequence typing (MLST) scheme for M. abscessus. Ninety-three isolates were analyzed, with 40 M. abscessus subsp. abscessus isolates, 47 M. abscessus subsp. bolletii isolates, and six isolates with no assigned subspecies. Forty-five isolates were obtained during five outbreaks, and 48 were sporadic isolates that were not associated with outbreaks. For MLST, seven housekeeping genes (argH, cya, glpK, gnd, murC, pta, and purH) were sequenced, and each isolate was assigned a sequence type (ST) from the combination of obtained alleles. The PFGE patterns of DraI-digested DNA were compared with the MLST results. All isolates were analyzable by both methods. Isolates from monoclonal outbreaks showed unique STs and indistinguishable or very similar PFGE patterns. Thirty-three STs and 49 unique PFGE patterns were identified among the 93 isolates. The Simpson's index of diversity values for MLST and PFGE were 0.69 and 0.93, respectively, for M. abscessus subsp. abscessus and 0.96 and 0.97, respectively, for M. abscessus subsp. bolletii. In conclusion, the MLST scheme showed 100% typeability and grouped monoclonal outbreak isolates in agreement with PFGE, but it was less discriminative than PFGE for M. abscessus. PMID:24899019

  13. Highly sensitive detection of S-nitrosylated proteins by capillary gel electrophoresis with laser induced fluorescence.

    Wang, Siyang; Circu, Magdalena L; Zhou, Hu; Figeys, Daniel; Aw, Tak Y; Feng, June

    2011-09-23

    S-nitrosylated proteins are biomarkers of oxidative damage in aging and Alzheimer's disease (AD). Here, we report a new method for detecting and quantifying nitrosylated proteins by capillary gel electrophoresis with laser induced fluorescence detection (CGE-LIF). Dylight 488 maleimide was used to specifically label thiol group (SH) after switching the S-nitrosothiol (S-NO) to SH in cysteine using the "fluorescence switch" assay. In vitro nitrosylation model-BSA subjected to S-nitrosoglutathione (GSNO) optimized the labeling reactions and characterized the response of the LIF detector. The method proves to be highly sensitive, detecting 1.3 picomolar (pM) concentration of nitrosothiols in nanograms of proteins, which is the lowest limit of detection of nitrosothiols reported to date. We further demonstrated the direct application of this method in monitoring protein nitrosylation damage in MQ mediated human colon adenocarcinoma cells. The nitrosothiol amounts in MQ treated and untreated cells are 14.8±0.2 and 10.4±0.5 pmol/mg of proteins, respectively. We also depicted nitrosylated protein electrophoretic profiles of brain cerebrum of 5-month-old AD transgenic (Tg) mice model. In Tg mice brain, 15.5±0.4 pmol of nitrosothiols/mg of proteins was quantified while wild type contained 11.7±0.3 pmol/mg proteins. The methodology is validated to quantify low levels of S-nitrosylated protein in complex protein mixtures from both physiological and pathological conditions. PMID:21820121

  14. Down-regulation of triose phosphate isomerase in Vineristine-resistant gastric cancer SGC7901 cell line identified by immobilized pH gradient two-dimensional gel electrophoresis and mierosequencing

    2002-01-01

    Objective:To exkplore new multidrug-resistance-related proteins in gastric SC7901 cells and clarify their mechanisms.Methods:Two-dimensional(2-D) polyacrylamide gel electrophoresis with immobilized pH gradients(IPG) was applied to compare the differential expression of multidrug-resistance-related proteins in gastric cancer SGC7901 cells and Vineristine-resistant SGC7901 cells (SGC7901/VCR) induced by vincristine sulfate.The 2-D gels were silver-stained.Then,preparative 2-D PAGE was performed.The differential proteins of PVDF membranes were cxcised and identified by N-terminal microsequencing.The mRNA expressions of differential proteins were detected in SGC 7901 cells and SGC7901/VCR cells by RT-PCR.Results:Approximatedly 680 protein sports were resolved on each 2-D gel by silver staining.Most protein spots showed no difference in composition,shape or density.25 proteins differed in abundance (6 higher in SGC7901/VCR cells;19 higher in 7901 cells);5 proteins were unique to one kind of cell or the othe(3 in SGC7901/VRC cells,2 in 7901 cells).One drug-resistance-related protein,which was down-regulated in SGC7901/VCR cells,was identified as trisephosphate isomerase(TPI),a glycolytic pathway enzyme.Conclusions:the results suggest that these differential proteins including TPI may be related to the Vincristine-resistant mechanism in human gastric cancer SGC7901/VCR cell line.

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

    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.

  16. Population dynamics of two antilisterial cheese surface consortia revealed by temporal temperature gradient gel electrophoresis

    Hasler Madlen

    2010-03-01

    Full Text Available Abstract Background Surface contamination of smear cheese by Listeria spp. is of major concern for the industry. Complex smear ecosystems have been shown to harbor antilisterial potential but the microorganisms and mechanisms involved in the inhibition mostly remain unclear, and are likely related to complex interactions than to production of single antimicrobial compounds. Bacterial biodiversity and population dynamics of complex smear ecosystems exhibiting antilisterial properties in situ were investigated by Temporal temperature gradient gel electrophoresis (TTGE, a culture independent technique, for two microbial consortia isolated from commercial Raclette type cheeses inoculated with defined commercial ripening cultures (F or produced with an old-young smearing process (M. Results TTGE revealed nine bacterial species common to both F and M consortia, but consortium F exhibited a higher diversity than consortium M, with thirteen and ten species, respectively. Population dynamics were studied after application of the consortia on fresh-produced Raclette cheeses. TTGE analyses revealed a similar sequential development of the nine species common to both consortia. Beside common cheese surface bacteria (Staphylococcus equorum, Corynebacterium spp., Brevibacterium linens, Microbacterium gubbeenense, Agrococcus casei, the two consortia contained marine lactic acid bacteria (Alkalibacterium kapii, Marinilactibacillus psychrotolerans that developed early in ripening (day 14 to 20, shortly after the growth of staphylococci (day 7. A decrease of Listeria counts was observed on cheese surface inoculated at day 7 with 0.1-1 × 102 CFU cm-2, when cheeses were smeared with consortium F or M. Listeria counts went below the detection limit of the method between day 14 and 28 and no subsequent regrowth was detected over 60 to 80 ripening days. In contrast, Listeria grew to high counts (105 CFU cm-2 on cheeses smeared with a defined surface culture

  17. DNA damaging effects of carbofuran and its main metabolites on mice by micronucleus test and single cell gel electrophoresis

    ZHOU Pei; LIU Baofeng; LU Yitong

    2005-01-01

    The DNA damaging effects of the carbamate pesticide carbofuran and its four metabolites (carbofuranphenol, 3-ketocarbofuran, 3-hydrocarbofuran and nitrosocarbofuran) on mice were evaluated by single cell gel electrophoresis (SCGE) assay and micronucleus test. KM mice were exposed to test compounds with different doses of 0.1, 0.2 and 0.4mg/kg through intraperitoneal injection two times with an internal of 24 h, and then killed by cervical dislocation 6 h after the second injection. In SCGE assay, isolated mice peripheral blood lymphocytes were employed to determine DNA damaging degree after a 1 h treatment by test compounds and a following electrophoresis. Carbofuran and carbofuranphenol showed negative results in both test and had no obvious toxicity. 3-hydrocarbofuran and nitrosocarbofuran were positive.3-ketocarbofuran could not induce micronucleus formation but caused significant DNA migration in SCGE test. These tests revealed that 3-ketocarbofuran, 3-hydrocarbofuran and nitrosocarbofuran are potential mutagesis and further research is needed.

  18. Electrophoresis of proteins and DNA on horizontal sodium dodecyl sulfate polyacrylamide gels

    Colombo Paolo; Cascone Eleonora; Bellavia Daniele; Duro Giovanni; Di Fiore Renata; Costa Maria A; Izzo Vincenzo; Gioviale Maria C; Barbieri Rainer

    2006-01-01

    Abstract An inexpensive Plexiglas apparatus which allows a simple and rapid preparation of horizontal polyacrylamide gels of different dimensions for different purposes, is described. Preparation of such gels is as easy and rapid as agarose gel preparation, and polymerized polyacrylamide gels are used to fractionate proteins or small DNA fragments using a common horizontal electrophoretic tank. This apparatus was used to electrophoretically fractionate proteins or DNA for immuno-blot analyses...

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

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

    2016-02-01

    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.

  20. Estimation of circular DNA size using gamma-irradiation and pulsed-field gel electrophoresis

    A method is described for estimating the size of large circular DNAs found within complex chromosomal DNA preparations. DNAs are treated with low levels of gamma-irradiation, sufficient to introduce a single double-stranded break per circle, and the resulting linear DNA is sized by pulsed-field electrophoresis and blot hybridization. The method is fast, reproducible, and very conveniently applied to the agarose-enclosed chromosomal DNA preparations commonly used in pulsed field electrophoresis

  1. Detection of a NotI polymorphism with the pmetH probe by pulsed-field gel electrophoresis.

    Julier, C; White, R.

    1988-01-01

    The pmetH probe, tightly linked to the locus for cystic fibrosis (CF), detected a NotI polymorphism with allele sizes of 1,000 and 550 kb that could be separated by pulsed-field gel electrophoresis. Both alleles were found on chromosomes bearing either the normal or the CF allele. Preliminary data showed that this polymorphism was not in strong linkage disequilibrium with the CF locus. Preliminary large-scale restriction mapping of the fragment showed that the NotI polymorphic site is 200-370...

  2. Studies of DNA damage in tumor cells induced by radiation exposure by means of single cell gel electrophoresis

    In order to detect the radiation sensitivity of tumor cells, the methods of single cell gel electrophoresis (SCGE) and 3H-TdR incorporation were applied to determine effects of radiation on single strand breaks of DNA in K562, SMMC-772 and HOS8603 cell lines. The experimental results proved that the cell migration distance was prolonged, DNA syntheses was restricted and the 3H-TdR incorporation rate was decreased in the above-mentioned 3 tumor cells lines after irradiation by 60Co γ rays (1-20 Gy)

  3. Explanation for the specification of dose estimation for the radiation victims in the early stage using single cell gel electrophoresis

    Based on the extensive research of literature,systematic study of the relevant laws and regulations related to the specification, the Specification of Dose Estimation for the Radiation Victims in the Early Stage Using Single Cell Gel Electrophoresis was enacted according to the principles about it. It can be used for the quantitative assay of DNA damage induced by whole body uniform irradiation in case of low linear energy transfer. To correctly implement this specification,contents in it were interpreted in this article. (authors)

  4. Genotypic Characterization of Salmonella enteritidis Phage Types by Plasmid Analysis, Ribotyping, and Pulsed-Field Gel Electrophoresis

    Ridley, A. M.; Threlfall, E J; Rowe, B.

    1998-01-01

    Pulsed-field gel electrophoresis (PFGE) was used to resolve XbaI and SpeI macrorestriction fragments from 60 defined phage type (PT) reference strains of Salmonella enteritidis. The level of discrimination was compared to that afforded by plasmid profile analysis and ribotyping. Twenty-eight distinct XbaI pulsed-field profiles (PFPs) were observed, although a single type, PFP X1, predominated. Absence of the 57-kb spv-associated fragment was observed for three PT reference strains, and the pr...

  5. Triton X-114 cloud point extraction to subfractionate blood plasma proteins for two-dimensional gel electrophoresis

    Jessen, Flemming; Wulff, Tune

    2015-01-01

    A simple and reproducible procedure for enrichment of a plasma protein subfraction suitable for two-dimensional polyacrylamide gel electrophoresis (2DE) was developed, using a Triton X-114-based cloud point extraction (CPE). Appropriate conditions for such a CPE procedure were found by SDS-PAGE to......-sterol acyltransferase, serum amyloid A, and serum paraoxonase/arylesterase 1, which are proteins of a hydrophobic nature, as in plasma they relate to lipoprotein particles. Thus, Triton X-114-based CPE is a simple plasma prefractionation tool, attractive for detailed 2DE studies of hydrophobic plasma proteins and their...

  6. Quantifying clustered DNA damage induction and repair by gel electrophoresis, electronic imaging and number average length analysis

    Sutherland, Betsy M.; Georgakilas, Alexandros G.; Bennett, Paula V.; Laval, Jacques; Sutherland, John C.; Gewirtz, A. M. (Principal Investigator)

    2003-01-01

    Assessing DNA damage induction, repair and consequences of such damages requires measurement of specific DNA lesions by methods that are independent of biological responses to such lesions. Lesions affecting one DNA strand (altered bases, abasic sites, single strand breaks (SSB)) as well as damages affecting both strands (clustered damages, double strand breaks) can be quantified by direct measurement of DNA using gel electrophoresis, gel imaging and number average length analysis. Damage frequencies as low as a few sites per gigabase pair (10(9)bp) can be quantified by this approach in about 50ng of non-radioactive DNA, and single molecule methods may allow such measurements in DNA from single cells. This review presents the theoretical basis, biochemical requirements and practical aspects of this approach, and shows examples of their applications in identification and quantitation of complex clustered damages.

  7. Quantifying clustered DNA damage induction and repair by gel electrophoresis, electronic imaging and number average length analysis

    Sutherland, Betsy M.; Georgakilas, Alexandros G.; Bennett, Paula V.; Laval, Jacques; Sutherland, John C

    2003-10-29

    Assessing DNA damage induction, repair and consequences of such damages requires measurement of specific DNA lesions by methods that are independent of biological responses to such lesions. Lesions affecting one DNA strand (altered bases, abasic sites, single strand breaks (SSB)) as well as damages affecting both strands (clustered damages, double strand breaks) can be quantified by direct measurement of DNA using gel electrophoresis, gel imaging and number average length analysis. Damage frequencies as low as a few sites per gigabase pair (10{sup 9} bp) can be quantified by this approach in about 50 ng of non-radioactive DNA, and single molecule methods may allow such measurements in DNA from single cells. This review presents the theoretical basis, biochemical requirements and practical aspects of this approach, and shows examples of their applications in identification and quantitation of complex clustered damages.

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

    Arnab Roy; Umesh Varshney; Debnath Pal

    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.

