Sample records for gel electrophoresis 2-d

  1. A Spot Segmentation Approach for 2D Gel Electrophoresis Images Based on 2D Histograms

    E-print Network

    Athens, University of

    A Spot Segmentation Approach for 2D Gel Electrophoresis Images Based on 2D Histograms Eleni@bioacademy.gr Abstract Spot-Segmentation, an essential stage of processing 2D gel electrophoresis images, remains approach to spot segmentation in 2D gel electrophoresis images. The proposed approach is based on 2D

  2. INDEPENDENT COMPONENT ANALYSIS OF SIMULATED 2D ELECTROPHORESIS GELS

    E-print Network

    Adali, Tulay

    INDEPENDENT COMPONENT ANALYSIS OF SIMULATED 2D ELECTROPHORESIS GELS Nicolle Correat, Haleh Safavit differentially expressed pro- teins in simulated two-dimensional electrophoresis (2DE) gels us- ing spatial in the spatial domain. 1. INTRODUCTION Two-dimensional electrophoresis (2DE) gel has been widely used to separate

  3. Silver Staining of 2D Electrophoresis Gels Thierry Rabilloud

    E-print Network

    Paris-Sud XI, Université de

    Silver Staining of 2D Electrophoresis Gels Thierry Rabilloud CEA-DSV-iRTSV/LCBM and UMR CNRS separation on polyacrylamide gels. It combines excellent sensitivity (in the low nanogram range) with the use spectrometry, quantification, polyacrylamide gels, protein visualisation, silver staining #12;1. Introduction

  4. 2-D gel electrophoresis: constructing 2D-gel proteome reference maps.

    PubMed

    Simula, Maria Paola; Notarpietro, Agata; Toffoli, Giuseppe; De Re, Valli

    2012-01-01

    Two-dimensional gel electrophoresis (2-DE) is the most popular and versatile method of protein separation among a rapidly growing array of proteomic technologies. Based on two independent biochemical characteristics of proteins, it combines isoelectric focusing, which separates proteins according to their isoelectric point (pI), and SDS-PAGE, which separates them further according to their molecular mass. An evolution of conventional 2-DE is represented by the 2D-Difference in Gel Electrophoresis (2D-DIGE) that allows sample multiplexing and achieving more accurate and sensitive quantitative proteomic determinations. The 2-DE separation permits the generation of protein maps of different cells or tissues and the study, by differential proteomics, of protein expression changes associated to the different states of a biological system. In order to identify the molecular bases of pathological processes, it is also useful to characterize the physiological protein homeostasis in healthy cells or tissues. On these grounds, the availability of detailed 2D reference maps could be very useful for proteomic studies. The protocol described in this chapter is based on the 2D-DIGE technology and has been applied to obtain the first 2-DE reference map of the human small intestine. PMID:22130991

  5. DETECTION AND SEGMENTATION IN 2D GEL ELECTROPHORESIS IMAGES Eirini Kostopoulou, Eleni Zacharia, and Dimitris Maroulis

    E-print Network

    Athens, University of

    DETECTION AND SEGMENTATION IN 2D GEL ELECTROPHORESIS IMAGES Eirini Kostopoulou, Eleni Zacharia: ikostop@di.uoa.gr, eezacharia@gmail.com, dmaroulis@di.uoa.gr ABSTRACT Analyzing 2D-gel electrophoresis presents an original approach to detecting and segmenting spots in 2D-gel electrophoresis images

  6. Unsupervised 2D gel electrophoresis image segmentation based on active contours

    E-print Network

    Athens, University of

    Unsupervised 2D gel electrophoresis image segmentation based on active contours Michalis A August 2011 Keywords: Segmentation Active contours 2D-gel electrophoresis images a b s t r a c in two-dimensional gel electrophoresis (2D-GE) images. The proposed segmentation scheme is the first

  7. Segmentation of 2D Gel Electrophoresis Spots Using a Markov Random Field

    E-print Network

    Corso, Jason J.

    Segmentation of 2D Gel Electrophoresis Spots Using a Markov Random Field Christopher S. Hoeflich based model was tested on actual 2D gel electrophoresis images. Keywords: Segmentation, Statistical) model and simulated annealing. The entire process of matching 2D gel electrophoresis images is widely

  8. ESTIMATION OF THIN PLATE SPLINE WARP PARAMETERS FROM PROTEIN SPOT POSITIONS IN 2D ELECTROPHORESIS GELS

    E-print Network

    - mation and a Thin Plate Spline transformation in the reg- istration of 2D gel electrophoresis imagesESTIMATION OF THIN PLATE SPLINE WARP PARAMETERS FROM PROTEIN SPOT POSITIONS IN 2D ELECTROPHORESIS GELS Lars Pedersen Informatics and Mathematical Modelling Richard Petersens Plads, Building 321

  9. Silver Staining of 2D Electrophoresis Gels Ccile Lelong, Mireille Chevallet, Sylvie Luche, Thierry Rabilloud

    E-print Network

    Paris-Sud XI, Université de

    1 Silver Staining of 2D Electrophoresis Gels Cécile Lelong, Mireille Chevallet, Sylvie Luche Cedex 9, France 1. Introduction Silver staining of polyacrylamide gels was introduced in 1979 by Switzer staining protocols for proteins in polyacrylamide gels can be found in the literature. However, all of them

  10. Gel Electrophoresis

    NSDL National Science Digital Library

    2007-04-19

    This interactive activity from the Dolan DNA Learning Center illustrates the process of gel electrophoresis, in which DNA fragments are separated by size as they migrate at different rates through a gel matrix.

  11. Gel Electrophoresis

    NSDL National Science Digital Library

    In the early days of DNA manipulation, DNA fragments were laboriously separated by gravity. In the 1970s, the powerful tool of DNA gel electrophoresis was developed. This process uses electricity to separate DNA fragments by size as they migrate through a gel matrix. This animation from Cold Spring Harbor Laboratory's Dolan DNA Learning Center presents Gel Electrophoresis through a series of illustrations of the processes involved.

  12. Gel Electrophoresis

    NSDL National Science Digital Library

    Julie Yu

    2007-01-01

    In this activity, learners simulate the process of DNA fingerprinting by using electricity to separate colored dyes. Learners use simple materials to assemble a comb (electrophoresis chamber) to hold the samples, make a 0.2% sodium bicarbonate buffer and 1% gel solution, connect a high voltage power supply, and prepare 5 different samples. Then learners test their model and observe each sample.

  13. Gel electrophoresis, 2D animationSite: DNA Interactive (www.dnai.org)

    NSDL National Science Digital Library

    2008-10-06

    In the early days of DNA manipulation, DNA fragments were laboriously separated by gravity. In the 1970s, the powerful tool of DNA gel electrophoresis was developed. This process uses electricity to separate DNA fragments by size as they migrate through a gel matrix.

  14. Investigation on sequential extraction of peanut allergens for subsequent analysis by ELISA and 2D gel electrophoresis

    Microsoft Academic Search

    Hubert Chassaigne; Marcel Brohe; Jrgen V. Nrgaard; Arjon J. van Hengel

    2007-01-01

    A two-step sequential extraction method of peanut proteins was proposed with the aim to investigate the protein composition and allergen content of peanut samples. The extraction procedure reported is fully compatible with subsequent analysis by enzyme-linked immunosorbent assays (ELISA) as well as 2D gel electrophoresis (2D PAGE). This sequential extraction method was used to study three different peanut varieties and

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

    ERIC Educational Resources Information Center

    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

  16. This work introduces a novel active contour-based scheme for unsupervised segmentation of protein spots in two-dimensional gel electrophoresis (2D-GE) images. The proposed segmentation scheme is the first to

    E-print Network

    Athens, University of

    of protein spots in two-dimensional gel electrophoresis (2D-GE) images. The proposed segmentation scheme: Segmentation; Active contours; 2D-gel electrophoresis images. * Corresponding author Tel.: +30-210-7275317 Fax-dimensional gel electrophoresis (2D-GE) [1]. In 2D-GE, an indicative portion of the total protein component

  17. Proteomic investigation of human burn wounds by 2D-difference gel electrophoresis and mass spectrometry

    PubMed Central

    Pollins, Alonda C.; Friedman, David B.; Nanney, Lillian B.

    2009-01-01

    Background In humans, thermal cutaneous injury represents a serious traumatic event that induces a host of dynamic alterations. Unfortunately the molecular mechanisms that underlie these serious perturbations remain poorly understood. We applied a global analysis method to identify dynamically changing proteins within the burn environment, which could eventually become biomarkers or targets for treatment. Materials and Methods Protein extracts of normal/unwounded skin and burn wounds were assayed by 2D difference gel electrophoresis (DIGE), a proteomic technology by which abundance levels of intact proteins (including isoforms) were simultaneously quantified from multiple samples with statistical confidence. Through unsupervised multivariate principal component analysis, protein expression patterns from individual samples were appropriately clustered into their correct temporal healing periods grouped into postburn periods of 13 days, 46 days or 710 days after injury. Forty-six proteins were subsequently selected for identification by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Results Proteins identified with differential temporal patterns of expression included predictable cytoskeletal proteins such as vimentin, and keratins 1, 5, 6, 16, and 17. Other candidate proteins with potential involvement in healing included HSP90, members of the serpin family (Serpin B1, SCCA1 & 2), haptoglobin, gelsolin, eIF4A1, IQGAP1, and TCTP. Conclusions We have utilized the combined technique, DIGE/MS, to capture new insights into cutaneous responses to burn trauma and subsequent processes of early wound healing in humans. This pilot study provides a proteomic snapshot of temporal events that can be used to weave together the interconnected processes that define the response to serious cutaneous injury. PMID:17604053

  18. Identification of methanococcus jannaschii proteins in 2-D gel electrophoresis patterns by mass spectrometry.

    SciTech Connect

    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.

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

    PubMed Central

    Morisawa, Hiraku; Hirota, Mikako; Toda, Tosifusa

    2006-01-01

    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. PMID:17018156

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

    PubMed Central

    Nebija, Dashnor; Noe, Christian R.; Urban, Ernst; Lachmann, Bodo

    2014-01-01

    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. PMID:24739811

  1. Quality control and stability studies with the monoclonal antibody, trastuzumab: application of 1D- vs. 2D-gel electrophoresis.

    PubMed

    Nebija, Dashnor; Noe, Christian R; Urban, Ernst; Lachmann, Bodo

    2014-01-01

    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. PMID:24739811

  2. Gel Electrophoresis and Photography

    E-print Network

    Simpson, Larry

    Gel Electrophoresis and Photography An Application Note UVP-AB-1000-02 #12;The GDS-8000 Gel on the overlayed scan. GEL ELECTROPHORESIS IMAGING, DOCUMENTATION AND ANALYSIS ... TODAY. #12;The introduction of the technique of electrophoresis in acrylamide or agarose gels was a major advance in nucleic acid technology

  3. Proteomic analysis of ovomucoid hypersensitivity in mice by two-dimensional difference gel electrophoresis (2D-DIGE).

    PubMed

    Hobson, D J; Rupa, P; Diaz, G J; Zhang, H; Yang, M; Mine, Y; Turner, P V; Kirby, G M

    2007-12-01

    There is a need to develop reliable methods to assess the safety of genetically modified and other novel foods. The aim of this study was to identify protein biomarkers of food allergy in mice exposed to ovomucoid (OVM), a major food allergen found in chicken egg white. BALB/c mice were repeatedly sensitized by gavage with OVM and cholera toxin (CT) and control mice were exposed to a mixture of amino acids with CT. At the endpoint, all mice were challenged intraperitoneally with OVM and alum. Type-1 hypersensitivity was confirmed in OVM-sensitized mice by observation of clinical signs of anaphylaxis and elevated levels of plasma histamine, OVM-specific IgE and OVM-specific IgG by ELISA. Differential protein expression was assessed in albumin-depleted plasma as well as in mesenteric lymph node, liver, spleen, and ileum by two-dimensional difference gel electrophoresis (2D-DIGE). Differentially expressed proteins were identified by liquid chromatography with tandem mass spectrometry. Plasma proteins overexpressed in OVM-sensitized mice included haptoglobin (41-fold), serum amyloid A (19-fold) and peroxiredoxin-2 (1.9-fold). Further validation of these plasma proteins in other animal models of food allergy with different food allergens is required to assess their potential as candidate biomarkers for use in evaluating the allergenicity of novel foods. PMID:17897766

  4. Gel Electrophoresis of Dyes

    NSDL National Science Digital Library

    Janice Stephens

    2011-01-01

    In this experiment related to plant biotechnology, learners discover how to prepare and load an electrophoresis gel. They will then run the gels in an electrophoresis system to separate several dyes that are of different molecular sizes and carry different charges. This technique is fundamental to many of the procedures used in biotechnology. This lesson guide includes background information for the educator, safety precautions, and questions with answers for learners. For safety reasons, adult supervision is recommended. Modifications for use with younger learners are described in a related PDF (see related resource).

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

    PubMed

    Yusenko, Maria V; Ruppert, Thomas; Kovacs, Gyula

    2010-01-01

    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. PMID:20440404

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

    PubMed Central

    Yusenko, Maria V.; Ruppert, Thomas; Kovacs, Gyula

    2010-01-01

    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. PMID:20440404

  7. Metal imaging in non-denaturating 2D electrophoresis gels by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) for the detection of metalloproteins.

    PubMed

    Becker, J Susanne; Lobinski, Ryszard; Becker, J Sabine

    2009-01-01

    Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was developed as a powerful analytical technique for metal imaging of 2D gels for the detection of metalloproteins in rat kidney after electrophoretic separation. Protein complexes, extracted with water, were separated in their native state in the first and second dimension by blue native gel electrophoresis (BN-PAGE). Essential and toxic metals, such as zinc, copper, iron, manganese and lead, were monitored by LA-ICP-MS after gel ablation by a focused laser beam in a way that the total surface of a selected fragment of the gel was totally ablated. The metal distribution of this part of the gel was then constructed by plotting the metal (isotope) signal intensity as a function of the x,y (isoelectric point, molecular mass) coordinates of the gel. The proteins at locations rich in metals were cut out, digested with trypsin and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). PMID:21305128

  8. SDS Polyacrylamide Gel Electrophoresis Gel Recipes

    E-print Network

    Pike, Linda J.

    SDS Polyacrylamide Gel Electrophoresis Gel Recipes % Acrylamide 5% 7.5% 10.% 12.5% 15% 18% 4 for running gel; ~10 ml for stacking gel Electrophoresis Buffer: 5X Buffer: 1 X Buffer 60 g Tris base 9 g Tris% Stacking Gel 30% Acrylamide (ml) 5.0 7.5 10.0 12.5 15.0 18.0 1.3 1% Bisacrylamide (ml) 7.8 5.8 3.9 3.1 3

  9. Copolymers For Capillary Gel Electrophoresis

    DOEpatents

    Liu, Changsheng (State College, PA); Li, Qingbo (State College, PA)

    2005-08-09

    This invention relates to an electrophoresis separation medium having a gel matrix of at least one random, linear copolymer comprising a primary comonomer and at least one secondary comonomer, wherein the comonomers are randomly distributed along the copolymer chain. The primary comonomer is an acrylamide or an acrylamide derivative that provides the primary physical, chemical, and sieving properties of the gel matrix. The at least one secondary comonomer imparts an inherent physical, chemical, or sieving property to the copolymer chain. The primary and secondary comonomers are present in a ratio sufficient to induce desired properties that optimize electrophoresis performance. The invention also relates to a method of separating a mixture of biological molecules using this gel matrix, a method of preparing the novel electrophoresis separation medium, and a capillary tube filled with the electrophoresis separation medium.

  10. Introduction to Agarose Gel Electrophoresis

    NSDL National Science Digital Library

    CLIMB: Cornell's Learning Initiative in Medicine and Bioengineering

    In this module, developed as part of Cornell's Learning Initiative in Medicine and Bioengineering (CLIMB), students are introduced to the concepts of gel electrophoresis without requiring all the equipment needed to run a full gel electrophoresis experiment. The goal is to have students understand how gels are made for DNA separation and how altering the composition can affect the experimental parameters. This module contains a teacher's guide, classroom activity, and suggestions for extended activities. This lab is a precursor to Cornells Institute for Biology Teachers labs entitled DNA Profiling Paternity Testing, which is linked within the teacher's guide. CLIMB is part of the NSF GK-12 program.

  11. Pre-Cast Gel Electrophoresis Guide

    E-print Network

    Kirschner, Marc W.

    Novex® Pre-Cast Gel Electrophoresis Guide Version B January 27, 2003 IM-1002 Novex® Pre-Cast Gel Electrophoresis Guide General information and protocols for using Novex® pre-cast gels www.invitrogen.com tech.............................................................................................................................28 Electrophoresis of Novex® Pre-Cast Gels

  12. Abstract--This work addresses the segmentation of two-dimensional polyacrylamide gel electrophoresis images

    E-print Network

    Athens, University of

    -PAGE Images, Overlapping Spots I. INTRODUCTION wo-dimensional polyacrylamide gel electrophoresis (2- D PAGEAbstract--This work addresses the segmentation of two- dimensional polyacrylamide gel electrophoresis images containing overlapping protein spots. A novel segmentation approach is proposed, which

  13. Gel Electrophoresis Lab: DNA Fingerprinting

    NSDL National Science Digital Library

    Ehlers, Megan

    This lab activity from the Biotechnology Alliance for Suncoast Biology Educators introduces the methods of RFLP analysis, or DNA fingerprinting, by using gel electrophoresis. Students will learn the role of restriction enzymes in DNA fingerprinting. Required materials, procedure and instructions are provided. This lesson plan may be downloaded in Microsoft Word document file format.