  9. An alkaline single-cell gel electrophoresis (comet assay for environmental biomonitoring with native rodents

    Juliana da Silva

    2000-03-01

    Full Text Available The main advantages of single-cell gel electrophoresis (SCG are its applicability to any eukaryotic organism and cell type, its low cost and the short time required to obtain results. These properties make the SCG assay particularly useful in screening for environmental genotoxicity. The present study describes a modified version of this technique for use in field work with native rodents and examines some factors which influence the outcome of the assay. Wild rodents (Ctenomys torquatus, "tuco-tuco" from a region close to a strip coal mine and from a region with no coal mines were used. Animals from the coal mining region had significantly more DNA damage than those from the control area. The use of this SCG technique for direct sampling in the field should facilitate environmental genotoxicity studies with natural populations, without the need to remove the animals from their habitat or to sacrifice them.A mais importante vantagem do Ensaio Cometa é a possibilidade do seu uso em qualquer organismo e tipo celular. Além de ser barato e rápido, se obtem resultados em poucas horas. Devido a isto é que ele vem sendo recomendado como teste inicial para monitoramento genotóxico ambiental. Neste trabalho investigaram-se adaptações na técnica para trabalhos de campo, com o uso de roedores endêmicos de região de mineração de carvão. O organismo utilizado foi o Ctenomys torquatus, capturado em duas diferentes áreas: Pelotas (região sem mineração de carvão-controle e Candiota (zona de mineração de carvão. Foi coletado sangue periférico de 18 roedores, que após marcação foram liberados. O sangue foi protegido da luz e mantido sob refrigeração, e processado in loco. As concentrações das agaroses e as condições alcalinas de lise e eletroforese foram modificadas a partir das metodologias sugeridas pelas revisões existentes. As amostras restantes foram mantidas em RPMI 1640 (1:10 (4ºC e pela nossa experiência podem ser

  10. Binding isotherms of sodium dodecyl sulfate to protein polypeptides with special reference to SDS-polyacylamide gel electrophoresis.

    Takagi, T; Tsujii, K; Shirahama, K

    1975-05-01

    To clarify the mode of interaction between sodium dodecyl sulfate (SDS) and protein polypeptides with special reference to SDS-polyacrylamide gel electrophoresis, the binding of SDS to several protein polypeptides was investigated by the equilibrium dialysis technique. Each of the binding isotherms was characterized by the presence of two phases: an initial gradual increase in the amount of binding to 0.3-0.6 g/g (first phase) and a subsequent steep increase to 1.2-1.5 g/g (second phase). The binding was completed at a concentration of SDS below the critical micelle concentration. Throughout the first and second phases, the isotherms obtained were different for each kind of protein. On the basis of experiments with bovine serum albumin and ribonuclease (EC 3.1.4.22], the isotherms were profoundly affected by the method used for modification of the sulfhydryl groups. The claim of Reynolds and Tanford (Proc. Natl, Acad. Sci. U.S., 66, 1002 (1970)) that the isotherms are virtually identical for many kinds of proteins was not supported by the present data. Changes in the gross and local conformations were examined with reference to the isotherms by measurements of CD spectrum, free boundary electrophoresis, and gel filtration. The results obtained were collectively interpreted based on the model of SDS-protein polypeptide complexes proposed by the present authors (J. Biochem., 75, 309 (1974)). PMID:1158859

  11. Influence of irradiation conditions on the measurement of DNA double-strand breaks by pulsed-field gel electrophoresis

    The induction of γ-ray induced DNA double-strand breaks (DSBs) determined by pulsed-field gel electrophoresis was compared in the DNA of intact Chinese hamster ovary (CHO) cells imbedded in agarose plugs, and in the isolated DNA of agarose-imbedded CHO cells that had been lysed before irradiation to determine whether these irradiation conditions would influence their measurement. DSB-induction in irradiated cells or isolated DNA was measured as the loss of DNA from the plug compared with the unirradiated control. Chromatin protein was completely digested by the lysis buffer and as such did not affect DNA migration upon electrophoresis, whereas concentrations of EDTA as low as 10-5 M affected the induction of DSBs in the isolated DNA. The gel plugs required several hours of washing with PBS to remove the contaminating EDTA from the lysis buffer. Once the residual EDTA was removed, DNA DSB induction as a function of dose was 70 times greater in isolated DNA than in the DNA of intact cells. (Author)

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

    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. PMID:14709007

  13. Analysis of a ribonuclease H digestion of N3'-->P5' phosphoramidate-RNA duplexes by capillary gel electrophoresis.

    DeDionisio, L; Gryaznov, S M

    1995-07-01

    Phosphodiester oligonucleotides (ODNs) and their analogs are presently being investigated as potential antisense therapeutics in the treatment of viral infections and various forms of cancer. here, we would like to report results from an investigation of activity for a ribonuclease H (RNase H) mediated RNA digestion assay in the duplexes formed by an ODN or the ODN analog, N3'-->P5' phosphoramidate (3'-phosphoramidate), and complimentary RNA strands. Capillary gel electrophoresis (CGE) proved to be an effective method for determining RNA hydrolysis in the presence of RNase H. RNA and an ODN or RNA and a 3'-phosphoramidate were hybridized in a Tris-HCl, MgCl2 buffer at room temperature (RT) and incubated with RNase H. Digestions were carried out at RT or at 37 degrees C. Control samples were unhybridized RNA with RNase H, RNA without RNase H, and duplexes (RNA-ODN or 3'-phosphoramidate) without RNase H. All controls were incubated in Tris-HCl, MgCl2 buffer, and sample aliquots were analyzed at various time intervals. A homodecamer, (dT)10, was used as an internal standard to determine the relative migration time of the RNA strand. The final digestion products for the duplexes and the various controls were monitored by CGE. In addition, polyacrylamide gel electrophoresis (PAGE) was used in conjunction with Stains-All (staining) and a densitometric analysis to verify CGE results. PMID:7581876

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

    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.

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

    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)

  16. Separation of nuclear protein complexes by blue native polyacrylamide gel electrophoresis

    Nováková, Zora; Hodný, Zdeněk; Man, P.; Hozák, Pavel

    Pavia: organizátor workhshopu, 2003. s. 1. [The Willhelm Bernhard Workshop - International Workshop on the Cell Nucleus /18./. 04.09.2003-09.09.2003, Pavia] Institutional research plan: CEZ:AV0Z5039906 Keywords : electrophoresis Subject RIV: EB - Genetics ; Molecular Biology

  17. 2D Gel-Based Multiplexed Proteomic Analysis during Larval Development and Metamorphosis of the Biofouling Polychaete Tubeworm Hydroides elegans

    Zhang, Yu

    2010-09-03

    Larval settlement and metamorphosis of a common biofouling polychaete worm, Hydroides elegans, involve remarkable structural and physiological changes during this pelagic to sessile habitat shift. The endogenous protein molecules and post-translational modifications that drive this larval transition process are not only of interest to ecologists but also to the antifouling paint industry, which aims to control the settlement of this biofouling species on man-made structures (e.g., ship hulls). On the basis of our recent proteomic studies, we hypothesize that rapid larval settlement of H. elegans could be mediated through changes in phosphorylation status of proteins rather than extensive de novo synthesis of proteins. To test this hypothesis, 2D gel-based multiplexed proteomics technology was used to monitor the changes in protein expression and phosphorylation status during larval development and metamorphosis of H. elegans. The protein expression profiles of larvae before and after they reached competency to attach and metamorphose were similar in terms of major proteins, but the percentage of phosphorylated proteins increased from 41% to 49% after competency. Notably, both the protein and phosphoprotein profiles of the metamorphosed individuals (adult) were distinctly different from that of the larvae, with only 40% of the proteins phosphorylated in the adult stage. The intensity ratio of all phosphoprotein spots to all total protein spots was also the highest in the competent larval stage. Overall, our results indicated that the level of protein phosphorylation might play a crucial role in the initiation of larval settlement and metamorphosis. © 2010 American Chemical Society.

  18. Denaturing gradient gel electrophoresis profiling of bacterial communities composition in Arabian Sea

    Singh, S.K.; Ramaiah, N.

    and sequenced indicated the preponderance of gammaproteobacteria, bacteroidetes and cyanobacteria. Non-metric dimensional scaling of the DGGE gels indicated that the spatial variations in BCC were prominent among the sampling locations. Temporal variations...

  19. 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

    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.

  20. Random amplified polymorphic DNA assay is less discriminant than pulsed-field gel electrophoresis for typing strains of methicillin-resistant Staphylococcus aureus.

    Saulnier, P; Bourneix, C; Prévost, G; Andremont, A

    1993-04-01

    Twenty-six strains of methicillin-resistant Staphylococcus aureus with different pulsed-field gel electrophoresis fingerprints were tested by random amplified polymorphic DNA assay with three primers, resulting in 15 to 20 different random amplified polymorphic DNA fingerprints. By summing the results for the three primers, the number of different fingerprints increased to 25, but two strains could not be differentiated. We conclude that pulsed-field gel electrophoresis remains the best method of typing methicillin-resistant S. aureus strains. PMID:8463406

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

    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.

  2. Restriction landmark cDNA scanning (RLCS): a novel cDNA display system using two-dimensional gel electrophoresis.

    Suzuki, H; Yaoi, T; Kawai, J; Hara, A; Kuwajima, G; Wantanabe, S

    1996-01-01

    We have developed a new method, designated restriction landmark cDNA scanning (RLCS), which displays many cDNA species quantitatively and simultaneously as two-dimensional gel spots. In this method cDNA species of uniform length were prepared for each mRNA species using restriction enzymes. After the restriction enzyme sites were radiolabeled as landmarks, the labeled fragments were subjected to high resolution two-dimensional gel electrophoresis. In analyses of cDNA samples from adult mouse liver and brain (cerebral cortex, cerebellum and brain stem) we detected approximately 500 and >1000 discrete gel spots respectively of various intensities at a time. The spot patterns of the three brain regions were very similar, although not identical, but were quite different from the pattern for the liver. RNA blot hybridization analysis using several cloned spot DNAs as probes showed that differences in intensity of the spots among RLCS profiles correlated well with expression levels of the corresponding mRNA species in the brain regions. Because the spots and their intensities reflect distinct mRNA species and their expression level respectively, the RLCS is a novel cDNA display system which provides a great deal of information and should be useful for systematic documentation of differentially expressed genes. PMID:8628652

  3. 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

    Garaizar, J.; Lopez-Molina, N.; Laconcha, I.; Baggesen, Dorte Lau; Rementeria, A.; Vivanco, A.; Audicana, A.; Perales, I.

    2000-01-01

    PCR fingerprinting interassay and intercenter reproducibility was low and was only increased when DNA samples were extracted at the same time and amplified with the same reaction mixtures. Reproducibility of IRS-PCR technique reached 100%, but discrimination was low (D = 0.52), The PFGE procedure......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 of.......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 of...

  4. Subpopulations of liver coated vesicles resolved by preparative agarose gel electrophoresis

    1986-01-01

    Rat liver clathrin coated vesicles (CVs) were separated into several distinct subpopulations using non-sieving concentrations of agarose, which allowed the separation of species differing primarily in surface charge. Using preparative agarose electrophoresis (Kedersha, N. L., and L. H. Rome, 1986, Anal. Biochem., in press), the CVs were recovered and analyzed for differences in morphology, coat protein composition, and stripped vesicle protein composition. Coat proteins from different populat...

  5. Molecular Fingerprinting of Dairy Microbial Ecosystems by Use of Temporal Temperature and Denaturing Gradient Gel Electrophoresis

    Ogier, J.-C.; Lafarge, V.; Girard, V.; A. Rault; Maladen, V.; Gruss, A; Leveau, J.-Y.; Delacroix-Buchet, A

    2004-01-01

    Numerous microorganisms, including bacteria, yeasts, and molds, constitute the complex ecosystem present in milk and fermented dairy products. Our aim was to describe the bacterial ecosystem of various cheeses that differ by production technology and therefore by their bacterial content. For this purpose, we developed a rapid, semisystematic approach based on genetic profiling by temporal temperature gradient electrophoresis (TTGE) for bacteria with low-G+C-content genomes and denaturing grad...

  6. Acrylamide gel electrophoresis of proteins, acid phosphatases and RN-ases from three potato varieties

    A. Kubicz; E. Wieczorek; B. Morawiecka

    2015-01-01

    Studies on variety differences in the protein and acid phosphatase patterns as well as ribunuclease activity distribution were carried out by disc electrophoresis on saline extracts of three varieties of the potato Solanum tuberosum (L.). The protein bands varied in number, position and relative abundance. One main zone of the acid phosphatase activity was detected consisting of 2-3 electrophoretically different bands. Variety differences were concerned with the number and relative abundance ...

  7. Proteome changes of lungs artificially infected with H-PRRSV and N-PRRSV by two-dimensional fluorescence difference gel electrophoresis

    Zhu Kongju

    2010-05-01

    Full Text Available Abstract Background Porcine reproductive and respiratory syndrome with PRRS virus (PRRSV infection, which causes significant economic losses annually, is one of the most economically important diseases affecting swine industry worldwide. In 2006 and 2007, a large-scale outbreak of highly pathogenic porcine reproductive and respiratory syndrome (PRRS happened in China and Vietnam. However little data is available on global host response to PRRSV infection at the protein level, and similar approaches looking at mRNA is problematic since mRNA levels do not necessarily predict protein levels. In order to improve the knowledge of host response and viral pathogenesis of highly virulent Chinese-type PRRSV (H-PRRSV and Non-high-pathogenic North American-type PRRSV strains (N-PRRSV, we analyzed the protein expression changes of H-PRRSV and N-PRRSV infected lungs compared with those of uninfected negative control, and identified a series of proteins related to host response and viral pathogenesis. Results According to differential proteomes of porcine lungs infected with H-PRRSV, N-PRRSV and uninfected negative control at different time points using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE and mass spectrometry identification, 45 differentially expressed proteins (DEPs were identified. These proteins were mostly related to cytoskeleton, stress response and oxidation reduction or metabolism. In the protein interaction network constructed based on DEPs from lungs infected with H-PRRSV, HSPA8, ARHGAP29 and NDUFS1 belonged to the most central proteins, whereas DDAH2, HSPB1 and FLNA corresponded to the most central proteins in those of N-PRRSV infected. Conclusions Our study is the first attempt to provide the complex picture of pulmonary protein expression during H-PRRSV and N-PRRSV infection under the in vivo environment using 2D-DIGE technology and bioinformatics tools, provides large scale valuable information for better

  8. Separation of 1–23-kb complementary DNA strands by urea–agarose gel electrophoresis

    Hegedüs, Éva; Kókai, Endre; Kotlyar, Alexander; Dombrádi, Viktor; Szabó, Gábor

    2009-01-01

    Double-stranded (ds), as well as denatured, single-stranded (ss) DNA samples can be analyzed on urea–agarose gels. Here we report that after denaturation by heat in the presence of 8 M urea, the two strands of the same ds DNA fragment of ∼1–20-kb size migrate differently in 1 M urea containing agarose gels. The two strands are readily distinguished on Southern blots by ss-specific probes. The different migration of the two strands could be attributed to their different, base composition-depen...