  14. Gel Electrophoresis Lab: Paternity Case

    NSDL National Science Digital Library

    This lab activity from the Biotechnology Alliance for Suncoast Biology Educators provides instructions for conducting a gel electrophoresis lab. Students will try to solve a paternity case with this activity by obtaining a DNA fingerprint from each potential father, the mother and the child. This activity may be downloaded in PDF file format. A data collection sheet and student questions are also included.

  15. Allison Lab Protocol: Gel Electrophoresis, 1/2008, Steve Allison Gel Electrophoresis of Nucleic Acids

    E-print Network

    German, Donovan P.

    Allison Lab Protocol: Gel Electrophoresis, 1/2008, Steve Allison Gel Electrophoresis of Nucleic the Polaroid camera to take a photo of the gel. · Note the electrophoresis conditions on the gel photo and tape Acids · Always wear gloves; ethidium bromide is a powerful mutagen! · For a 1.5% gel in the small gel

  16. Conducting polymer electrodes for gel electrophoresis.

    PubMed

    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 ?-conjugated polymers such as poly(3,4-ethylenedioxythiophene) (PEDOT) can be placed between traditional metal electrodes and an electrolyte to mitigate electrolysis in liquid (capillary electroosmosis/electrophoresis) systems. In this report, we extend our previous result to gel electrophoresis, and show that electrodes containing PEDOT can be used with a commercial polyacrylamide gel electrophoresis system with minimal impact to the resulting gel image or the ionic transport measured during a separation. PMID:24586761

  17. Comparative proteomics using 2-D gel electrophoresis and mass spectrometry as tools to dissect stimulons and regulons in bacteria with sequenced or partially sequenced genomes

    PubMed Central

    Hernndez, Magdalena; Martnez-Batallar, Gabriel; Contreras, Sandra; del Carmen Vargas, Mara; Mora, Jaime

    2005-01-01

    We propose two-dimensional gel electrophoresis (2-DE) and mass spectrometry to define the protein components of regulons and stimulons in bacteria, including those organisms where genome sequencing is still in progress. The basic 2-DE protocol allows high resolution and reproducibility and enables the direct comparison of hundreds or even thousands of proteins simultaneously. To identify proteins that comprise stimulons and regulons, peptide mass fingerprint (PMF) with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS) analysis is the first option and, if results from this tool are insufficient, complementary data obtained with electrospray ionization tandem-MS (ESI-MS/MS) may permit successful protein identification. ESI-MS/MS and MALDI-TOF-MS provide complementary data sets, and so a more comprehensive coverage of a proteome can be obtained using both techniques with the same sample, especially when few sequenced proteins of a particular organism exist or genome sequencing is still in progress. PMID:16145578

  18. Fluorescence detection for gel and capillary electrophoresis

    SciTech Connect

    Hogan, B.

    1992-07-21

    First, an indirect fluorescence detection system for the separation of proteins via gel electrophoresis. Quantities as low as 50 nanograms of bovine serum albumin and soybean trypsin inhibitor are separated and detected visually without the need for staining of the analytes. This is very similar to levels of protein commonly separated with gel electrophoresis.

  19. Development of a non-denaturing 2D gel electrophoresis protocol for screening in vivo uranium-protein targets in Procambarus clarkii with laser ablation ICP MS followed by protein identification by HPLC-Orbitrap MS.

    PubMed

    Xu, Ming; Frelon, Sandrine; Simon, Olivier; Lobinski, Ryszard; Mounicou, Sandra

    2014-10-01

    Limited knowledge about in vivo non-covalent uranium (U)-protein complexes is largely due to the lack of appropriate analytical methodology. Here, a method for screening and identifying the molecular targets of U was developed. The approach was based on non-denaturing 1D and 2D gel electrophoresis (ND-PAGE and ND-2D-PAGE (using ND-IEF as first dimension previously described)) in conjunction with laser ablation inductively coupled plasma mass spectrometry (LA-ICP MS) for the detection of U-containing proteins. The proteins were then identified by bore HPLC-Orbitrap MS/MS. The method was applied to the analysis of cytosol of hepatopancreas (HP) of a model U-bioaccumulating organism (Procambarus clarkii). The imaging of uranium in 2D gels revealed the presence of 11 U-containing protein spots. Six protein candidates (i.e. ferritin, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, cytosolic manganese superoxide dismutase (Mn-SOD), glutathione S transferase D1 and H3 histone family protein) were then identified by matching with the data base of crustacea Decapoda species (e.g. crayfish). Among them, ferritin was the most important one. This strategy is expected to provide an insight into U toxicology and metabolism. PMID:25059147

  20. Edinburgh Research Explorer Native gel electrophoresis of human telomerase distinguishes

    E-print Network

    Millar, Andrew J.

    Edinburgh Research Explorer Native gel electrophoresis of human telomerase distinguishes active Bihan, T & Harrington, L 2012, 'Native gel electrophoresis of human telomerase distinguishes active and investigate your claim. Download date: 16. Jun. 2014 #12;Native gel electrophoresis of human telomerase

  1. Lights, Camera, Action! Systematic Variation in Difference Gel Electrophoresis

    E-print Network

    Lights, Camera, Action! ­ Systematic Variation in Difference Gel Electrophoresis Kimberly F Abstract Two-dimensional Difference Gel Electrophoresis (DIGE) circumvents many of the prob- lems classical, single-dye gel electrophoresis images. Department of Statistics, Carnegie Mellon University

  2. Alignment of two-dimensional electrophoresis gels Guihua Shi, Tianzi Jiang *, Wanlin Zhu, Bing Liu, Huizhi Zhao

    E-print Network

    Jiang,Tianzi

    Alignment of two-dimensional electrophoresis gels Guihua Shi, Tianzi Jiang *, Wanlin Zhu, Bing Liu-dimensional electrophoresis is a major separating technique for proteins in proteomics. Alignment of gel images is critical a novel iterative closest point (ICP) method for 2D-gel electrophoresis image alignment. The paper seeks

  3. Improved method for identification of low abundance proteins using 2D-gel electrophoresis, MALDI-TOF and TOF/TOF

    EPA Science Inventory

    Introduction: Differential protein expression studies have been routinely performed in our laboratory to determine the health effects of environmentally-important chemicals. In this abstract, improvements in the in-gel protein digestion, MALDI plate spotting and data acquisition...

  4. A Simple Vertical Slab Gel Electrophoresis Apparatus.

    ERIC Educational Resources Information Center

    Carter, J. B.; And Others

    1983-01-01

    Describes an inexpensive, easily constructed, and safe vertical slab gel kit used routinely for sodium dodecyl sulphate-polyacrylamide gel electrophoresis research and student experiments. Five kits are run from a single transformer. Because toxic solutions are used, students are given plastic gloves and closely supervised during laboratory

  5. Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology

    Microsoft Academic Search

    Gerard Muyzer; Kornelia Smalla

    1998-01-01

    Here, the state of the art of the application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology will be presented. Furthermore, the potentials and limitations of these techniques will be discussed, and it will be indicated why their use in ecological studies has become so important. Abbreviations: ARDRA - amplified ribosomal DNA restriction

  6. Pulsed field gel electrophoresis for bifidobacterium.

    PubMed

    Jimnez, Esther; Gmez, Marta; Moles, Laura

    2015-01-01

    Pulse Field Gel Electrophoresis (PFGE), unlike conventional electrophoresis, can resolve DNA fragments greater than 30 kb and is a highly discriminatory molecular typing method. Here we describe a PFGE protocol for bifidobacteria characterized by a short lysis time determined by the addition of lysis reagents to the initial cell suspension, a reduced incubation period of the plugs in proteinase K, and an improved washing plug step with preheating of the buffer in a shaking incubator. PMID:25862062

  7. Two-dimensional Gel Electrophoresis (2DE)

    NASA Astrophysics Data System (ADS)

    K?odzi?ska, Ewa; Buszewski, Bogus?aw

    The chemical compounds, which are present in the environment, increasingly cause bad effects on health. The most serious effects are tumors and various mutations at the cellular level. Such compounds, from the analytical point of view, can serve the function of biomarkers, constituting measurable changes in the organism's cells and biochemical processes occurring therein. The challenge of the twenty-first century is therefore searching for effective and reliable methods of identification of biomarkers as well as understanding bodily functions, which occur in living organisms at the molecular level. The irreplaceable tool for these examinations is proteomics, which includes both quality and quantity analysis of proteins composition, and also makes it possible to learn their functions and expressions. The success of proteomics examinations lies in the usage of innovative analytical techniques, such as electromigration technique, two-dimensional electrophoresis in polyacrylamide gel (2D PAGE), liquid chromatography, together with high resolution mass spectrometry and bio-informatical data analysis. Proteomics joins together a number of techniques used for analysis of hundreds or thousands of proteins. Its main task is not the examination of proteins inside the particular tissue but searching for the differences in the proteins' profile between bad and healthy tissues. These differences can tell us a lot regarding the cause of the sickness as well as its consequences. For instance, using the proteomics analysis it is possible to find relatively fast new biomarkers of tumor diseases, which in the future will be used for both screening and foreseeing the course of illness. In this chapter we focus on two-dimensional electrophoresis because as it seems, it may be of enormous importance when searching for biomarkers of cancer diseases.