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

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

    2002-01-01

    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 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...

  10. Investigating freshwater periphyton community response to uranium with phospholipid fatty acid and denaturing gradient gel electrophoresis analyses

    Periphyton communities can be used as monitors of ecosystem health and as indicators of contamination in lotic systems. Measures of biomass, community structure, and genetic diversity were used to investigate impacts of uranium (U) exposure on periphyton. Laboratory exposures of periphyton in river water amended with 238U were performed for 5 days, followed by 2 days of U depuration in unamended river water. Productivity as measured by biomass was not affected by concentrations up to 100 μg 238U L-1. Phospholipid fatty acid (PLFA) profiles and denaturing gradient gel electrophoresis (DGGE) banding patterns revealed no changes in community or genetic structure related to U exposure. We suggest that the periphyton community as a whole was not significantly impacted by exposures of 238U up to a concentration of 100 μg L-1. These findings have significance for the assessment and prediction of U impacts on aquatic ecosystems

  11. Detection of Lesch-Nyhan syndrome carriers: Analysis of hair roots for HPRT by agarose gel electrophoresis and autoradiography

    Flat agarose gel electrophoresis and autoradiography were used to analyze hypoxanthine phosphoribosyltransferase (HPRT) and adenine phosphoribosyltransferase (APRT) activity in individual hair roots collected from the scalps of females to determine the presence of HPRT-deficient cells. Autoradiographs of hair-root lysates of normal homozygous females contained two well-separated dark zones representing HPRT and APRT activities. In contrast, some hair roots from carriers of HPRT deficiency contained two zones of activity with the same relative proportion of APRT and HPRT as hair roots of normal homozygotes, while others contained decreased amounts of HPRT activity. These hair roots consisted of HPRT+ and HPRT- cells. In addition, some hair roots from heterozygous carriers contained APRT but no HPRT activity. Such hair roots consisted of HPRT- cells only. (author)

  12. Diagnosis of an outbreak of Salmonella typhimurium in chinchillas (Chinchilla lanigera) by pulsed-field gel electrophoresis.

    Gornatti Churria, Carlos D; Vigo, Germán B; Origlia, Javier; Campos, Josefina; Caffer, María; Píscopo, Miguel; Herrero Loyola, Miguel; Petruccelli, Miguel; Pichel, Mariana

    2014-01-01

    Adult chinchillas (Chinchilla lanigera) that had suddenly died in a commercial farm located in La Plata City, Buenos Aires Province, Argentina, in July 2012 were macroscopically, histopathologically, and microbiologically examined. Salmonella enterica serovar Typhimurium (S. Typhimurium) was isolated from the liver, spleen, heart, lungs, kidneys and intestines from each of the five animals evaluated. The five strains were susceptible to ampicillin, cephalotin, cefotaxime, nalidixic acid, gentamicin, streptomycin, chloramphenicol, fosfomycin, nitrofurantoin and trimethoprim-sulfamethoxazole, and resistant to tetracycline. Each of the five S. Typhimurium isolates was analyzed by XbaI- pulsed-field gel electrophoresis (PFGE), showing an identical electrophoretic profile with 15 defined bands, which was found to be identical to pattern ARJPXX01.0220 of the PulseNet Argentine National database of Salmonella PFGE patterns. This is the first work describing the postmortem diagnosis of an outbreak of salmonellosis in chinchillas by using molecular methods such as PFGE. PMID:25444129

  13. Time-based distribution of Staphylococcus saprophyticus pulsed field gel-electrophoresis clusters in community-acquired urinary tract infections

    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.

  14. POLYACRYLAMIDE GEL ELECTROPHORESIS AS A TOOL FOR THE TAXONOMIC IDENTIFICATION OF SNAKES FROM THE ELAPIDAE AND VIPERIDAE FAMILIES

    A. M. SOARES

    1998-01-01

    Full Text Available Polyacrylamide gel electrophoresis (PAGE for basic proteins may be a useful tool for the characterization of whole snake venoms and for the taxonomic classification of snakes of the Elapidae and Viperidae families. However, due to the close proximity of molecular weights among toxins bearing different net charges, sodium dodecyl sulfate (SDS - PAGE was not able to provide an efficient differentiation. This article reports the electrophoretic analysis of several venoms from the genera Micrurus, Bothrops, Bothriopsis, Crotalus and Lachesis and shows a typical and distinctive electrophoretic profile for each species, with intraspecific and geographic variations. Even in cases in which extreme morphological similarities were present, such as between B. jararacussu and B. pirajai ("Bahia jararacuçu", differentiation could be evidenced by PAGE. This simple and sensitive procedure may be applied to similar cases involving basic toxins.

  15. Quantitation of yeast total proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer for uniform loading.

    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. PMID:26796977

  16. Assessing the stability of cystatin/cysteine proteinase complexes using mildly-denaturing gelatin-polyacrylamide gel electrophoresis.

    Michaud, D; Cantin, L; Raworth, D A; Vrain, T C

    1996-01-01

    A method for assessing the stability of cystatin/cysteine proteinase complexes using mildly-denaturing gelatin-polyacrylamide gel electrophoresis (gelatin-PAGE) is described. As suggested by the use of well-known cystatins (human stefins A and B, and oryzacystatins I and II) and the plant cysteine proteinase papain, the ability of cystatin/cysteine proteinase complexes to remain stable during electrophoresis is associated with the degree of affinity between the enzyme and the inhibitor (and inversely associated with the Ki values), at least with the disulfide bond-lacking cystatins. Complexes with Ki values > or = 10(-8) M (weak interactions) are partly or completely dissociated under the conditions used, while those with lower Ki values (strong interactions) remain stable. As shown by the differential effects of two plant cystatins, oryzacystatins I and II, against a cysteine proteinase present in crude (complex) extracts from a plant pest -- the two-spotted spider mite (Tetranychus urticae Koch), the gelatin-PAGE procedure is suitable for studying the ability of cystatins to form highly stable complexes with cysteine proteinases, without the need for prior purification steps. Considering the well-recognized potential of proteinase inhibitors for pest and pathogen control, this analytical approach will be useful for rapidly assessing the respective potential of various cystatins for protection of plants, animals, and humans. PMID:8907521

  17. Major proteins in normal human lymphocyte subpopulations separated by fluorescence-activated cell sorting and analyzed by two-dimensional gel electrophoresis

    Madsen, P S; Hokland, M; Ellegaard, J; Hokland, P; Ratz, G P; Celis, A

    1988-01-01

    We have compared the overall patterns of protein synthesis of normal human lymphocyte subpopulations taken from five volunteers using high resolution two-dimensional gel electrophoresis. The lymphocytes were isolated using density gradient centrifugation, labeled with subtype-specific MoAbs, and ...

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

    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

  19. A comparison of BOX-PCR and pulsed-field gel electrophoresis to determine genetic relatedness of enterococci from different environments

    Aims: The genetic relatedness of enterococci from poultry litter to enterococci from nearby surface water and groundwater in the Lower Fraser Valley regions of British Columbia, Canada was determined. Methods and Results: BOX-PCR and Pulsed-Field Gel Electrophoresis (PFGE) were used to subtype en...

  20. A comparison of non-typhoidal Salmonella from humans and food animals using pulsed-field gel electrophoresis and antimicrobial susceptibility patterns

    Salmonellosis is one of the most important foodborne diseases affecting humans. To characterize the relationship between Salmonella causing human infections and their food animal reservoirs, we compared pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility patterns of non-typhoida...

  1. Analysis of bacterial communities in soil by use of denaturing gradient gel electrophoresis and clone libraries, as influenced by different reverse primers

    Brons, Jolanda; van Elsas, J.D.

    2008-01-01

    To assess soil bacterial diversity, PCR systems consisting of several slightly different reverse primers together with forward primer F968-GC were used along with subsequent denaturing gradient gel electrophoresis (DGGE) or clone library analyses. In this study, a set of 13 previously used and novel

  2. 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

    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…

  3. Proteomics analysis in mature seed of four peanut cultivars using two-dimensional gel electrophoresis reveals distinct differential expression of storage, anti-nutritive, and allergenic proteins

    Protein profiles of total seed proteins isolated from mature seeds of four peanut cultivars, New Mexico Valencia C (NM Valencia C), Tamspan 90, Georgia Green, and NC-7, were studied using two-dimensional gel electrophoresis coupled with nano electrospray ionization liquid chromatography tandem mass ...

  4. Pulsed-Field Gel Electrophoresis of Genomic Digests Demonstrates Linkages among Food, Food Handlers, and Patrons in a Food-Borne Salmonella javiana Outbreak in Massachusetts

    Lee, Roger; Peppe, Joseph; George, Harvey

    1998-01-01

    A total of 66 isolates of Salmonella javiana isolated from food, food handlers, and patrons that were epidemiologically linked to an outbreak of gastroenteritis were analyzed by pulsed-field gel electrophoresis. Analysis with restriction endonucleases XbaI and SpeI supported the epidemiologic association and suggested a pathway of transmission among food, food handlers, and patrons.

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

    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…

  6. Investigations into the oxygen effect during the radiolysis of serum albumin with the help of SDS-polyacrylamide gel electrophoresis

    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)

  7. Isolation of DNA fragments containing replication forks by two dimensional agarose gel electrophoresis

    Escherichia coli is used as a model system to study DNA repair processes that promote survival after damage from alkylating agents and other processes that affect mutation resulting from such damage. In order to test the hypothesis that the induced incision repair process is able to repair this damage, a two-dimensional gel system has been developed that allows resolution of DNA fragments containing replication forks from the rest of the genome

  8. Polymerization of SDS-PAGE gel by gamma irradiation and its use for characterization by electrophoresis of a protein [rapid communication

    Kim, Dong-Ho; Kim, Jin-Hee; Seo, Ji-Hyun; Lee, Ju-Woon; Lim, Sang-Yong; Lee, Ho-Joon; Byun, Myung-Woo

    2005-12-01

    An SDS-PAGE gel strip was polymerized using a gamma irradiation process and used for electrophoresis. The relative mobility (Rf) and resolution of marker proteins were determined. Polymerization was induced by gamma irradiation in an acrylamide and N'-methylene bisacrylamide mixture with and without the polymerization initiators, ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TEMED). The calibration curves of log 10 molecular weight of the protein versus the distance of the migration showed higher correlations in the gamma irradiated gel than in that of the APS-TEMED polymerized control. The Rf value of the protein was increased in the gel polymerized by gamma-irradiation.

  9. Investigation of a 2D two-point maximum entropy regularization method for signal-to-noise ratio enhancement: application to CT polymer gel dosimetry

    This study presents a new method of image signal-to-noise ratio (SNR) enhancement by utilizing a newly developed 2D two-point maximum entropy regularization method (TPMEM). When utilized as an image filter, it is shown that 2D TPMEM offers unsurpassed flexibility in its ability to balance the complementary requirements of image smoothness and fidelity. The technique is evaluated for use in the enhancement of x-ray computed tomography (CT) images of irradiated polymer gels used in radiation dosimetry. We utilize a range of statistical parameters (e.g. root-mean square error, correlation coefficient, error histograms, Fourier data) to characterize the performance of TPMEM applied to a series of synthetic images of varying initial SNR. These images are designed to mimic a range of dose intensity patterns that would occur in x-ray CT polymer gel radiation dosimetry. Analysis is extended to a CT image of a polymer gel dosimeter irradiated with a stereotactic radiation therapy dose distribution. Results indicate that TPMEM performs strikingly well on radiation dosimetry data, significantly enhancing the SNR of noise-corrupted images (SNR enhancement factors >15 are possible) while minimally distorting the original image detail (as shown by the error histograms and Fourier data). It is also noted that application of this new TPMEM filter is not restricted exclusively to x-ray CT polymer gel dosimetry image data but can in future be extended to a wide range of radiation dosimetry data

  10. Acrylamide gel electrophoresis of proteins, acid phosphatases and RN-ases from three potato varieties

    A. Kubicz

    2015-05-01

    Full Text Available Studies on variety differences in the protein and acid phosphatase patterns as well as ribunuclease activity distribution were carried out by disc electrophoresis on saline extracts of three varieties of the potato Solanum tuberosum (L.. The protein bands varied in number, position and relative abundance. One main zone of the acid phosphatase activity was detected consisting of 2-3 electrophoretically different bands. Variety differences were concerned with the number and relative abundance of these bands. RNase activity was detected in 4 main zones, in some of them additional subbands were visible. Differences between the three examined varieties were reflected in the occurence of the particular activity zones or their subbands.

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

    Steven Carberry

    2013-12-01

    Full Text Available 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.

  12. Application of fluorescence two-dimensional difference in-gel electrophoresis as a proteomic biomarker discovery tool in muscular dystrophy research.

    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

  13. The effect of DNA concentration on mobility in pulsed field gel electrophoresis.

    Doggett, N A; Smith, C. L.; Cantor, C R

    1992-01-01

    The effect of DNA concentration on pulsed field gel electrophoretic mobility was studied for human genomic DNA prepared in agarose inserts at 8-800 micrograms/ml and digested to completion with Not I. An eighth of each 100 microliter insert was used to produce DNA loads of 0.1 to 10 micrograms per lane. The mobility of single copy restriction fragments, as detected by hybridization, was largely concentration independent when DNA concentrations were 80 micrograms/ml or less. However, at DNA co...