  8. Warping of electrophoresis gels using generalisations of dynamic programming

    E-print Network

    Glasbey, Chris

    Warping of electrophoresis gels using generalisations of dynamic programming Chris Glasbey a single track in a 1-D electrophoresis gel, such as the pulsed-field gel electrophoresis (PFGE) shown in Fig 1(a), with another track. Gel tracks need to be aligned with tracks in a reference database

  9. DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.

    SciTech Connect

    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.

  10. Monitoring intracellular protein profiles with two-dimensional gel electrophoresis.

    PubMed

    Dykstra, K H; Wang, H Y

    1992-01-01

    Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) is a method of separating complex protein mixtures, such as whole cell extracts, on the basis of protein isoelectric point and molecular weight. In bioprocess engineering, conventional 2D PAGE has tremendous potential to yield detailed information on the intracellular effect of various process conditions. It has been used in our work to examine global intracellular changes occurring in a typical cycloheximide fermentation and to look at the feedback regulatory behavior of cycloheximide biosynthesis. Application of the technique for bioprocess monitoring will require that the time necessary for preparation of a 2D electropherogram be substantially shortened. This may be accomplished by performing the separation on a miniature scale or eventually by use of capillary electrophoresis for one or more of the separations. Advantages and disadvantages of these two approaches are discussed. PMID:1368457

  11. The Genetic Science Learning Center: Gel Electrophoresis

    NSDL National Science Digital Library

    Gel Electrophoresis, designed and run by the University of Utah, is an interactive program that allows the student to learn and practice basic techniques that molecular biologists use every day. This program is an interactive animated procedure that allows the user to "see" DNA strands and instructs the student or user on the basics of DNA.

  12. The repton model of gel electrophoresis

    E-print Network

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

    1996-12-04

    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.

  13. ANALYSIS OF TWO-DIMENSIONAL ELECTROPHORESIS GEL IMAGES

    E-print Network

    ANALYSIS OF TWO-DIMENSIONAL ELECTROPHORESIS GEL IMAGES Lars Pedersen Informatics and Mathematical of proteomics. The subject is Analysis of Two-dimensional Electrophoresis Gel Images. This work was carried out analysis of two-dimensional gel electrophoresis (2DGE) images. 2DGE is the leading technique to separate

  14. Analysis of Inorganic Polyphosphates by Capillary Gel Electrophoresis

    E-print Network

    Church, George M.

    Analysis of Inorganic Polyphosphates by Capillary Gel Electrophoresis Andrew Lee and George M This paper describes the development of a method that uses capillary gel electrophoresis (CGE) to analyze mix)n, by capillary gel electrophoresis. Samples of condensed inorganic phosphate are typically mixtures of (Pi

  15. High reproducibility of large-gel two-dimensional electrophoresis.

    PubMed

    Challapalli, Kiran Kumar; Zabel, Claus; Schuchhardt, Johannes; Kaindl, Angela M; Klose, Joachim; Herzel, Hanspeter

    2004-09-01

    Two-dimensional gel electrophoresis (2-DE) facilitates the separation of thousands of proteins from highly complex protein mixtures and has become a central method in proteomics in recent years. In the present study, we examined the technical variability of large 2-DE gels with respect to sample preparation, electrophoresis procedure, data acquisition, and biological variation by analyzing a disease (Huntington's disease) and control state with a commercially available software package, PROTEOMWEAVER trade mark. Scatter plots and correlation coefficients were obtained to quantify both technical and biological variation. Even 2-DE gels run separately in both dimensions yielded correlation coefficients around 0.88 and deviations from the mean close to 20% for low-intensity spots. This indicates a high technical reproducibility of the 2-DE procedure developed in our laboratory. Variability within a biological condition was low and comparable to technical variation (at least 0.87). Two-dimensional (2-D) gels obtained from samples of different biological conditions (health vs. disease) achieved a variability similar to intracondition and technical variability. These findings highlight the importance of multiple gel and spot-by-spot comparisons to identify biological significant changes. Minor errors introduced by technical and biological variation allow a comparison of all gels within a study which facilitates the tackling of complex biological problems. PMID:15349946

  16. Seperation of proteins using cetyltrimethylammonium bromide discontinuous gel electrophoresis

    Microsoft Academic Search

    Robert E. Akins; Rocky S. Tuan

    1994-01-01

    The gel electrophoresis system presented here allows the separation of proteins with the concomitant retention of detectable\\u000a native activities. The system, referred to as CAT gel electrophoresis. uses the detergent cetyltrimethylammonium bromide in\\u000a combination with a discontinuous gel matrix to resolve protein mixtures into discrete bands. Many proteins retain detectable\\u000a levels of native activity after CAT electrophoresis, and gel bands

  17. Using Ultra-Zoom Gels for High-Resolution Two-Dimensional Polyacrylamide Gel Electrophoresis

    Microsoft Academic Search

    Sjouke Hoving; Hans Voshol; Jan van Oostrum

    \\u000a Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is one of the core Technologiestogether with mass spectrometryof\\u000a proteome research. It is the only method currently available that is able to simultaneously separate the thousands of proteins\\u000a found in biological samples. The method originates from the seminal work of OFarrell and Klose in the 1970s (1,2). The main drawback of the original method

  18. Protein Electrophoresis in the Biology Classroom Using "Safe" Gels.

    ERIC Educational Resources Information Center

    Atkins, Thomas

    1991-01-01

    Describes the use of electrophoresis in the high school utilizing two new gels that are easy to pour, do not require degassing, can be used on a horizontal gel electrophoresis, and are not neurotoxic or carcinogenic health hazards. Large diagrams and written instructions are used to present the protocol of setting up the electrophoresis. (PR)

  19. Gel Electrophoresis of Gold-DNA Nanoconjugates

    PubMed Central

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

    2007-01-01

    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. PMID:18401452

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

    ERIC Educational Resources Information Center

    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)

  1. Gel Electrophoresis on a Budget to Dye For

    NSDL National Science Digital Library

    Julie H. Yu

    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

  2. Biotechnology Laboratory: Gel Electrophoresis of Dyes

    NSDL National Science Digital Library

    A portion of the Partnership for Plant Genomics Education, hosted by the University of California-Davis, this PDF presents a student activity where students will use agarose gel electrophoresis to separate several different dyes. The lab is described as a ??precursor to DNA separations? and thus provides an important step in the subject matter. The lab provides for students: detailed instructions, background information, and a quiz and group questions. Answers to the questions, and also the general objective of the lab, are provided for the instructor. Overall, the lab is introductory in nature and perfect for any science classroom.

  3. Hybrid slab-microchannel gel electrophoresis system

    DOEpatents

    Balch, Joseph W. (Livermore, CA); Carrano, Anthony V. (Livermore, CA); Davidson, James C. (Livermore, CA); Koo, Jackson C. (San Ramon, CA)

    1998-01-01

    A hybrid slab-microchannel gel electrophoresis system. The hybrid system permits the fabrication of isolated microchannels for biomolecule separations without imposing the constraint of a totally sealed system. The hybrid system is reusable and ultimately much simpler and less costly to manufacture than a closed channel plate system. The hybrid system incorporates a microslab portion of the separation medium above the microchannels, thus at least substantially reducing the possibility of non-uniform field distribution and breakdown due to uncontrollable leakage. A microslab of the sieving matrix is built into the system by using plastic spacer materials and is used to uniformly couple the top plate with the bottom microchannel plate.

  4. Procedure for Mobility Gel Electrophoresis Prepare Linearized Plasmid DNA

    E-print Network

    Turro, Claudia

    Procedure for Mobility Gel Electrophoresis Prepare Linearized Plasmid DNA 1. Purified pUC18 plasmid C (deactivating enzyme) 3. Follow with the gel extraction kit (it doubles as an enzymatic clean a Test Gel Gel Conditions 1. .75% agarose gel 2. TBE buffer (pH = 8.3) 3. 50 uM DNA 4. 10 mM Sodium

  5. Peptide separations by slab gel electrophoresis in pluronic F127 polymer liquid crystals.

    PubMed

    Rill, Randolph L; Al-Sayah, Mohammad A

    2004-05-01

    Proteomics and peptidomics could benefit from simple methods for high-resolution separation of oligopeptides analogous to slab gel electrophoresis of proteins. Gels of Pluronic F127 copolymer surfactant were investigated as media for slab gel electrophoresis of oligopeptides using a trypsin digest of myoglobin. Concentrated solutions of Pluronic F127 are fluid at low temperatures (gel-like micellar liquid crystal upon warming. Nucleic acids are well separated by electrophoresis in these gels as previously shown by Rill and Liu. Good separations of myoglobin tryptic peptides were accomplished by electrophoresis on slab gels of 24% Pluronic F127 or 15% polyacrylamide using the alkaline Laemmli buffer system (without sodium dodecyl sulfate). Labeling of peptides with the succinimidyl ester of Cascade Yellow (CY) prior to electrophoresis allowed sensitive detection with blacklight illumination at 365 nm. Labeled tryptic peptides were identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry. An inverse dependence of electrophoretic mobility on mass of peptides with charge Z = -1 was observed in both media. Two-dimensional (2-D) electrophoresis of myoglobin peptides on polyacrylamide, then on Pluronic media, at pH 8.3 indicated that the primary separation mechanism of most peptides was the same in both media. A few off-diagonal spots indicated that some peptides were preferentially retarded in Pluronic gels, perhaps due to hydrophobic effects. The ease of gel preparation and peptide recovery are advantages of Pluronic F127 gels for oligopeptide electrophoresis. The two media can be combined conveniently for 2-D electrophoresis, providing means to facilitate protein identification and peptidomics. PMID:15174045

  6. Original article Two-dimensional gel electrophoresis of membrane

    E-print Network

    Paris-Sud XI, Université de

    Original article Two-dimensional gel electrophoresis of membrane proteins from ectomycorrhizal-dimensional polyacrylamide gels. Gels with limited back- ground staining and streaking and with clearly efficacité et leur compatibilité avec l'obtention de gels d'électro- phorèse bidimensionnelle. Une fraction

  7. Inexpensive and Safe DNA Gel Electrophoresis Using Household Materials

    ERIC Educational Resources Information Center

    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

  8. Hybrid slab-microchannel gel electrophoresis system

    DOEpatents

    Balch, J.W.; Carrano, A.V.; Davidson, J.C.; Koo, J.C.

    1998-05-05

    A hybrid slab-microchannel gel electrophoresis system is described. The hybrid system permits the fabrication of isolated microchannels for biomolecule separations without imposing the constraint of a totally sealed system. The hybrid system is reusable and ultimately much simpler and less costly to manufacture than a closed channel plate system. The hybrid system incorporates a microslab portion of the separation medium above the microchannels, thus at least substantially reducing the possibility of non-uniform field distribution and breakdown due to uncontrollable leakage. A microslab of the sieving matrix is built into the system by using plastic spacer materials and is used to uniformly couple the top plate with the bottom microchannel plate. 4 figs.

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

    Microsoft Academic Search

    Frank Gibson; Leigh Anderson; Gyorgy Babnigg; Mark Baker; Matthias Berth; Pierre-Alain Binz; Andy Borthwick; Phil Cash; Billy W Day; David B Friedman; Donita Garland; Howard B Gutstein; Christine Hoogland; Neil A Jones; Alamgir Khan; Joachim Klose; Angus I Lamond; Peter F Lemkin; Kathryn S Lilley; Jonathan Minden; Nicholas J Morris; Norman W Paton; Michael R Pisano; John E Prime; Thierry Rabilloud; David A Stead; Chris F Taylor; Hans Voshol; Anil Wipat; Andrew R Jones

    2009-01-01

    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

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

    PubMed

    Slater, Gary W

    2009-06-01

    DNA gel electrophoresis has been the most important experimental tool to separate DNA fragments for several decades. The introduction of PFGE in the 1980s and capillary gel electrophoresis in the 1990s made it possible to study, map and sequence entire genomes. Explaining how very large DNA molecules move in a gel and why PFGE is needed to separate them has been an active field of research ever since the launch of the journal Electrophoresis. This article presents a personal and historical overview of the development of the theory of gel electrophoresis, focusing on the reptation model, the band broadening mechanisms, and finally the factors that limit the read length and the resolution of electrophoresis-based sequencing systems. I conclude with a short discussion of some of the questions that remain unanswered. PMID:19517509

  11. SEPARATION AND IDENTIFICATION OF SOYBEAN LEAF PROTEINS BY TWO-DIMENSIONAL GEL ELECTROPHORESIS AND MASS SPECTROMETRY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To establish a proteomic reference map for soybean leaves, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 260 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted las...

  12. Dna electrophoresis in photopolymerized polyacrylamide gels on a microfluidic device

    E-print Network

    Lo, Chih-Cheng

    2009-05-15

    DNA gel electrophoresis is a critical analytical step in a wide spectrum of genomic analysis assays. Great efforts have been directed to the development of miniaturized microfluidic systems (lab-on-a-chip systems) to perform low-cost, high...

  13. ELECTROPHORESIS GEL BUFFER RECIRCULATOR FOR UNDER 20 DOLLARS

    EPA Science Inventory

    Procedures requiring extended periods of electrophoresis frequently require recirculation of the get buffer in order to reduce gel artifacts. ere we describe a recirculation device which can be built inexpensively and will fit many different model get boxes....

  14. Gel Electrophoresis on a Budget to Dye for

    ERIC Educational Resources Information Center

    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

  15. Gel Electrophoresis--The Easy Way for Students

    ERIC Educational Resources Information Center

    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

  16. Gel Electrophoresis of Gold-DNA Nano-Conjugates

    SciTech Connect

    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.

  17. THERMAL DETECTION OF DNA AND PROTEINS DURING GEL ELECTROPHORESIS

    SciTech Connect

    R. JOHNSTON

    2000-08-01

    This is the final report of a three-year, Laboratory-Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The goal of this project was to try to detect unstained, untagged, unlabeled DNA bands in real-time during gel electrophoresis using simple thermal measurements. The technical and ES&H advantages to this approach could potentially be quite significant, especially given the extreme importance of gel electrophoresis to a wide variety of practical and research fields. The project was unable to demonstrate sufficient thermal sensitivity to detect DNA bands. It is clear that we still do not understand the gel electrophoresis phenomenon very well. The temperature control techniques developed during the course of this project have other useful applications.

  18. The instantaneous monitoring of polyacrylamide gels during electrophoresis.

    PubMed Central

    Elliott, A

    1976-01-01

    The advantages of being able to see protein zones in a gel during electrophoresis (and hence before staining) are pointed out, and a method is described which depends on local increments of refractive index in these zones. The use of local increments of refractive index in polyacrylamide gels for measuring protein concentrations in zones during electrophoresis is briefly considered; it is found that such increments are greater than would be expected from the amount of protein when sodium dodecyl sulphate is present. The enhancement depends on conditions and time of running. This makes quantitative estimates difficult, but the sensitivity of detection of protein zones by observations based on refractive-index changes is greatly increased by this property of sodium dodecyl sulphate. Methods are described for making optically uniform gels (both with uniform and with graded concentrations of polyacrylamide), necessary for observation of small changes in refractive index. A simple dark-field system of observation is described. Examples are given showing protein samples observed with the system during electrophoresis and compared with the same gel stained with Coomassie Blue after completion of the run. Under optimal conditions the optical method is comparable in sensitivity with staining. With the proteins of lower mol.wt. (approx. 15000), the optical method is not so sensitive, becoming less sensitive with longer running time. This loss of sensitivity is greatly decreased by using more concentrated polyacrylamide gels, and graded gels are therefore more suitable for optical observation than are uniform gels. The observation of protein zones during electrophoresis adds nothing to the time needed for making a stained gel and gives much information long before it can be obtained from the stained gel. Images PLATE 1 PLATE 2 PMID:1008832

  19. Sample collection system for gel electrophoresis

    DOEpatents

    Olivares, Jose A.; Stark, Peter C.; Dunbar, John M.; Hill, Karen K.; Kuske, Cheryl R.; Roybal, Gustavo

    2004-09-21

    An automatic sample collection system for use with an electrophoretic slab gel system is presented. The collection system can be used with a slab gel have one or more lanes. A detector is used to detect particle bands on the slab gel within a detection zone. Such detectors may use a laser to excite fluorescently labeled particles. The fluorescent light emitted from the excited particles is transmitted to low-level light detection electronics. Upon the detection of a particle of interest within the detection zone, a syringe pump is activated, sending a stream of buffer solution across the lane of the slab gel. The buffer solution collects the sample of interest and carries it through a collection port into a sample collection vial.