  14. Caracterização de cepas de referência de Leptospira sp utilizando a técnica de pulsed field gel electrophoresis Characterization of Leptospira sp reference strains using the pulsed field gel electrophoresis technique

    Lívia Machry

    2010-04-01

    Full Text Available INTRODUÇÃO: A leptospirose é uma zoonose endêmica, mundialmente distribuída, causada por bactérias do gênero Leptospira. Este gênero compreende espécies patogênicas e saprofíticas, com mais de 200 sorovares distintos, dificultando sua caracterização. A técnica de pulsed field gel electrophoresis tem sido empregada como uma ferramenta para auxiliar nesta caracterização. Os objetivos deste trabalho foram padronizar a técnica de PFGE, determinar os perfis moleculares das cepas de referência utilizadas pelo Laboratório de Referência Nacional para Leptospirose/Centro Colaborador da Organização Mundial de Saúde para Leptospirose e criar um banco de dados com estes perfis. MÉTODOS: Foram analisadas, por PFGE, dezenove cepas utilizando a enzima de restrição NotI. RESULTADOS: Cada cepa apresentou um perfil único que pode ser considerado como uma identidade genômica específica, com exceção dos sorovares Icterohaemorrhagiae e Copenhageni, cujos perfis foram indistinguíveis. CONCLUSÕES: Dessa forma, foi possível a criação de um banco de perfis moleculares que está sendo utilizado no Laboratório para a comparação e identificação de cepas isoladas de quadros clínicos.INTRODUCTION: Leptospirosis is an endemic zoonosis of worldwide distribution, caused by bacteria of the genus Leptospira. This genus includes pathogenic and saprophytic species, with more than 200 different serovars, thus making it difficult to characterize. The technique of pulsed field gel electrophoresis has been used as a tool to aid in this characterization. The aims of this study were to standardize the PFGE technique, determine the molecular profiles of reference strains used at the National Reference Laboratory for Leptospirosis/World Health Organization Collaborating Center for Leptospirosis and create a database with these profiles. METHODS: Nineteen strains were analyzed by means of PFGE, using the restriction enzyme NotI. RESULTS: Each strain

  15. DNA double-strand breaks measured in individual cells subjected to gel electrophoresis

    Microscopic examination of individual mammalian cells embedded in agarose, subjected to electrophoresis, and stained with a fluorescent DNA-binding dye provides a novel way of measuring DNA damage and more importantly, of assessing heterogeneity in DNA damage within a mixed population of cells. With this method, DNA double-strand breaks can be detected in populations of cells exposed to X-ray doses as low as 5 Gy. The radiation dose-response relationship for initial formation of double-strand breaks was identical for cell lines irradiated in G1, regardless of their sensitivity to killing by ionizing radiation. However, for cells irradiated in S phase, DNA migration was significantly reduced. For Chinese hamster V79 cells, Chinese hamster ovary cells, WiDr human colon carcinoma cells, and L5178Y-R mouse lymphoblastoid cells, S-phase DNA appeared to be about 3 times less sensitive to X-ray damage than DNA from other phases of the cell cycle. However, for the very radiosensitive L5178Y-S cells, the migration of replicating DNA was reduced only slightly. For Chinese hamster V79 and Chinese hamster ovary cells, damage was repaired at a similar rate in all cells of the population, and 85% of the breaks were rejoined within 2 h after irradiation. The radiosensitive L5178Y-S cells repaired damage more slowly than V79 or Chinese hamster ovary cells; 2 h after exposure to 50 Gy, approximately 50% of the damage was still present

  16. Detection of Sperm DNA Damage in Workers Exposed to Benzene by Modified Single Cell Gel Electrophoresis

    Bo SONG; Zhi-ming CAI; Xin LI; Li-xia DENG; Qiao ZHANG; Lu-kang ZHENG

    2005-01-01

    Objective To assess the effect of benzene on sperm DNA damageMethods Twenty-seven benzene-exposed workers were selected as exposed groupand 35 normal sperm donors as control group. Air concentration of benzene series inworkshop was determined by gas chromatography. As an internal exposure dose ofbenzene, the concentration of trans, trans-muconic acid (ttMA) was determined byhigh performance liquid chromatography. DNA was detected by modified single cellgel electrophoresis (SCGE).Results The air concentrations of benzene, toluene and xylene at the workplace were86.49 ± 2.83 mg/m3, 97.20 ±3.52 mg/m3 and 97.45 ±2.10 mg/m3, respectively.Urinary ttMA in exposed group (1.040 ± 0.617 mg/L) was significantly higher thanthat of control group (0.819 ± 0.157 mg/L). The percentage of head DNA, determinedby modified SCGE method, significantly decreased in the exposed group (n=13, 70.18%± 7.36%) compared with the control (n=16, 90.62% ± 2.94%)(P<0.001).Conclusion The modified SCGE method can be used to investigate the damage ofsperm DNA. As genotoxin and reprotoxins, benzene had direct effect on the germ cellsduring the spermatogenesiss.

  17. Identification of stable plant cystatin/nematode proteinase complexes using mildly denaturing gelatin/polyacrylamide gel electrophoresis.

    Michaud, D; Cantin, L; Bonadé-Bottino, M; Jouanin, L; Vrain, T C

    1996-08-01

    The biochemical interactions between two cystatins from rice seeds, oryzacystatin I (OCI) and oryzacystatin II (OCII), and the cysteine proteinases from three plant parasitic nematodes, Meloidogyne hapla, M. incognita and M. javanica, were assessed using standard protease assays and mildly denaturing gelatin/polyacrylamide gel electrophoresis (gelatin/PAGE). Activity detected in extracts of preparasitic second-stage larvae (J2) from M. hapla was optimal at pH 5.5 and was inhibited in vitro by the cysteine proteinase inhibitors trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane, hen egg cystatin, OCI, and OCII. As demonstrated by class-specific activity staining, all the activity measured between pH 3.5 and pH 7.5 was accounted for by a major proteinase form, Mhp1, and two minor forms, Mhp2 and Mhp3. Mhps were also detected in extracts and excretions of parasitic J2 and adult females, indicating their continuous expression throughout development of M. hapla, and their possible involvement in the extracellular degradation of proteins. Interestingly, the two plant cysteine proteinase inhibitors OCI and OCII showed different degrees of affinity for the major proteinase form, Mhp1. Both inhibitors almost completely inactivated this proteinase in native conditions but, unlike OCII, OCI conserved a high affinity for Mhp1 during mildly denaturing gelatin/PAGE, showing the differential stabilities of the OCI/Mhp1 and OCII/Mhp1 complexes. In contrast to Mhp1, the major cysteine proteinases detected in the two closely related species M. incognita and M. javanica were strongly inhibited by OCII, while the inhibition of OCI was partly prevented during electrophoresis. This species-related efficiency of plant cystatins against nematode cysteine proteinases could have practical implications when planning their use to control nematodes of the genus Meloidogyne. PMID:8874065

  18. Comparisons of Pasteurella multocida lipopolysaccharides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine relationship between group B and E hemorrhagic septicemia strains and serologically related group A strains.

    Rimler, R B

    1990-01-01

    Lipopolysaccharides (LPSs) purified from 16 reference somatic serotypes of Pasteurella multocida were examined and compared by discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Resolution of LPS patterns in a gel was optimum when sample wells were cast separately from the stacking gel and the running gel consisted of 15% T (total monomer) polyacrylamide and 4 M deionized urea. Band patterns of P. multocida LPSs in a gel differed from control Salmonella minnesota wild-ty...

  19. Single-based resolution for oligodeoxynucleotides and their phosphorothioate modifications by replaceable capillary gel electrophoresis.

    Chen, Rong; Luo, Xuefang; Di, Xin; Li, Ying; Sun, Yuqing; Hu, Yuzhu

    2006-11-01

    A replaceable capillary gel electrophoretic (replaceable CGE) method was developed for the separation of two sets of model compounds of single-stranded oligodeoxynucleotide mixtures (18-20 mers), phosphodiester oligodeoxynucleotides (PO-ODNs) and their phosphorothioate modifications (PS-ODNs), with equal sequences differing in a single base. Polyethylene glycol (PEG) 35000 was chosen as the sieving matrix. It was confirmed that PEG polymer solution less influenced resolutions of the PS-ODNs compared with those of the PO-ODNs, while acetonitrile used as an additive in the system improved the separation significantly. It was also noticed that the effect of temperature on separation was much larger than that of denaturant urea. PMID:16809077

  20. Evaluation of radio-induced DNA damage and their repair in human lymphocytes by comet assay or single cell gel electrophoresis; Avaliacao do dano radioinduzido no DNA e reparo em linfocitos humanos pelo metodo do cometa (single cell gel electrophoresis)

    Nascimento, Patricia A. do; Suzuki, Miriam F.; Okazaki, Kayo [Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, SP (Brazil)

    1997-12-01

    The comet assay, also called single cell gel electrophoresis technique, permits to evaluate quantitatively DNA breakage induced by chemical and physical agents at the level of the single cell. The present paper refers to the construction of dose-response curves to DNA damage and repair studies in human peripheral lymphocytes, utilizing the comet assay for the radiosensitivity analysis. So, the blood samples were obtained from healthy donors (40-50 year old), irradiated in a {sup 60} Co source (GAMMACEL 220) with doses of 0.17, 0.25, 0.57, 1.10, 2.12 and 4.22 Gy (0.59 Gy/min.) and processed 1 and 24 hours after the exposition. Results obtained showed a increase in the total lenght of comet (DNA migration) as a function of radiation dose in samples processed 1 and 24 hours after the treatment. The DNA lesion in irradiated lymphocytes with 4.22 Gy (means value of 101.4 {mu}m) were 3.4 times higher than in the untreated lymphocytes (mean value of 30 {mu}m) instead of 24 hours after the irradiation were 1.5 times higher (mean value of 46.3 {mu}m). This reduction on DNA repair occurred in these cells. It was also possible visualized the presence of subpopulations of the cells with different sensitivity and repair capacity to ionizing radiation in these donors. (author). 8 refs., 3 figs.

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

    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.

  2. Single-cell gel electrophoresis applied to the analysis of UV-C damage and its repair in human cells

    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 109 daltons are detected. (Author)

  3. Use of single cell micro-gel electrophoresis to detect the cellular radiosensitivity of two fibroblast strains

    Single cell micro-gel electrophoresis (SCGE) and MTT assay were used to determine the relationships between radiation-induced initial DNA double-strand breaks (DSBs), the ability of cells to repair DSBs and cellular radiosensitivity in both AT5BIVA and GM637 cell lines. The results demonstrated that AT5BIVA cell line was significantly more radiosensitive than GM637. The initial DSBs in both AT5BIVA and GM637 lines increased with the rise of irradiation dose, and showed significant dependence on dose. At a given dose of 20 Gy, radiation-induced initial DSBs in AT5BIVA cell line was significantly higher than those in GM637. The ability of AT5BIVA cell line to repair DSBs was more powerful than that of GM637. The results suggested that the radiation-induced initial DSBs in cells and the ability of cells to repair DSBs both correlated with cellular radiosensitivity to a certain extent, and could be used as potential predictors of intrinsic radiosensitivity of cells

  4. Detection of heavy-ion-induced DNA double-strand breaks using static-field gel electrophoresis

    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 rather misrepaired or unrepaired DNA lesions are related to cellular inactivation. (orig.)

  5. Analysis of microbial diversity on deli slicers using polymerase chain reaction and denaturing gradient gel electrophoresis technologies.

    Koo, O K; Mertz, A W; Akins, E L; Sirsat, S A; Neal, J A; Morawicki, R; Crandall, P G; Ricke, S C

    2013-02-01

    Cross-contamination of pathogenic and spoilage bacteria from food-contact surfaces to food products is a serious public health issue. Bacteria may survive and attach to food-contact surfaces by residual food components and/or background bacteria which may subsequently transfer to other food products. Deli slicers, generally used for slicing ready-to-eat products, can serve as potential sources for considerable bacterial transfer. The objective of this study was to assess the extent and distribution of microbial diversity of deli slicers by identification of pathogenic and background bacteria. Slicer-swab samples were collected from restaurants in Arkansas and Texas in the United States. Ten surface areas for each slicer were swabbed using sterile sponges. Denaturing gradient gel electrophoresis (DGGE) was applied to investigate the fingerprint of samples, and each band was further identified by sequence analysis. Pseudomonads were identified as the dominant bacteria followed by Enterobacteriaceae family, and lactic acid bacteria such as Lactococcus lactis and Streptococcus thermophilus were also found. Bacterial distribution was similar for all surface areas, while the blade guard exhibited the greatest diversity. This study provides a profile of the microbial ecology of slicers using DGGE to develop more specific sanitation practices and to reduce cross-contamination during slicing. PMID:23121623

  6. Monitoring of the microbial communities involved in the soy sauce manufacturing process by PCR-denaturing gradient gel electrophoresis.

    Tanaka, Yasushi; Watanabe, Jun; Mogi, Yoshinobu

    2012-08-01

    Soy sauce is a traditional seasoning produced through the fermentation of soybeans and wheat using microbes. In this study, the microbial communities involved in the soy sauce manufacturing process were analyzed by PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE). The bacterial DGGE profile indicated that the bacterial microbes in the koji were Weissella cibaria (Weissella confusa, Weissella kimchii, Weissella salipiscis, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus iners, or Streptococcus thermophilus), Staphylococcus gallinarum (or Staphylococcus xylosus), and Staphylococcus kloosii. In addition to these bacteria, Tetragenococcus halophilus was also detected in the mash during lactic acid fermentation. The fungal DGGE profile indicated that the fungal microbes in the koji were not only Aspergillus oryzae but also several yeasts. In the mash, Zygosaccharomyces rouxii appeared in the early fermentation stage, Candida etchellsii (or Candida nodaensis) and Candida versatilis were detected at the middle fermentation stage, and Candida etchellsii was detected at the mature fermentation stage. These results suggest that the microbial communities present during the soy sauce manufacturing process change drastically throughout its production. This is the first report to reveal the microbial communities involved in the soy sauce manufacturing process using a culture-independent method. PMID:22475947

  7. Analysis of microbial communities in doenjang, a Korean fermented soybean paste, using nested PCR-denaturing gradient gel electrophoresis.

    Kim, Tae-Woon; Lee, Jun-Hwa; Kim, Sung-Eon; Park, Min-Hee; Chang, Hae Choon; Kim, Hae-Yeong

    2009-05-31

    Doenjang is a traditional Korean fermented soybean paste that provides a major source of protein. The microbial diversity of 10 samples of doenjang (5 commercially manufactured products and 5 homemade products) was investigated using nested PCR-denaturing gradient gel electrophoresis (DGGE). In the first step, the nearly complete 16S rRNA and 18S rRNA genes were amplified using universal primers. Subsequently, these products were used as a template in a nested PCR to obtain fragments suitable for DGGE. The bacterial DGGE profile targeting the V3 region of the 16S rRNA gene indicated that lactic acid bacteria such as Leuconostoc mesenteroide, Tetragenococcus halophilus, and Enterococcus faecium were the predominant species. However, bands corresponding to Bacillus species, known to be the main organisms in doenjang, were not detected under the conditions described above. When selective PCR was conducted using a primer specific for Bacillus species, Bacillus subtilis and B. licheniformis were detected in several doenjang samples. In analysis of fungi, Mucor plumbeus, Aspergillus oryzae, and Debaryomyces hansenii were the most common species in the doenjang samples. On the basis of DGGE, a few differences in community structure were found for different samples. Also, cluster analysis of the DGGE profile revealed that the microbial diversity did not differ clearly between commercially manufactured and homemade products. The nested PCR-DGGE technique was used for the first time in this study to asses a microbial community in doenjang and proved to be effective in profiling microbial diversity. PMID:19324443

  8. Denaturing gradient gel electrophoresis (DGGE as a powerful novel alternative for differentiation of epizootic ISA virus variants.