  20. Extensions of Gel Electrophoresis with Proteins

    NSDL National Science Digital Library

    Mr. Brian McClain (Amos P. Godby High School)

    2006-04-01

    This inquiry activity is intended to help familiarize students with the procedure of agarose electrophoresis and to make them aware of the types of proteins within tissue samples. This inquiry activity was developed by a K-12 science teacher in the American Physiological Society?s 2006 Frontiers in Physiology Program. The NSES Standards addressed by this activity are current as of the year of development. For more information on the Frontiers in Physiology Program, please visit www.frontiersinphys.org.

  1. SYPRO Ruby Protein Gel Stain Advanced staining technology for 2-D gels and proteomics

    E-print Network

    Lebendiker, Mario

    expression comparisons Staining is compatible with mass spectrometry or microsequencing Simple staining protein can be recovered from the gel and accurately identified using MALDI-TOF mass spectrometry.3SYPRO Ruby Protein Gel Stain Advanced staining technology for 2-D gels and proteomics Detection

  2. Quantification of Polyacrylamide Gel Electrophoresis for Analysis of Whey Proteins

    Microsoft Academic Search

    D. F. Darling; D. W. Butcher

    1976-01-01

    Polyacrylamide gel electrophoresis of whey proteins has been quantified by standardization of the separation and staining procedure. During each electro- phoresis experiment, a standard solution of whey proteins was separated and stained under the same conditions as the test material. In this way, proteins in the standard solution were subjected to iden- tical processing conditions as the test samples. Densitometric

  3. Polymerase Chain Reaction (PCR) and Gel Electrophoresis Powerpoint

    NSDL National Science Digital Library

    The HURI SURI project is developing a regional biotechnology workforce pipeline by expanding and supporting biotechnology research experiences for Jamestown Community College (JCC) undergraduates and disseminating these research experiences and materials to area high school teachers and students. This 15 slide presentation provides an introduction to DNA and explains polymerase chain reactions and gel electrophoresis. Many diagrams are included to help explain the concepts.

  4. Single cell gel electrophoresis assay: methodology and applications

    Microsoft Academic Search

    E Rojas; M. C Lopez; M Valverde

    1999-01-01

    The single cell gel electrophoresis or Comet assay is a sensitive, reliable, and rapid method for DNA double- and single-strand breaks, alkali-labile sites and delayed repair site detection, in eukariotic individual cells. Given its overall characteristics, this method has been widely used over the past few years in several different areas. In this paper we review the studies published to

  5. Solid friction in gel electrophoresis S. F. Burlatskya)

    E-print Network

    Deutch, John

    Solid friction in gel electrophoresis S. F. Burlatskya) and John M. Deutch Department of Chemistry 1995 We study the influence of solid frictional forces acting on polymer chains moving in a random environment. We show that the total reduction in the chain tension resulting from the small friction between

  6. An Investigation on Gel Electrophoresis with Quantum Dots End-labeled DNA

    E-print Network

    Chen, Xiaojia

    2009-05-15

    Invented in the 1950s, gel electrophoresis has now become a routine analytical method to verify the size of nucleic acids and proteins in molecular biology labs. Conventional gel electrophoresis can successfully separate DNA fragments from several...

  7. Analysis of Bacterial Communities in Seagrass Bed Sediments by Double-Gradient Denaturing Gradient Gel Electrophoresis

    E-print Network

    Sherman, Tim

    -Gradient Denaturing Gradient Gel Electrophoresis of PCR-Amplified 16S rRNA Genes J.B. James1 , T.D. Sherman2 and R denaturing gradient gel electrophoresis (DG- DGGE) was used to generate banding patterns from

  8. GEL ELECTROPHORESIS 2009 1 Minority Science Programs School of Biological Sciences University of California, Irvine

    E-print Network

    Rose, Michael R.

    GEL ELECTROPHORESIS 2009 1 Minority Science Programs ­ School the DNA encodes just by looking at the tube. Gel electrophoresis is one of the techniques scientists use to look at the DNA they have. This technique separates DNA molecules by size. How gel electrophoresis

  9. A Comparative Study of Adaptive Registration Methods for Two Dimensional Gel Electrophoresis Images

    E-print Network

    Paris-Sud XI, Université de

    A Comparative Study of Adaptive Registration Methods for Two Dimensional Gel Electrophoresis Images Huamei Xin, Yuemin Zhu, Longfei Wu Abstract--Two-dimensional gel electrophoresis (2DGE) images play-dimensional gel electrophoresis (2DGE) is currently the leading proteomic technique for analyzing, visualizing

  10. University of Newcastle upon Tyne GELI: A data model for the representation of a gel electrophoresis

    E-print Network

    Newcastle upon Tyne, University of

    of a gel electrophoresis experiment F. Gibson, A. Wipat, M. Pocock and N. Morris. TECHNICAL REPORT SERIES January, 2006 A data model for the representation of a gel electrophoresis experiment Frank Gibson, Anil and further analysis, and provide a standard repository format for proteomics. Gel electrophoresis can form

  11. Microchip DNA electrophoresis with automated whole-gel scanning detection.

    PubMed

    Lo, Roger C; Ugaz, Victor M

    2008-12-01

    Gel electrophoresis continues to play an important role in miniaturized bioanalytical systems, both as a stand alone technique and as a key component of integrated lab-on-a-chip diagnostics. Most implementations of microchip electrophoresis employ finish-line detection methods whereby fluorescently labeled analytes are observed as they migrate past a fixed detection point near the end of the separation channel. But tradeoffs may exist between the simultaneous goals of maximizing resolution (normally achieved by using longer separation channels) and maximizing the size range of analytes that can be studied (where shorter separation distances reduce the time required for the slowest analytes to reach the detector). Here we show how the miniaturized format can offer new opportunities to employ alternative detection schemes that can help address these issues by introducing an automated whole-gel scanning detection system that enables the progress of microchip-based gel electrophoresis of DNA to be continuously monitored along an entire microchannel. This permits flexibility to selectively observe smaller faster moving fragments during the early stages of the separation before they have experienced significant diffusive broadening, while allowing the larger slower moving fragments to be observed later in the run when they can be better resolved but without the need for them to travel the entire length of the separation channel. Whole-gel scanning also provides a continuous and detailed picture of the electrophoresis process as it unfolds, allowing fundamental physical parameters associated with DNA migration phenomena (e.g., mobility, diffusive broadening) to be rapidly and accurately measured in a single experiment. These capabilities are challenging to implement using finish-line methods, and make it possible to envision a platform capable of enabling separation performance to be rapidly screened in a wide range of gel matrix materials and operating conditions, even allowing separation and matrix characterization steps to be performed simultaneously in a single self-calibrating experiment. PMID:19023477

  12. Plasmid replication in Xenopus eggs and egg extracts: a 2D gel electrophoretic analysis.

    PubMed Central

    Hyrien, O; Mchali, M

    1992-01-01

    We have examined the replication patterns of ribosomal DNA plasmids in vivo and in vitro using Xenopus eggs. Plasmids carrying different parts of the Xenopus ribosomal DNA sequence were allowed to replicate either in vitro in an egg extract or in vivo after microinjection into unfertilized eggs. The replication intermediates were analyzed by the 2D gel electrophoretic technique of Brewer and Fangman (1), using original or modified electrophoresis conditions. With standard electrophoresis conditions, the patterns obtained for restriction fragments larger than 5 kb were unreliable because of artefactually distorted Y arcs and unrecognizable bubble arcs. Interpretable patterns could nevertheless be obtained using suitably modified electrophoresis parameters. Under these conditions, replication was found to initiate and terminate at multiple, random locations on each plasmid both in vivo and in vitro. However, only one or very few of these potential initiation sites are used during the replication of an individual plasmid molecule. We discuss the possible artefacts and misinterpretations that can result when the 2D electrophoresis parameters are not adapted to the size of the fragment examined. We also discuss the relevance of the random replication mode to the mechanisms and the control of DNA replication in eukaryotes. Images PMID:1349740

  13. Thermally reversible gels in electrophoresis. I - Matrix characterization

    NASA Technical Reports Server (NTRS)

    Righetti, Pier Giorgio; Snyder, Robert S.