    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.

  9. Proteomic analysis of docetaxel resistance in human nasopharyngeal carcinoma cells using the two-dimensional gel electrophoresis method.

    Peng, Xingchen; Gong, Fengming M; Ren, Min; Ai, Ping; Wu, ShaoYong; Tang, Jie; Hu, XiaoLin

    2016-09-01

    Docetaxel-based chemotherapy has been recommended for advanced nasopharyngeal carcinoma (NPC). However, treatment failure often occurs because of acquired drug resistance. In this study, a docetaxel-resistant NPC cell line CNE-2R was established with increasing doses of docetaxel for more than 6 months. Two-dimensional gel electrophoresis and ESI-Q-TOF-MS were used to compare the differential expression of docetaxel-resistance-associated proteins between human NPC CNE-2 cells and docetaxel-resistant CNE-2R cells. As a result, 24 differentially expressed proteins were identified, including 11 proteins with increased expression and 13 proteins with decreased expression. These proteins function in diverse biological processes such as metabolism, signal transduction, calcium ion binding, immune response, proteolysis, and so on. Among these, α-enolase (ENO1), significantly upregulated in CNE-2R, was selected for detailed analysis. Inhibition of ENO1 by shRNA restored CNE-2R cells' sensitivity to docetaxel. Moreover, overexpression of ENO1 could facilitate the development of acquired resistance of docetaxel in CNE-2 cells. Western blot and reverse-transcription PCR data of clinical samples confirmed that α-enolase was upregulated in docetaxel-resistant human NPC tissues. Finding such proteins might improve interpretation of the molecular mechanisms leading to the acquisition of docetaxel chemoresistance. PMID:27333594

  10. Investigating the fate of activated sludge extracellular proteins in sludge digestion using sodium dodecyl sulfate polyacrylamide gel electrophoresis.

    Park, Chul; Helm, Richard F; Novak, John T

    2008-12-01

    The fate of activated sludge extracellular proteins in sludge digestion was investigated using three different cation-associated extraction methods and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Extraction methods used were the cation exchange resin (CER) method for extracting calcium (Ca2+) and magnesium (Mg2+), sulfide extraction for removing iron, and base treatment (pH 10.5) for dissolving aluminum. Extracellular polymeric substances extracted were then subjected to SDS-PAGE, and the resultant protein profiles were examined before and after sludge digestion. The SDS-PAGE results showed that three methods led to different SDS-PAGE profiles for both undigested and digested sludges. The results further revealed that CER-extracted proteins remained mainly undegraded in anaerobic digestion, but were degraded in aerobic digestion. While the fate of sulfide- and base-extracted proteins was not clear for aerobic digestion, their changes in anaerobic digestion were elucidated. Most sulfide-extracted proteins were removed by anaerobic digestion, while the increase in protein band intensity and diversity was observed for base-extracted proteins. These results suggest that activated sludge flocs contain different fractions of proteins that are distinguishable by their association with certain cations and that each fraction undergoes different fates in anaerobic and aerobic digestion. The proteins that were resistant to degradation and generated during anaerobic digestion were identified by liquid chromatography tandem mass spectrometry. Protein identification results and their putative roles in activated sludge and anaerobic digestion are discussed in this study. PMID:19146099

  11. Frequencies of virulence genes and pulse field gel electrophoresis fingerprints in Escherichia coli isolates from canine pyometra.

    Maluta, Renato P; Borges, Clarissa A; Beraldo, Lívia G; Cardozo, Marita V; Voorwald, Fabiana A; Santana, André M; Rigobelo, Everlon C; Toniollo, Gilson H; Avila, Fernando A

    2014-11-01

    Escherichia coli is the most common bacterial agent isolated from canine pyometra. The frequencies of 24 virulence genes and pulsed field gel electrophoresis (PFGE) profiles were determined for 23 E. coli isolates from cases of canine pyometra in Brazil. The frequencies of virulence genes were 91.3% fimH, 91.3% irp-2, 82.6% fyuA, 56.5% iroN, 47.8% traT, 39.1% usp, 34.8% sfaD/E, 34.8% tsh, 30.4% papC, 30.4% hlyA, 26.1% papGIII, 26.1% cnf-1, 21.7% papE/F, 21.7% iss, 17.4% iutA, 17.4% ompT, 17.4% cvaC, 17.4% hlyF, 17.4% iucD, 13.0% iucC, 13.0% astA, 4.3% papGII, 0% afaB/C and 0% papGI. The high frequency of yersiniabactin (fyuA and irp2) and salmochelin (iroN) genes suggests that iron uptake systems might be important in the pathogenesis of canine pyometra. PFGE profiles of 19 isolates were heterogeneous, confirming that E. coli isolates from canine pyometra are unlikely to be epidemic clones. PMID:25201253

  12. Top-Down, Bottom-Up, and Side-to-Side Proteomics with Virtual 2-D Gels

    Ogorzalek Loo, Rachel R.

    2004-01-01

    Intact protein masses can be measured directly from immobilized pH gradient (IPG) isoelectric focusing (IEF) gels loaded with mammalian and prokaryotic samples, as demonstrated here with murine macrophage and Methanosarcina acetivorans cell lysates. Mass accuracy and resolution is improved by employing instruments which decouple the desorption event from mass measurement; e.g., quadrupole time-of-flight instruments. MALDI in-source dissociation (ISD) is discussed as a means to pursue top-do...

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

    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)

  14. DNA double-strand break induction in yeast by X-rays and α-particles measured by pulsed-field gel electrophoresis

    Pulsed-field gel electrophoresis was used to separate chromosomes of Saccharomyces cerevisiae 211 *B after irradiation with X-rays and α-particles. After electrophoresis, gels were stained with ethidium bromide, placed on a UV-transilluminator and photographed with a digitizing camera connected to a personal computer. Linearity between DNA amount and fluorescence was demonstrated. Fluoroescence intensity for the band with the lowest electrophoretic mobility was found to decrease exponentially with dose. Based on the known size of the native DNA molecules, double-strand break yields were found to be (8.2 ± 0.4) and (14.8 ± 0.5)10-12 (g/mol)-1Gy-1 for 80 keV X-rays and 3.5 MeV 241Am α-particles respectively which gives a relative biological effectiveness of 1.8 ± 0.1. (author)

  15. Detection of DNA double-strand breaks in synchronous cultures of CHO cells by means of asymmetric field inversion gel electrophoresis

    A pulsed field gel electrophoresis technique, asymmetric field inversion gel electrophoresis (AFIGE), was used to evaluate induction by X-rays of DNA damage in CHO cells. The fraction of DNA activity released from the plug (FAR) was used as a measure for the amount of radiation-induced DNA damage, predominantly DNA double-strand breaks (dsb) (Stamato and Denko 1990), and was determined at various stages of growth and phases of the cell cycle in a range of doses between zero and 70 Gy. It is concluded that caution needs to be exercised before differences observed in the FAR between different cell lines or between various phases of the cell cycle after exposure to a given dose of radiation are interpreted as suggesting differences in the induction of DNA dsb. (author)

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

    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...

  17. Identification of coagulase-negative staphylococci by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and rRNA restriction patterns.

    Pennington, T H; Harker, C.; Thomson-Carter, F

    1991-01-01

    A total of 1,417 staphylococcal and micrococcal strains were collected from the beards and scalps of 10 subjects over a period of 8 months. Sixteen strains identified as Staphylococcus epidermidis with an API system had distinctive yellow colonies on nutrient agar plates and sodium dodecyl sulfate-polyacrylamide gel electrophoresis whole-cell polypeptide profiles similar to those of Staphylococcus capitis; this identification was confirmed by analysis of rRNA gene restriction patterns.

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

    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...... species difficult to identify by SDS-PAGE or by urea-IEF in the case of cold smoked products. (C) 2000 Elsevier Science Ltd. All rights reserved....

  19. Selective Discrimination of Listeria monocytogenes Epidemic Strains by a Mixed-Genome DNA Microarray Compared to Discrimination by Pulsed-Field Gel Electrophoresis, Ribotyping, and Multilocus Sequence Typing

    Borucki, Monica K.; Kim, So Hyun; Call, Douglas R.; Smole, Sandra C.; Pagotto, Franco

    2004-01-01

    Listeria monocytogenes can cause serious illness in humans, and subsequent epidemiological investigation requires molecular characterization to allow the identification of specific isolates. L. monocytogenes is usually characterized by serotyping and is subtyped by using pulsed-field gel electrophoresis (PFGE) or ribotyping. DNA microarrays provide an alternative means to resolve genetic differences among isolates, and unlike PFGE and ribotyping, microarrays can be used to identify specific g...

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

    HADIWIYONO; JAKA WIDADA; SITI SUBANDIYAH; MARK FEGAN

    2011-01-01

    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 solan...

  1. Temporal study of staphylococcal species on the skin of human subjects in isolation and clonal analysis of Staphylococcus capitis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

    Maggs, A F; Pennington, T H

    1989-01-01

    The staphylococcal skin floras of an isolated group of subjects were studied for 1 year. A wide variation in isolation patterns was found for different species. Staphylococcus intermedius, previously thought to be of veterinary origin, was found to be part of the resident flora of some subjects, and this may indicate a wider role for it in clinical infection. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of S. capitis isolates indicated persistent skin coloniza...

  2. Molecular identification of bacteria from a coculture by denaturing gradient gel electrophoresis of 16S ribosomal DNA fragments as a tool for isolation in pure cultures.

    Teske, A; Sigalevich, P; Cohen, Y.; Muyzer, G.

    1996-01-01

    Molecular information about the bacterial composition of a coculture capable of sulfate reduction after exposure to oxic and microoxic conditions was used to identify and subsequently to isolate the components of the mixture in pure culture. PCR amplification of 16S ribosomal DNA fragments from the coculture, analyzed by denaturing gradient gel electrophoresis, resulted in two distinct 16S ribosomal DNA bands, indicating two different bacterial components. Sequencing showed that the bands wer...

  3. Antimicrobial resistance, virulence-associated genes, and pulsed-field gel electrophoresis profiles of Salmonella enterica subsp. enterica serovar Typhimurium isolated from piglets with diarrhea in Korea

    Hur, Jin; Choi, Yoon Young; Park, Jong Ho; Jeon, Byung Woo; Lee, Hee Soo; Kim, Ae Ran; Lee, John Hwa

    2011-01-01

    Salmonella enterica subsp. enterica serovar Typhimurium was isolated from diarrheic piglets in 2 periods, 2000–2001 (n = 25) and 2005–2006 (n = 17). To compare the characteristics of the isolates collected during the 2 periods, all isolates were tested for antimicrobial resistance, the presence of virulence genes, and pulsed-field gel electrophoresis (PFGE) patterns. All 42 isolates were resistant to at least 1 of the 20 antimicrobials tested, and 39 (93%) were resistant to 2 or more antimicr...

  4. Comparison of an Automated Repetitive-Sequence-Based PCR Microbial Typing System with Pulsed-Field Gel Electrophoresis for Molecular Typing of Vancomycin-Resistant Enterococcus faecium▿ †

    Chuang, Yu-Chung; Wang, Jann-Tay; CHEN, MEI-LING; Chen, Yee-Chun

    2010-01-01

    Vancomycin-resistant Enterococcus faecium (VRE) has become an important health care-associated pathogen because of its rapid spread, limited therapeutic options, and possible transfer of vancomycin resistance to more-virulent pathogens. In this study, we compared the ability to detect clonal relationships among VRE isolates by an automated repetitive-sequence-based PCR (Rep-PCR) system (DiversiLab system) to pulsed-field gel electrophoresis (PFGE), the reference method for molecular typing of...

  5. Dynamics of the microbial community responsible for traditional sour cassava starch fermentation studied by denaturing gradient gel electrophoresis and quantitative rRNA hybridization

    Ampe, Frédéric; Sirvent, A.; Zakhia, N.

    2001-01-01

    The microbial community developing during the spontaneous fermentation of sour cassava starch was investigated by cultivation-independent methods. Denaturing gradient gel electrophoresis (DGGE) of partially amplified 16S rDNA followed by sequencing of the most intense bands showed that the dominant organisms were all lactic acid bacteria (LAB), mainly close relatives of #Bifidobacterium minimum$, #Lactococcus lactis$, #Streptoccocus$ sp., #Enteroccucus saccharolyticus$ and #Lactobacillus plan...

  6. Molecular Fingerprinting by PCR-Denaturing Gradient Gel Electrophoresis Reveals Differences in the Levels of Microbial Diversity for Musty-Earthy Tainted Corks ▿

    Prat, Chantal; Ruiz-Rueda, Olaya; Trias, Rosalia; Anticó, Enriqueta; Capone, Dimitra; Sefton, Mark; Bañeras, Lluís

    2009-01-01

    The microbial community structure of cork with marked musty-earthy aromas was analyzed using denaturing gradient gel electrophoresis of amplified ribosomal DNA. Cork stoppers and discs were used for DNA extraction and were analyzed by using selective primers for bacteria and fungi. Stoppers clearly differed from discs harboring a different fungal community. Moreover, musty-earthy samples of both types were shown to have a specific microbiota. The fungi Penicillium glabrum and Neurospora spp. ...

  7. Influence of Diet on the Proteome of Drosophila Melanogaster as Assessed by Two-Dimensional Gel Electrophoresis and Capillary Liquid Chromatography–Mass Spectrometry: The Hamburger Effect Revisited

    Culwell, Thomas F.; Thulin, Craig D.; Merrell, Karen J.; Graves, Steven W.

    2008-01-01

    Proteomic biomarker discovery has been called into question. Diamandis hypothesized that seemingly trivial factors, such as eating a hamburger, may cause sufficient proteomic change as to confound proteomic differences. This has been termed the hamburger effect. Little is known about the variability of complex proteomes in response to the environment. Two methods—two-dimensional gel electrophoresis (2DGE) and capillary liquid chromatography–electrospray ionization time-of-flight mass spectrom...