    1988-01-01

    Two series of thermally reversible hydrogen-bonded gels have been characterized: (5 pct) PVA-(4 pct) PEG and (5 pct) PVA-(0.04 pct) borate gels. They both have extremely low melting points (16-17 C) and could be of potential interest for recovery of proteins after preparative electrophoresis. The PVA-borate gels can be exploited in the pH range 7-11 by progressively increasing the borate content in the pH interval 8 to 7 and concomitantly decreasing the borate levels in the pH zone 8 to 11. It is hypothesized that the low melting point of these gels is due to the fact that they are sparingly and sparsely hydrogen bonded along the PVA chain: on the average, 1 OH group out of 3 or 4 OH groups in the PVA polymer should be engaged in H-bond formation.

  14. SDS-PAGE (Sodium dodecyl sulphate Poly acrylamide gel electrophoresis) 1. Glass cassette and casting stand

    E-print Network

    Schmidt, Marius

    SDS-PAGE (Sodium dodecyl sulphate Poly acrylamide gel electrophoresis) 1. Glass cassette the wells with dH2O. Here gel cassette is ready to go. 3. Electrophoresis Module assembly Place the gel between the glass plates with a piece of paper. 2. Gel preparation and casting Place the comb between

  15. Deployment of Freestanding Polyacrylamide Gel Electrophoresis Platform for Point-of-Care Diagnostic Applications

    E-print Network

    Sekhon, Jasjeet S.

    Deployment of Freestanding Polyacrylamide Gel Electrophoresis Platform for Point-of-Care Diagnostic polyacrylamide gel (fsPAG) electrophoresis is proposed as a cheap and robust platform for the diagnosis of preserving reagents within the gels; and the conditions under which the gels are dehydrated and rehydrated

  16. SDS capillary gel electrophoresis of proteins in microfabricated channels

    PubMed Central

    Yao, Shao; Anex, Deon S.; Caldwell, W. Brett; Arnold, Don W.; Smith, Katherine B.; Schultz, Peter G.

    1999-01-01

    Analysis of variations in the concentrations or structures of biomolecules (e.g., mRNAs, proteins, peptides, natural products) that occur either naturally or in response to environmental or genetic perturbations can provide important insight into complex biological processes. Many biological samples are mixtures that require a separation step before quantitation of variations in the individual components. Two-dimensional denaturing gel electrophoresis has been used very effectively to separate complex mixtures of proteins, but it is time consuming and requires considerable amounts of sample. Microchannel-based separations have proven very effective in rapidly separating small amounts of nucleic acids; more recently, isoelectric focusing of proteins also has been adapted to the microchannel format. Here, we describe microchannel-based SDS capillary gel electrophoresis of proteins and demonstrate the speed and high resolution it provides. This development is an important step toward the miniaturization and integration of multidimensional and array separation methods for complex protein mixtures. PMID:10318890

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

    PubMed Central

    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 utility still exist. This review discusses the principles of 2-DE as well as both recent methodological advances and new applications. PMID:24735559

  18. nature biotechnology volume 26 number 8 august 2008 863 Guidelines for reporting the use of gel electrophoresis

    E-print Network

    Cai, Long

    electrophoresis in proteomics To the editor: We wish to alert your readers to the MIAPE Gel Electrophoresis (MIAPE-dimensional gel electrophoresis in a proteomics experiment. Developed through a joint effort between the gel-GE-compliant submission2. Gel electrophoresis facilitates the separation of protein (or peptide) mixtures, usually

  19. Effectiveness and limitation of two-dimensional gel electrophoresis in bacterial membrane protein proteomics and perspectives.

    PubMed

    Bunai, Keigo; Yamane, Kunio

    2005-02-01

    Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using isoelectric focusing and SDS-PAGE in the first and second dimensions, respectively, is an established means of simultaneously separating over 1000 proteins and two new types have recently been developed. These procedures have significant shortcomings such as low load ability and poor separation of hydrophobic, acidic and alkaline proteins. We therefore modified the protocols to analyze the Bacillus subtilis membrane proteome. The 2D-PAGE techniques effectively separated membrane proteins having one and two transmembrane segments but not those with more than four. Compared with new LC/MS/MS procedures that are independent of electrophoretic separation, 2D-PAGE can globally analyze and quantify proteins at various stages of the cell cycle when labeled with isotopes such as 35S-methionine or the stable isotope, 15N. PMID:15652812

  20. HIGH THROUGHPUT PROTEIN IDENTIFICATION USING 2-DIMENSIONAL DIFFERENCE GEL ELECTROPHORESIS AND ROBOTIC SPOT PICKING FOR ALUMINUM TOLERANCE-RELATED MAIZE ROOT TIP PROTEINS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two-dimensional difference gel electrophoresis (2-D DIGE) is the most effective method utilized to carry out gel-based quantitative proteomics. Unfortunately, the most popular image analysis software used to process DIGE images (DeCyder) produces picking coordinates in a format that is incompatible...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  3. LLaannggeerrhhaannss LLaabb PPrroottooccoollss Agarose Gel Electrophoresis.docx revised 11/2/12 by JW pg 1 of 3

    E-print Network

    Langerhans, Brian

    LLaannggeerrhhaannss LLaabb PPrroottooccoollss Agarose Gel Electrophoresis.docx revised 11/2/12 by JW pg 1 of 3 Agarose Gel Electrophoresis Protocol (Making, Loading, Running, & Viewing) CAUTION: Wear Agarose Gel Electrophoresis.docx revised 11/2/12 by JW pg 2 of 3 Running the Gel 1. After the gel has

  4. Two-dimensional gel electrophoresis in bacterial proteomics.

    PubMed

    Curreem, Shirly O T; Watt, Rory M; Lau, Susanna K P; Woo, Patrick C Y

    2012-05-01

    Two-dimensional gel electrophoresis (2-DE) is a gel-based technique widely used for analyzing the protein composition of biological samples. It is capable of resolving complex mixtures containing more than a thousand protein components into individual protein spots through the coupling of two orthogonal biophysical separation techniques: isoelectric focusing (first dimension) and polyacrylamide gel electrophoresis (second dimension). 2-DE is ideally suited for analyzing the entire expressed protein complement of a bacterial cell: its proteome. Its relative simplicity and good reproducibility have led to 2-DE being widely used for exploring proteomics within a wide range of environmental and medically-relevant bacteria. Here we give a broad overview of the basic principles and historical development of gel-based proteomics, and how this powerful approach can be applied for studying bacterial biology and physiology. We highlight specific 2-DE applications that can be used to analyze when, where and how much proteins are expressed. The links between proteomics, genomics and mass spectrometry are discussed. We explore how proteomics involving tandem mass spectrometry can be used to analyze (post-translational) protein modifications or to identify proteins of unknown origin by de novo peptide sequencing. The use of proteome fractionation techniques and non-gel-based proteomic approaches are also discussed. We highlight how the analysis of proteins secreted by bacterial cells (secretomes or exoproteomes) can be used to study infection processes or the immune response. This review is aimed at non-specialists who wish to gain a concise, comprehensive and contemporary overview of the nature and applications of bacterial proteomics. PMID:22610887

  5. Two-dimensional gel electrophoresis in proteomics: past, present and future Thierry Rabilloud1,2

    E-print Network

    Paris-Sud XI, Université de

    Two-dimensional gel electrophoresis in proteomics: past, present and future Thierry Rabilloud1;Abstract Two-dimensional gel electrophoresis has been instrumental in the birth and developments. In this review, a historical perspective is made, starting from the days where two-dimensional gels were used

  6. Analysis of protein glycation using fluorescent phenylboronate gel electrophoresis

    PubMed Central

    Pereira Morais, Marta P.; Marshall, Dominic; Flower, Stephen E.; Caunt, Christopher J.; James, Tony D.; Williams, Robert J.; Waterfield, Nicholas R.; van den Elsen, Jean M. H.

    2013-01-01

    Glycated proteins are important biomarkers for age-related disorders, however their analysis is challenging because of the complexity of the protein-carbohydrate adducts. Here we report a method that enables the detection and identification of individual glycated proteins in complex samples using fluorescent boronic acids in gel electrophoresis. Using this method we identified glycated proteins in human serum, insect hemolymph and mouse brain homogenates, confirming this technique as a powerful proteomics tool that can be used for the identification of potential disease biomarkers. PMID:23531746

  7. Comparison of protein expression profiles between three Perkinsus spp., protozoan parasites of molluscs, through 2D electrophoresis and mass spectrometry.