  8. High-voltage and gel electrophoresis for the determination of radionuclidic impurities in fission molybdenum (Mo-99) including data processing, documentation- and information system

    The purpose of this work was to find a better method for the determination of radionuclidic impurities in fission molybdenum using high voltage - and gel electrophoresis. Using Whatman chromatography paper could be achieved good results under following conditions: voltage 20 V/cm, electric current 2 mA, electrolyte 0.1 M α-hydroxyisobutiric acid and separation time 30 minutes. Under these conditions, Ru-103 and Br-82 could be separated in 30 minutes. I-131 could be separated also but its separation using the distillation method showed better results. The separation of Ba-140 and La-140 needed 45 minutes. A gel electrophoresis vessel has been designed for the experiment using gel electrophoresis. The experiments have been carried out under following conditions: voltage 30 V/cm, electric current 5 mA, anode electrolyte 0.1 N HCl, cathode electrolyte 0.05 M (NH4)2H-citrate and separation time 4 hours. Under these conditions, Ru-103, Br-82 and I-131 could be separated but Ba-140 and La-140 could not be separated. Two computer programs have been made in this work namely 'Program for Radiochromatography Scanner' and 'Radionuclidic Impurities Determination of Fission Molybdenum'. Microsoft visual basic for MS-DOS and Windows have been used for making these computer programs. These computer programs were very useful for further experiments and they contain data processing, documentation- and information system. (author)

  9. 2D-gel based proteomics unravels neurogenesis and energetic metabolism dysfunction of the olfactory bulb in CUMS rat model.

    Cheng, Ke; Li, Juan; Yang, Deyu; Yang, Yongtao; Rao, Chenglong; Zhang, Shuxiao; Wang, Wei; Guo, Hua; Fang, Liang; Zhu, Dan; Han, Yu; Xie, Peng

    2016-10-15

    Major depression is a devastating psychiatric disease worldwide currently. A reduced olfactory sensitivity in MDD patients was well evidenced. We previously interrogated the mechanism of decreasing hippocampus neurogenesis in CUMS rat model of depression. The Olfactory Bulb (OB) is crucial part of the olfactory system which functions in post-developmental neurogenesis. However, the mechanism of the dysfunction of OB induced by CUMS is still largely unknown. Herein, by using the chronic unpredictable mild stress (CUMS) rat model of depression, differential protein expression between the OB proteomes of CUMS and control group was interrogated through two-dimensional electrophoresis coupling with matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry. Twenty nine differential protein expression was analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway over-representation and Ingenuity pathways analysis (IPA). Seven identified differential proteins were selected for Western blotting validation. This study provides insight that neurogenesis and Energy metabolism disorder is involved in OB dysfunction induced by CUMS. PMID:27340088

  10. Monitoring of microbial community structure and succession in the biohydrogen production reactor by denaturing gradient gel electrophoresis (DGGE)

    XING; Defeng; REN; Nanqi; GONG; Manli; LI; Jianzheng; LI; Q

    2005-01-01

    To study the structure of microbial communities in the biological hydrogen production reactor and determine the ecological function of hydrogen producing bacteria, anaerobic sludge was obtained from the continuous stirred tank reactor (CSTR) in different periods of time, and the diversity and dynamics of microbial communities were investigated by denaturing gradient gel electrophoresis (DGGE). The results of DGGE demonstrated that an obvious shift of microbial population happened from the beginning of star-up to the 28th day, and the ethanol type fermentation was established. After 28 days the structure of microbial community became stable, and the climax community was formed. Comparative analysis of 16S rDNA sequences from reamplifying and sequencing the prominent bands indicated that the dominant population belonged to low G+C Gram-positive bacteria (Clostridium sp. And Ethanologenbacterium sp.), β- proteobacteria (Acidovorax sp.), γ-proteobacteria (Kluyvera sp.), Bacteroides (uncultured bacterium SJA-168), and Spirochaetes (uncultured eubacterium E1-K13), respectively. The hydrogen production rate increased obviously with the increase of Ethanologenbacterium sp., Clostridium sp. And uncultured Spirochaetes after 21 days, meanwhile the succession of ethanol type fermentation was formed. Throughout the succession the microbial diversity increased however it decreased after 21 days. Some types of Clostridium sp. Acidovorax sp., Kluyvera sp., and Bacteroides were dominant populations during all periods of time. These special populations were essential for the construction of climax community. Hydrogen production efficiency was dependent on both hydrogen producing bacteria and other populations. It implied that the co-metabolism of microbial community played a great role of biohydrogen production in the reactors.

  11. Serum sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of patients with membranous nephropathy and focal and segmental glomerulosclerosis.

    Pant, Pragya; Singh, R G; Singh, Santosh K; Singh, Vijay P; Doley, Prodip K; Sivasankar, M

    2016-01-01

    Diagnosis of membranous nephropathy (MN) and focal and segmental glomerulo- sclerosis (FSGS) needs a renal biopsy, which is an invasive procedure with potentially serious complications. Proteomics may be applied for the development of a biomarker for these diseases which will obviate the need of biopsy. Serum sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) analysis gives an idea of the various proteins with different molecular weights (MWs) in a given sample. This study was conducted to analyze proteins with different MWs in patients with MN and FSGS and to compare the two groups with regard to their protein profile. This was a comparative, experimental study performed from June 2013 to July 2014 in the Department of Nephrology, Sir Sunderlal Hospital, Banaras Hindu University, Varanasi. Twenty-three histologically diagnosed cases of primary MN and 25 cases of FSGS were included in the study. Patients were categorized as having mild, moderate, and severe proteinuria with 24 h urinary protein levels of <4, 4- 8 and ≥8 g/24 h, respectively. SDS-PAGE analysis was performed by the method of Laemmli and revealed a significantly higher number of patients with FSGS (80%) having a protein corresponding to 29 kDa MW, than those with MN (39.1%) (P = 0.004). Protein of 5 kDa MW was present in a significantly higher number of patients with moderate (80%) and severe (100%) proteinuria than those with mild proteinuria (25%) (P <0.001). Thus, protein of MW 29 kDa may be a marker for FSGS and needs further characterization. Similarly, 5 kDa protein, present in patients with moderate and severe proteinuria, might be either contributing to or be a marker of severe illness. PMID:27215247

  12. Diversity and dynamics of antibiotic-resistant bacteria in cheese as determined by PCR denaturing gradient gel electrophoresis.

    Flórez, Ana Belén; Mayo, Baltasar

    2015-12-01

    This work reports the composition and succession of tetracycline- and erythromycin-resistant bacterial communities in a model cheese, monitored by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Bacterial 16S rRNA genes were examined using this technique to detect structural changes in the cheese microbiota over manufacturing and ripening. Total bacterial genomic DNA, used as a template, was extracted from cultivable bacteria grown without and with tetracycline or erythromycin (both at 25 μg ml(-1)) on a non-selective medium used for enumeration of total and viable cells (Plate Count agar with Milk; PCA-M), and from those grown on selective and/or differential agar media used for counting various bacterial groups; i.e., lactic acid bacteria (de Man, Rogosa and Sharpe agar; MRSA), micrococci and staphylococci (Baird-Parker agar; BPA), and enterobacteria (Violet Red Bile Glucose agar; VRBGA). Large numbers of tetracycline- and erythromycin-resistant bacteria were detected in cheese samples at all stages of ripening. Counts of antibiotic-resistant bacteria varied widely depending on the microbial group and the point of sampling. In general, resistant bacteria were 0.5-1.0 Log10 units fewer in number than the corresponding susceptible bacteria. The PCR-DGGE profiles obtained with DNA isolated from the plates for total bacteria and the different bacterial groups suggested Escherichia coli, Lactococcus lactis, Enterococcus faecalis and Staphylococcus spp. as the microbial types resistant to both antibiotics tested. This study shows the suitability of the PCR-DGGE technique for rapidly identifying and tracking antibiotic resistant populations in cheese and, by extension, in other foods. PMID:26241491

  13. Identification of ataxia telangiectasia heterozygotes, a cancer-prone population, using the single-cell gel electrophoresis (Comet) assay.

    Djuzenova, C S; Schindler, D; Stopper, H; Hoehn, H; Flentje, M; Oppitz, U

    1999-06-01

    Heterozygotes of ataxia telangiectasia (AT) may comprise up to 1% of the general population. Because these individuals have no clinical expression of AT but may be highly radiosensitive and strongly predisposed for several forms of cancer, identification of AT carriers represents a considerable interest in cancer epidemiology and radiotherapy. We report a new approach for the in vitro identification of AT-heterozygotes based on the evaluation of the radiosensitivity and DNA damage repair ability of peripheral blood mononuclear cells using the single-cell gel electrophoresis (Comet) assay. The assay was performed on cells isolated from four different groups of individuals: (1) apparently healthy donors (n = 10); (2) patients with breast cancer showing a normal reaction to radiotherapy (n = 10); (3) a group of obligate AT carriers (parents of AT-homozygotes, n = 20); and (4) AT-homozygotes (n = 4). Cells irradiated with 3 Gy of x-rays were assayed for three parameters: (1) the initial and (2) residual DNA damage and (3) the kinetics of DNA damage repair. Both AT-heterozygotes' and AT-homozygotes' cells were found to be highly sensitive to x-irradiation. Quantitative evaluation of the single-cell electrophoregrams revealed that the average initial DNA damage in AT-heterozygous and AT-homozygous cells was almost three times higher than that in control non-AT cells. In addition, the DNA repair process in irradiated AT carrier cells was almost three times slower, and the extent of irreparable DNA damage in these cells was three times greater than in controls. Simultaneous assessment of the three parameters enabled correct identification of all tested AT carriers. This method seems to be a sensitive and useful tool for populational studies as a rapid prescreening test for a mutated AT status. The approach can also be extended for prediction of the in vivo radiosensitivity, which would enable optimization of individual radiotherapy schedules. PMID:10378512

  14. Bulk and rhizosphere soil bacterial communities studied by denaturing gradient gel electrophoresis: plant-dependent enrichment and seasonal shifts revealed.

    Smalla, K; Wieland, G; Buchner, A; Zock, A; Parzy, J; Kaiser, S; Roskot, N; Heuer, H; Berg, G

    2001-10-01

    The bacterial rhizosphere communities of three host plants of the pathogenic fungus Verticillium dahliae, field-grown strawberry (Fragaria ananassa Duch.), oilseed rape (Brassica napus L.), and potato (Solanum tuberosum L.), were analyzed. We aimed to determine the degree to which the rhizosphere effect is plant dependent and whether this effect would be increased by growing the same crops in two consecutive years. Rhizosphere or soil samples were taken five times over the vegetation periods. To allow a cultivation-independent analysis, total community DNA was extracted from the microbial pellet recovered from root or soil samples. 16S rDNA fragments amplified by PCR from soil or rhizosphere bacterium DNA were analyzed by denaturing gradient gel electrophoresis (DGGE). The DGGE fingerprints showed plant-dependent shifts in the relative abundance of bacterial populations in the rhizosphere which became more pronounced in the second year. DGGE patterns of oilseed rape and potato rhizosphere communities were more similar to each other than to the strawberry patterns. In both years seasonal shifts in the abundance and composition of the bacterial rhizosphere populations were observed. Independent of the plant species, the patterns of the first sampling times for both years were characterized by the absence of some of the bands which became dominant at the following sampling times. Bacillus megaterium and Arthrobacter sp. were found as predominant populations in bulk soils. Sequencing of dominant bands excised from the rhizosphere patterns revealed that 6 out of 10 bands resembled gram-positive bacteria. Nocardia populations were identified as strawberry-specific bands. PMID:11571180

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

    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 gamma-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)

  16. 2DBase: 2D-PAGE database of Escherichia coli

    Vijayendran, Chandran; Burgemeister, Sebastian; Friehs, Karl; Niehaus, Karsten; Flaschel, Erwin

    2007-01-01

    We present a web-based integrated proteome database, termed 2DBase of Escherichia coli which was designed to store, compare, analyse, and retrieve various information obtained by 2D polyacrylamide gel electrophoresis and mass spectrometry. The main objectives of this database are (1) to provide the features for query and data-mining applications to access the stored proteomics data (2) to efficiently compare the specific protein spots present in the comparable proteome maps and (3) to analyse...

  17. Synthesis of 1D, 2D, and 3D ZnO Polycrystalline Nanostructures Using the Sol-Gel Method

    Yung-Kuan Tseng

    2012-01-01

    Full Text Available This study employed various polyol solvents to synthesize zinc oxide polycrystalline nanostructures in the form of fibers (1D, rhombic flakes (2D, and spheres (3D. The synthetic process primarily involved the use of zinc acetate dihydrate in polyol solutions, which were used to derive precursors of zinc alkoxides. Following hydrolysis at 160°C, the zinc alkoxide particles self-assembled into polycrystalline nanostructures with different morphologies. Following calcination at 500°C for 1 h, polycrystalline ZnO with good crystallinity was obtained. FE-SEM explored variations in surface morphology; XRD was used to analyze the crystalline structures and crystallinity of the products, which were confirmed as ZnO wurtzite structures. FE-TEM verified that the ZnO nanostructures were polycrystalline. Furthermore, we employed TGA/DSC to observe the phase transition. According to the results of property analyses, we proposed models of the relevant formation mechanisms. Finally, various ZnO structures were applied in the degradation of methylene blue to compare their photocatalytic efficiency.

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

    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.

  19. 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

    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 125I, 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

  20. DNA double-strand break measurement in mammalian cells by pulsed-field gel electrophoresis: an approach using restriction enzymes and gene probing

    DNA samples prepared from human SP3 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)

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

    Rehbein, H.; Kundiger, R.; Yman, I.M.; Ferm, M.; Etienne, M.; Jerome, M.; Craig, A.; Mackie, I.; Jessen, Flemming; Martinez, I.; Mendes, R.; Smelt, A.; Luten, J.; Pineiro, C.; Perez-Martin, R.

    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...

  2. Recent advances in preparative electrophoresis

    Mosher, Richard A.; Thormann, Wolfgang; Egen, Ned B.; Couasnon, Pascal; Sammons, David W.

    1987-01-01

    Various approaches for preparative electrophoresis, and three new instruments for preparative electrophoresis are discussed. Consideration is given to isoelectric focusing, isotachophoresis, and zone electrophoresis, three gel-based electrophoresis methods. The design, functions, and performance of the Elphor VaP 21 device of Hannig (1982), the shear-stabilized BIOSTREAM separator of Thompson (1983), and the recycling isoelectric focusing device are described.

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

    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

  4. Simplification and improvement of protein detection in two-dimensional electrophoresis gels with SERVA HPE™ lightning red.

    Griebel, Anja; Obermaier, Christian; Westermeier, Reiner; Moche, Martin; Büttner, Knut

    2013-07-01

    A new fluorescent amino-reactive dye has been tested for both labelling proteins prior to electrophoretic separations and between the two steps of two-dimensional electrophoresis. A series of experiments showed, that the labelling of lysines with this dye is compatible with all standard additives used for sample preparation, including reducing substances and carrier ampholytes. Using this dye for pre-labelling considerably simplifies the electrophoresis and detection workflow and provides highly sensitive and quantitative visualisation of proteins. PMID:23786184

  5. Exploitation of the Dose/Time-Response Relationship for a New Measure of DNA Repari in the Single-Cell Gel Electrophoresis (Comet) Assay

    The comet assay (also called the single-cell gel electrophoresis assay) has been widely used for detecting DNA damage and repair in individual cells. Since the conventional methods of evaluating comet assay data using frequency statistics are unsatisfactory we developed a new quantitative measure of DNA damage/repair that is based on all information residing in the dose/time-response curves of a comet experiment. Blood samples were taken from 25 breast cancer patients before undergoing radiotherapy. The comet assay was performed under alkaline conditions using isolated lymphocytes

  6. A subtle calculation method for nanoparticle’s molar extinction coefficient: The gift from discrete protein-nanoparticle system on agarose gel electrophoresis

    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.

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

    Hammerum, Anette Marie; Fussing, Vivian; Aarestrup, Frank Møller; Wegener, Henrik Caspar

    2000-01-01

    isolates yielded 40 types whereas PFGE-typing yielded 57 types discriminated by differences in more than three bands. By molecular typing, both clonal spread of E. faecium as well as horizontal transmission of Tn1546 between animals and humans was supported. Furthermore, it was found that the population of......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...

  8. Sensitive and simultaneous analysis of five transgenic maizes using multiplex polymerase chain reaction, capillary gel electrophoresis, and laser-induced fluorescence

    García-Cañas, Virginia; González García, Ramón; Cifuentes, Alejandro

    2004-01-01

    The benefits of using multiplex polymerase chain reaction (PCR) followed by capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) for the simultaneous detection of five transgenic maizes (Bt11, T25, MON810, GA21, and Bt176) are demonstrated. The method uses a hexaplex PCR protocol to amplify the five mentioned transgenic amplicons plus the zein gene used as reference, followed by a CGE-LIF method to analyze the six DNA fragments. CGE-LIF was demonstrated very useful and info...

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

    Shaw, A; Christiansen, Gunna; Birkelund, Svend

    1999-01-01

    Chlamydia trachomatis is an obligate intracellular bacterium causing human ocular and genital disease. The lymphokine interferon gamma (IFN-gamma) is an important immune effector exerting antimicrobial effects towards several intracellular parasites, the chlamydia included. IFN-gamma has been......]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...

  10. Random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE) and phage-typing in the analysis of a hospital outbreak of Salmonella enteritidis

    Skibsted, U.; Baggesen, Dorte Lau; Dessau, R.;

    1998-01-01

    Isolates of Salmonella Enteritidis from 81 patients from Herlev Hospital or from Copenhagen County were analysed by random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE) and phage-typing. Fourteen polymorphic markers from five decamer primers unambiguously placed...... (both PFGE type I) formed the fifth group. The sixth group of four isolates was not phage typeable and was PFGE type III. Forty outbreak-related isolates of phage-type 6 were resolved into three strains. No diversity of phage-type 6 was found among isolates unrelated to the outbreak. It is concluded...

  11. Comparison of protein patterns after two-dimensional gel electrophoresis from leaves of in vitro cultures and seedlings of Rubus chamaemorus L.

    Barbara Thiem

    2014-02-01

    Full Text Available Proteins from leaves of Rubus chamaemorus propagated in vitro were subjected to miniaturized 2-D electrophoresis. The 2-DE patterns of proteins showed qualitative differences between plants propagated in vitro and control seedlings. More proteins of a high molecular weight were observed in leaves of plants from in vitro culture. A two-dimensional map of proteins from leaves provides detailed data concerning both polymorphism and protein patterns of this species. This makes it possible to start constructing a protein map of R. chamaemorus. The reasons for qualitative differences are discussed.

  12. The selective fabrication of large-area highly ordered TiO2 nanorod and nanotube arrays on conductive transparent substrates via sol-gel electrophoresis

    Large-area free-standing arrays of TiO2 nanorods and nanotubes were selectively synthesized on transparent conducting indium tin oxide substrates using sol-gel electrophoresis and anodic alumina (AAO) thin film templates. The effect of sol-gel ageing on the growth of TiO2 was explained, providing a tailored ability to produce nanotubes and nanorods. An annular tungsten base electrode, stemming from the anodization of the AAO template, was found to be crucial to the growth of nanotubes. This was supported by a study of substrate annealing in a reducing atmosphere. The work can be readily adapted for the fabrication of free-standing arrays of other metal, metal oxide, and complex oxide nanorod and nanotube arrays on conducting substrates.

  13. Proteins pattern alteration in AZT-treated K562 cells detected by two-dimensional gel electrophoresis and peptide mass fingerprinting

    Mignogna Giuseppina

    2006-03-01

    Full Text Available Abstract In this study we report the effect of AZT on the whole protein expression profile both in the control and the AZT-treated K562 cells, evidenced by two-dimensional gel electrophoresis and peptide mass fingerprinting analysis. Two-dimensional gels computer digital image analysis showed two spots that appeared up-regulated in AZT-treated cells and one spot present only in the drug exposed samples. Upon extraction and analysis by peptide mass fingerprinting, the first two spots were identified as PDI-A3 and stathmin, while the third one was proved to be NDPK-A. Conversely, two protein spots were present only in the untreated K562 cells, and were identified as SOD1 and HSP-60, respectively.

  14. Phorbol diester-induced phosphorylation of nuclear matrix proteins in HL60 promyelocytes. Possible role in differentiation studied by cationic detergent gel electrophoresis

    Immortal HL60 promyelocytes are induced to differentiate to mortal adherent cells by a variety of agents which activate protein kinase C, including 12-O-tetradecanoylphorbol 13-acetate (TPA). In order to investigate the mechanism of this effect, we incubated HL60 cells with [32P]orthophosphate with or without TPA and extracted their proteins with the cationic detergent benzyldimethyl-n-hexadecylammonium chloride prior to electrophoresis in a discontinuous polyacrylamide gel system in the first dimension. In this system, proteins migrate toward the cathode as a function of their molecular weight, and they are separated from other radioactive components which can obscure the pattern of protein phosphorylation on sodium dodecyl sulfate (SDS) gels. SDS gel electrophoresis was used in the second dimension, resulting in the clear resolution of a large number of proteins. TPA caused many changes in the pattern of protein phosphorylation in intact cells. Two proteins which prominently increased their incorporation of 32P were investigated in particular, and they were both found to be retained in the nuclear matrix following successive extraction of cells with Triton, digestion with DNase and RNase, and extraction with 2 M NaCl. These proteins migrated with apparent molecular weights of 80,000 and 33,000 on SDS gels, and are designated NP80 and NP33, respectively. NP80 was half-maximally phosphorylated after 7 min exposure to TPA, and half-maximally phosphorylated by 10 nM TPA. NP80 co-migrated with a faint Coomassie Blue-stained protein, and NP33 co-migrated with a more prominent protein. Several proteins incorporated less 32P when the cells were exposed to TPA, including one which was extracted from nuclei with the core histones and which co-migrated with histone H2A

  15. Protein mapping by combined 2-D electrophoresis and mass spectrometry: an approach to identify the proteome changes in muscle of diabetic rats and treatment with Cynodon dactylon L

    Sivanesan Ravikumar

    2012-02-01

    Full Text Available Objective: A proteomics approach used to understand the diabetes mellitus-related changes in muscle proteome composition during diabetes mellitus and treatment with Cynodon dactylon. This approach may provide way to investigate the muscle function associated with diabetes and metabolic protein mechanism responsible for this disease and also new targets of diabetes diagnosis and effective treatment. Methods: We used comparative proteomic techniques to identify muscle proteins associated with diabetes mellitus in alloxan-induced rats by using 2-dimensional electrophoresis (2-DE, matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF mass spectrometry (MS and peptide mass fingerprint (PMF analysis. The rats (four groups; six animals each group were made diabetic by single intraperitoneal (i.p. alloxan injection (150 mg/kg b.w. After 72 h alloxan injection, the treatment was with C.dactylon leaf extract for 15 days. The differential protein expressions were analyzed in alloxan-induced diabetic rats and those treated with C.dactylon leaf extract. Results: In this experiment, two proteins, i.e. fructose-bis-phosphate aldolase and glyceraldehyde-3-phosphate dehydrogenase were up-regulated in diabetic treated group. Conclusion: This result suggests that, up-regulated proteins were involved in glucose metabolism and also in transcriptional control of nuclear membrane fusion. These proteins were up-regulated by the effect of C.dactylon extract in muscle tissues playing a critical role in glucose metabolism which in turn control and reduce the severity of diabetes mellitus. [J Exp Integr Med 2012; 2(1.000: 61-70

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

    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.

  17. Protection of DNA by herb mixture in HL-60 cells exposed to γ-rays; analysed by micronuclei formation and single cell gel electrophoresis

    A herb mixture(Paeonia Radix, Cnidii Rhizoma and Angelica gigantis Radix; HIM-I) was designed to protect gastrointestine, hematopoietic organs and immune system against radiation damage. In the present study, the protective effect of HIM-I and P.P-I(water extract of HIM-I added with 20% of its polysaccaride fraction) on DNA damage in HL-60 cells exposed to 60Co γ-rays was evaluated using micronuclei formtion and alkaline single cell gel electrophoresis(comet assay). The frequency of micronuclei was decreased in groups treated with water extract, methanol fraction, polysaccaride fraction and P.P-I before exposure to 200 cGy of γ-rays. In alkaline single cell gel electrophoresis, the DNA migration was decreased in groups treated with water extract, methanol fraction, polysaccaride fraction and P.P-I before exposure to 50, 100 and 200 cGy of γ-rays. These results indicated that HIM-I and P.P-I might protect DNA damage induced by γ-rays. Therefore, HIM-I and P.P-I might be a useful radioprotector, especially since it is a relatiely nontoxic product

  18. 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

    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.

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

    Mackie, I.; Craig, A.; Etienne, M.; Jerome, M.; Fleurence, J.; Jessen, Flemming; Smelt, A.; Kruijt, A.; Yman, I.M.; Ferm, M.; Martinez, I.; Perez-Martin, R.; Pineiro, C.; Rehbein, H.; Kundiger, R.

    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 on...

  20. Random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE) and phage-typing in the analysis of a hospital outbreak of Salmonella enteritidis

    Skibsted, U.; Baggesen, Dorte Lau; Dessau, R.; Lisby, G.

    1998-01-01

    Isolates of Salmonella Enteritidis from 81 patients from Herlev Hospital or from Copenhagen County were analysed by random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE) and phage-typing. Fourteen polymorphic markers from five decamer primers unambiguously placed...... that RAPD is useful as a tool in investigations of microbial outbreaks in its own right, or to supplement phage-typing and PFGE of Salmonella Enteritidis.......Isolates of Salmonella Enteritidis from 81 patients from Herlev Hospital or from Copenhagen County were analysed by random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE) and phage-typing. Fourteen polymorphic markers from five decamer primers unambiguously placed...... (both PFGE type I) formed the fifth group. The sixth group of four isolates was not phage typeable and was PFGE type III. Forty outbreak-related isolates of phage-type 6 were resolved into three strains. No diversity of phage-type 6 was found among isolates unrelated to the outbreak. It is concluded...

  1. Comparison of ethanol-soluble proteins from different rye (Secale cereale) varieties by two-dimensional electrophoresis

    Radzikowski, Louise; Nesic, Ljiljana; Hansen, H.B.; Jacobsen, Susanne; Søndergaard, Ib

    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...... 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, that the...... 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...

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

    Rehbein, H.; Kundiger, R.; Yman, I.M.; Ferm, M.; Etienne, M.; Jerome, M.; Craig, A.; Mackie, I.; Jessen, Flemming; Martinez, I.; Mendes, R.; Smelt, A.; Luten, J.; Pineiro, C.; Perez-Martin, R.

    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...... incorrect, and with SDS-PAGE a similar result was obtained. It was concluded that methods, as now developed, are suitable for checking the species declaration of fishery products. (C) 1999 Elsevier Science Ltd. All rights reserved...

  3. Combining blue native polyacrylamide gel electrophoresis with liquid chromatography tandem mass spectrometry as an effective strategy for analyzing potential membrane protein complexes of Mycobacterium bovis bacillus Calmette-Guérin

    Li Weijun

    2011-01-01

    Full Text Available Abstract Background Tuberculosis is an infectious bacterial disease in humans caused primarily by Mycobacterium tuberculosis, and infects one-third of the world's total population. Mycobacterium bovis bacillus Calmette-Guérin (BCG vaccine has been widely used to prevent tuberculosis worldwide since 1921. Membrane proteins play important roles in various cellular processes, and the protein-protein interactions involved in these processes may provide further information about molecular organization and cellular pathways. However, membrane proteins are notoriously under-represented by traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE and little is known about mycobacterial membrane and membrane-associated protein complexes. Here we investigated M. bovis BCG by an alternative proteomic strategy coupling blue native PAGE to liquid chromatography tandem mass spectrometry (LC-MS/MS to characterize potential protein-protein interactions in membrane fractions. Results Using this approach, we analyzed native molecular composition of protein complexes in BCG membrane fractions. As a result, 40 proteins (including 12 integral membrane proteins, which were organized in 9 different gel bands, were unambiguous identified. The proteins identified have been experimentally confirmed using 2-D SDS PAGE. We identified MmpL8 and four neighboring proteins that were involved in lipid transport complexes, and all subunits of ATP synthase complex in their monomeric states. Two phenolpthiocerol synthases and three arabinosyltransferases belonging to individual operons were obtained in different gel bands. Furthermore, two giant multifunctional enzymes, Pks7 and Pks8, and four mycobacterial Hsp family members were determined. Additionally, seven ribosomal proteins involved in polyribosome complex and two subunits of the succinate dehydrogenase complex were also found. Notablely, some proteins with high hydrophobicity or multiple transmembrane

  4. Serum proteins analysis by capillary electrophoresis

    Uji, Yoshinori; Okabe, Hiroaki

    2001-01-01

    The purpose of this study was to evaluate the efficacy of multi-capillary electrophoresis instrument in clinical laboratory. An automated clinical capillary electrophoresis system was evaluated for performing serum proteins electrophoresis and immuno-fixation electrophoresis by subtraction. In this study the performance of capillary electrophoresis was compared with the cellulose acetate membrane electrophoresis and agarose gel immunofixation electrophoresis for serum proteins. The results of...

  5. Antibody-linked polymerase assay on protein blots: a novel method for identifying polymerases following SDS-polyacrylamide gel electrophoresis.

    van der Meer, J.; Dorssers, L; Zabel, P

    1983-01-01

    We describe a method for correlating polymerase activity with a particular polypeptide band in an SDS-polyacrylamide gel which does not require renaturation of the SDS-denatured enzyme. The method involves the following steps: (i) transfer of proteins from an SDS-polyacrylamide gel onto nitrocellulose; (ii) incubation with excess antiserum raised against a partially purified polymerase preparation to link one Fab site of an antibody molecule to the denatured enzyme on the nitrocellulose; (iii...

  6. Proteomics of renal disorders: Urinary proteome analysis by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry

    Kumar, Yadunanda; Venkata Uppuluri, Nageshwar Rao; Babu, Kishore; Phadke, Kishore; Kumar, Prasanna; Ballal, Sudarshan; Tatu, Utpal

    2002-01-01

    The proteomes of urinary samples from patients with different renal conditions were analysed by two dimensional electrophoresis and MALDI-TOF technology. Samples from three different renal conditions, namely kidney failure, nephrotic syndrome and microalbuminuria, were included in the analysis. Apart from the presence of albumin, the profiles of protein spots found in these urine samples were quite distinct. While kidney failure patients showed predominantly low molecular weight proteins, the...

  7. Efficient extraction of proteins from recalcitrant plant tissue for subsequent analysis by two-dimensional gel electrophoresis.

    Parkhey, Suruchi; Chandrakar, Vibhuti; Naithani, S C; Keshavkant, S

    2015-10-01

    Protein extraction for two-dimensional electrophoresis from tissues of recalcitrant species is quite problematic and challenging due to the low protein content and high abundance of contaminants. Proteomics in Shorea robusta is scarcely conducted due to the lack of a suitable protein preparation procedure. To establish an effective protein extraction protocol suitable for two-dimensional electrophoresis in Shorea robusta, four procedures (borate buffer/trichloroacetic acid extraction, organic solvent/trichloroacetic acid precipitation, sucrose/Tris/phenol, and organic solvent/phenol/sodium dodecyl sulfate) were evaluated. Following these, proteins were isolated from mature leaves and were analyzed for proteomics, and also for potential contaminants, widely reported to hinder proteomics. The borate buffer/trichloroacetic acid extraction had the lowest protein yield and did not result in any banding even in one-dimensional electrophoresis. In contrast, organic solvent/phenol/sodium dodecyl sulfate extraction allowed the highest protein yield. Moreover, during proteomics, organic solvent/phenol/sodium dodecyl sulfate extracted protein resolved the maximum number (144) of spots. Further, when proteins were evaluated for contaminants, significant (77-95%) reductions in the nucleic acids, phenol, and sugars were discernible with refinement in extraction procedure. Accumulated data suggested that the organic solvent/phenol/sodium dodecyl sulfate extraction was the most effective protocol for protein isolation for proteomics of Shorea robusta and can be used for plants that have a similar set of contaminants. PMID:26257211

  8. Inhibition of H2O2-induced DNA damage in single cell gel electrophoresis assay (comet assay by castasterone isolated from leaves of centella asiatica

    Nishi Sondhi

    2010-06-01

    Full Text Available Brassinosteroids (BRs are a large group of polyhydroxy steroids, which regulate numerous aspects of plant growth and development, including stem elongation, leaf bending, tracheary element differentiation, stress protection and photomorphogenesis. Recent studies indicate antigenotoxic and anticancerous activities of these compounds. The role of natural BRs in H2O2 (hydrogen peroxide -induced DNA damage in human lymphocytes is still unknown. The present study reports the presence of Castasterone from leaves of Centella asiatica, an important medicinal herb commonly used as a memory enhancer and immunomodulator. CA50 fraction isolated from Centella asiatica was characterized as Castasterone by electrospray ionization mass spectral data with standard Castasterone. An attempt has been made to study antigenotoxic activity of the isolated Castasterone against H2O2 -induced DNA damage in human blood lymphocytes using Single cell gel electrophoresis assay (Comet Assay. Castasterone at 10–9 M concentration proved to be effective in diminishing the DNA damage by 89.42 %.

  9. ASTRO Research Fellow Presentation - A comparison of the comet assay and pulsed-field gel electrophoresis as a predictive assay for radiosensitivity in human fibroblasts

    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 60Co 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

  10. Multiplex polymerase chain reaction-capillary gel electrophoresis: a promising tool for GMO screening--assay for simultaneous detection of five genetically modified cotton events and species.

    Nadal, Anna; Esteve, Teresa; Pla, Maria

    2009-01-01

    A multiplex polymerase chain reaction assay coupled to capillary gel electrophoresis for amplicon identification by size and color (multiplex PCR-CGE-SC) was developed for simultaneous detection of cotton species and 5 events of genetically modified (GM) cotton. Validated real-time-PCR reactions targeting Bollgard, Bollgard II, Roundup Ready, 3006-210-23, and 281-24-236 junction sequences, and the cotton reference gene acp1 were adapted to detect more than half of the European Union-approved individual or stacked GM cotton events in one reaction. The assay was fully specific (labeling in one step. Thus, it is an easy and inexpensive tool for initial screening, to be complemented with quantitative assays if necessary. PMID:19610365

  11. Investigation of an outbreak of human salmonellosis caused by Salmonella enterica ssp enterica serovar Infantis by use of pulsed field gel electrophoresis

    Wegener, Henrik Caspar; Baggesen, Dorte Lau

    Analysis of chromosomal DNA restriction patterns produced by pulsed field gel electrophoresis (PFGE) was used to investigate an outbreak of human salmonellosis caused by Salmonella enterica ssp. enterica serovar Infantis (S. Infantis) involving more than 500 registered human cases. The outbreak had...... been tentatively traced back to a single pig slaughterhouse. A total of 135 isolates from various sources produced 21 different PFGE patterns with the restriction endonuclease XbaI. All human isolates from the outbreak belonged to a single type, the 'EPI-type', whereas human isolates recovered before...... and after the outbreak belonged to several different types. All isolates investigated from the suspect pig slaughterhouse and its supplier pig herds belonged to the EPI-type. Isolates from pork from the central meat market in Copenhagen, which received most of the carcasses from the suspect...

  12. Analysis of phosphate-accumulating organisms cultivated under different carbon sources with polymerase chain reaction-denaturing gradient gel electrophoresis assay

    YU Shui-li; LIU Ya-nan; JING Guo-lin; ZHAO Bing-jie; GUO Si-yuan

    2005-01-01

    To investigate the microbial communities of microorganisms cultivated under different carbon sources, three sequencing batch reactors were operated. They were supplied with sewage, glucose and sodium acetate as carbon sources respectively and showed high phosphorus removal performance. The results of denaturing gradient gel electrophoresis(DGGE) of polymerase chain reaction-amplified (PCR) 16S rDNA fragments demonstrated that β-protebacteria, Actinomyces sp. and γ-protebacteria only exited in 1 # reactor. The microbiological diversity of 1 # reactor exceeded the other two reactors. Flavobacterium, Bacillales, Actinomyces, Actinobacteridae and uncultured bacteria(AF527584, AF502204, AY592749, AB076862, AJ619051, AF495454 and AY133070) could be detected in the biological phosphorus removal reactors.

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

    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.

  14. Pulsed-field gel electrophoresis, virulence determinants and antimicrobial susceptibility profiles of type Ia group B streptococci isolated from humans in Brazil

    Ana Beatriz de A Corrêa

    2009-07-01

    Full Text Available Group B streptococci (GBS infections occur worldwide. Although serotyping has been used for epidemiologic purposes, this does not accurately characterize enough members of a genetically heterogeneous bacterial population. The aims of this work were to evaluate the genetic diversity of 45 type Ia GBS strains isolated in Brazil by pulsed-field gel electrophoresis as well as to evaluate antimicrobial susceptibility profiles and identify virulence genes. Twenty-four strains were assigned to cluster A. All strains under study contained the hylB and scpB genes. The bca gene was detected in only 10 strains and none of the streptococci carried the bac gene. Thirty-nine strains were resistant to tetracycline.

  15. 2-D GEL ELECTROPHORESIS MAP OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS TREATED WITH QUERCUS INFECTORIA GALL EXTRACT

    Dayang Fredalina Basri; Lee Seng Aik; Radhiah Khairon; Mariati Abdul Rahman

    2013-01-01

    The widespread outbreak of Methicillin-Resistant Staphylococcus Aureus (MRSA) has caused clinical and epidemiological concern in hospital environment. The emergence of Vancomycin-Intermediate S. Aureus (VISA) and, more recently, Vancomycin-Resistant S. Aureus (VRSA) has further alarmed clinician and scientist worlwide. The objective of this study is to determine the optimum concentration of sample protein from MRSA after treatment with acetone extract from Quercus infectoria gall. Comparison ...

  16. Effect of bromodeoxyuridine on radiation-induced DNA damage and repair based on DNA fragment size using pulsed-field gel electrophoresis

    We have used biphasic linear ramping pulsed-field gel electrophoresis (PFGE) to understand the effect of incorporation of bromodeoxyuridine (BrdUrd) on radiation-induced DNA damage and repair. This technique permits a determination of the fragment size distribution produced immediately after irradiation as well as during the repair period. We found that incorporation of BrdUrd increased the induction and decreased the repair of radiation damage. The fragment size distribution was consistent with a random breakage model. When we found that significantly more damage was detected after irradiation of deproteinized DNA compared to intact cells, we studied the effects of BrdUrd incorporation on the radiation response of cells or DNA at various phases of preparation for electrophoresis: cells adherent to the culture dish (A), trypsinized cells (B), agarose-embedded cells (C) and deproteinized DNA (D). Although there was a general tendency to detect more damage when irradiation was performed later in the preparation process, steps B and C were the only successive steps which were significantly different. These findings demonstrate that incorporation of BrdUrd randomly increases the induction of radiation damage and decreases its repair at the level of 200 kbp to 5 Mbp fragments. Furthermore, they confirm that the amount of damage detected depends upon the conditions of the cells or DNA at the time of irradiation. 34 refs., 5 figs., 2 tabs

  17. [Urine protein analysis with the sodium-dodecyl-sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE) in healthy cats and cats with kidney diseases].

    Meyer-Lindenberg, A; Wohlsein, P; Trautwein, G; Nolte, I

    1997-03-01

    In this investigation, the value of urine protein analysis by means of molecular-weight related sodium dodecyl-polyacryl gradient gel electrophoresis (SDS-PAGE) was examined with regard to its applicability and diagnostic significance in nephropathy in the cat. A total of 87 cats was included in the study, 30 of them that were clinically healthy served as the control group. The urine protein pattern of this group had, besides the band representing the market albumin, and additional broad band within the size of the marker transferrin. In some cases, weak bands were present within the range of the Tamm-Horsfall-protein and immunoglobulin G. Micromolecular protein bands were not demonstrable. The remaining 57 animals had a histologically proven nephropathy. Thirty-eight cats had elevated urea and/or creatinine values in the plasma (group 1), and 19 animals had values within the reference range (group 2). The urine protein pattern as evidenced by SDS-urine electrophoresis was altered in all cats with histologically proven nephropathy, and it is thus concluded that with this technique a nephropathy can be diagnosed very early and prior to changes of plasma urea and creatinine (group 2). Moreover, in most of the cases, the nephrological changes can be classified as glomerular or tubulo-interstitial (group 1 and group 2). However, it is not possible to draw exact conclusions concerning the underlying morphological changes, nor can the severity of the disease be correctly assessed. PMID:9123982

  18. Polyclonal infections due to Mycobacterium avium complex in patients with AIDS detected by pulsed-field gel electrophoresis of sequential clinical isolates.

    Slutsky, A M; Arbeit, R D; Barber, T W; Rich, J; von Reyn, C F; Pieciak, W; Barlow, M A; Maslow, J N

    1994-01-01

    Invasive infection with organisms of the Mycobacterium avium complex (MAC) is common among patients with advanced human immunodeficiency virus infection. In previous studies, we analyzed multiple individual colonies of MAC isolated from specimens obtained at the same time and observed that 14 to 20% of patients are simultaneously infected with more than one strain. In this study, we examined sequential isolates from 12 patients with AIDS who had two or more MAC isolates available from clinical specimens collected more than 1 week apart; the intervals between the first and last specimens ranged from 8 to 192 (median, 46) days. For each isolate, restriction digests of genomic DNA were analyzed by pulsed-field gel electrophoresis; DNA was prepared by using a protocol, described here in detail, which had been optimized for conditions of bacterial growth and lysis. The pulsed-field gel electrophoresis analysis identified four patients (33%) infected with two different MAC strains. Both M. avium and M. intracellulare were cultured from blood specimens from two patients. In each of the four patients, the second strain was identified from a culture taken within 14 days of the initial study isolate, and in three of these patients, the first strain was detected again in a subsequent culture. These observations suggest that the presence of two different strains among isolates from sequential cultures may reflect ongoing polyclonal infection. We conclude that polyclonal infection with MAC is common among patients with AIDS. The identification of such infections may be critical in the development of effective treatments. Images PMID:7929773

  19. Protein composition of wheat gluten polymer fractions determined by quantitative two-dimensional gel electrophoresis and tandem mass spectrometry

    Flour proteins from the US bread wheat Butte 86 were extracted in 0.5% SDS using a two-step procedure with and without sonication and further separated by size exclusion chromatography into monomeric and polymeric fractions. Proteins in each fraction were analyzed by quantitative two-dimensional gel...

  20. 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

    Shaw, AC; Rossel Larsen, M; Roepstorff, P; Holm, A; Christiansen, Gunna; Birkelund, Svend

    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...... and future data accessible for interlaboratory comparison, we constructed a 2-D PAGE database on the World Wide Web....... 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 these...