    PubMed

    Fernndez-Boo, S; Chicano-Glvez, E; Alhama, J; Barea, J L; Villalba, A; Cao, A

    2014-05-01

    The genus Perkinsus includes protozoan parasites of a wide range of marine molluscs worldwide, some of which have been responsible for heavy mollusc mortalities and dramatic economic losses. This study was performed with the aim of increasing the knowledge of Perkinsus spp. proteome. Proteins extracted from in vitro cultured cells of three species of this genus, P. marinus, P. olseni and P. chesapeaki, were analysed using 2D electrophoresis. Four gels from each species were produced. Qualitative and quantitative comparisons among gels were performed with Proteamweaver software. Cluster analysis grouped the four gels of each Perkinsus sp.; furthermore, P. marinus and P. olseni gels were grouped in a cluster different from P. chesapeaki. Around 2000 spots of each species were considered, from which 213 spots were common to the 3 species; P. chesapeaki and P. marinus shared 310 spots, P. chesapeaki and P. olseni shared 315 spots and P. marinus and P. olseni shared 242 spots. A number of spots were exclusive of each Perkinsus species: 1161 spots were exclusive of P. chesapeaki, 1124 of P. olseni and 895 of P. marinus. A total of 84 spots, including common and species-specific ones, were excised from the gels and analysed using MALDI-TOF and nESI-IT (MS/MS) techniques. Forty-two spots were successfully sequenced, from which 28 were annotated, most of them clustered into electron transport, oxidative stress and detoxification, protein synthesis, carbohydrate metabolism, signal transduction, metabolic process and proteolysis. PMID:24607654

  8. Top-down, bottom-up, and side-to-side proteomics with virtual 2-D gels

    NASA Astrophysics Data System (ADS)

    Ogorzalek Loo, Rachel R.; Hayes, Richard; Yang, Yanan; Hung, Frank; Ramachandran, Prasanna; Kim, Nuri; Gunsalus, Robert; Loo, Joseph A.

    2005-02-01

    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-down sequencing for protein identification. Methods have been developed to enzymatically digest all proteins in an IEF gel simultaneously, leaving the polyacrylamide gel attached to its polyester support. By retaining all gel pieces and their placement relative to one another, sample handling and tracking are minimized, and comparison to 2-D gel images is facilitated. MALDI-MS and MS/MS can then be performed directly from dried, matrix-treated IPG strips following whole-gel trypsin digestion, bottom-up methodology. Side-to-side proteomics, highlighting the link between virtual and classical 2-D gel electrophoresis, is introduced to describe a method whereby intact masses are measured from one side (the IEF gel), while proteins are identified based on analyses performed from the other side (the SDS-PAGE gel).

  9. Separation of radiolabelled glycosaminoglycan oligosaccharides by polyacrylamide-gel electrophoresis.

    PubMed

    Hampson, I N; Gallagher, J T

    1984-08-01

    Glycosaminoglycan oligosaccharides generated by treatment of biosynthetically radiolabelled dermatan sulphate and hyaluronic acid with chondroitin AC lyase or testicular hyaluronidase may be resolved into a series of discrete bands by polyacrylamide-gel electrophoresis. Bands were identified by fixation in glacial acetic acid containing 20% (w/v) 2,5-diphenyloxazole followed by fluorography. The bands represented glycans which differed in size by one disaccharide unit. For the larger oligosaccharides (decasaccharides and above) of similar charge: mass ratio, there was a linear relationship between electrophoretic mobility and log Mr. However, the smaller species showed anomalous migration patterns. Consideration of the structures of the fragments produced by the different enzyme treatments suggests that copolymeric and homopolymeric oligosaccharides may be separated by polyacrylamide-gel electrophoresis. There are many potential applications of this technique, foremost amongst them being studies on the molecular size heterogeneity and patterns of enzyme-mediated depolymerization of native glycosaminoglycan chains and investigations into rates of polymer chain elongation and post-polymerization modification reactions so essential to glycosaminoglycan function. PMID:6477495

  10. Microscopic Dynamics of Quasi-2D Dense Colloidal Gels

    NASA Astrophysics Data System (ADS)

    Lohr, Matthew; Yodh, Arjun

    2011-03-01

    In this work, we investigate the microscopic dynamics of quasi-2D dense attractive colloidal systems. We confine bidisperse polystyrene spheres between glass coverslips in a suspension of water and 2,6-lutidine; as we increase the temperature of the sample into a critical regime, lutidine wets the colloids, creating a strong attractive interaction ( > 4kT). We specifically study suspensions in the "dense gel" regime, i.e., at a volume fraction high enough that the attractive particles form a spanning cluster, yet just low enough that there exists some structural heterogeneity larger than the individual particle size. We track the particle locations via bright-field video microscopy and analyze the dynamics of the system in order to compare them to lower-volume-fraction gel states and higher-volume-fraction glassy states. In doing so, we pinpoint the similarities and differences in the mechanisms for dynamic arrest in low-density colloidal gels and high-density colloidal glasses.

  11. Accounting for Spot Matching Uncertainty in the Analysis of Proteomics Data from Two-dimensional Gel Electrophoresis

    E-print Network

    Maitra, Ranjan

    -dimensional Gel Electrophoresis Volodymyr Melnykov , Ranjan Maitra and Dan Nettleton Abstract Two-dimensional gel electrophoresis is a biochemical technique that combines isoelectric focusing and SDS- polyacrylamide gel model 1 Introduction Two-dimensional gel electrophoresis (2DGE) is one of the oldest and most commonly

  12. Two-dimensional gel electrophoresis in proteomics: a tutorial Thierry Rabilloud 1,2

    E-print Network

    Paris-Sud XI, Université de

    1 Two-dimensional gel electrophoresis in proteomics: a tutorial Thierry Rabilloud 1,2 , Cécile)-4-38-78-44-99 e-mail: Thierry.Rabilloud@ cea.fr #12;2 Abstract Two-dimensional electrophoresis of proteins has of the process, from sample preparation to in-gel detection of proteins, commenting the constraints and caveats

  13. Detection of Polymorphisms of Human DNA by Gel Electrophoresis as Single-Strand Conformation Polymorphisms

    Microsoft Academic Search

    Masato Orita; Hiroyuki Iwahana; Hiroshi Kanazawa; Kenshi Hayashi; Takao Sekiya

    1989-01-01

    We developed mobility shift analysis of single-stranded DNAs on neutral polyacrylamide gel electrophoresis to detect DNA polymorphisms. This method follows digestion of genomic DNA with restriction endonucleases, denaturation in alkaline solution, and electrophoresis on a neutral polyacrylamide gel. After transfer to a nylon membrane, the mobility shift due to a nucleotide substitution of a single-stranded DNA fragment could be detected

  14. On-Chip Native Gel Electrophoresis-Based Immunoassays for Tetanus Antibody and Toxin

    E-print Network

    Herr, Amy E.

    On-Chip Native Gel Electrophoresis-Based Immunoassays for Tetanus Antibody and Toxin Amy E. Herr device, we have developed a microanalytical platform for performing electrophoresis- based immunoassays. The microfluidic immunoassays are performed by gel electrophoretic separation and quanti- tation of bound

  15. Gel-Electrophoresis and Diffusion of Ring-Shaped DNA

    E-print Network

    Uri Alon; David Mukamel

    1997-02-18

    A model for the motion of ring-shaped DNA in a gel is introduced and studied by numerical simulations and a mean-field approximation. The ring motion is mediated by finger-shaped loops (hernias) that move in an amoeba-like fashion around the gel obstructions. This constitutes an extension of previous reptation tube treatments. It is shown that tension is essential for describing the dynamics in the presence of hernias. It is included in the model as long range interactions over stretched DNA regions. The mobility of ring-shaped DNA is found to saturate much as in the well-studied case of linear DNA. Experiments in polymer gels, however, show that the mobility drops exponentially with the DNA ring size. This is commonly attributed to dangling-ends in the gel that can impale the ring. The predictions of the present model are expected to apply to artificial 2D obstacle arrays (W.D. Volkmuth, R.H. Austin, Nature 358,600 (1992)) which have no dangling-ends. In the zero-field case an exact solution of the model steady-state is obtained, and quantities such as the average ring size are calculated. An approximate treatment of the ring dynamics is given, and the diffusion coefficient is derived. The model is also discussed in the context of spontaneous symmetry breaking in one dimension.

  16. Pulsed-Field Gel Electrophoresis of Yersinia pestis.

    PubMed

    Revazishvili, Tamara; Johnson, Judith A

    2015-01-01

    Yersinia pestis is a human pathogen and can cause serious disease. Biosafety level 3 (BSL3) is required when handling this microorganism and all work requires a biological safety cabinet. For pulsed-field gel electrophoresis (PFGE), dedicated BSL3 PFGE equipment or a documented procedure that ensures that all viable bacteria are inactivated is required. All plasticware and glassware that comes into contact with the cultures should be disinfected/sterilized or disposed of in a safe manner, according to the guidelines of institution. This includes decontamination of pipettes, spatulas, etc. that were in contact with the cell suspensions or plugs. Disinfection of reusable plug molds should be done before they are washed; the disposable plug molds, including the tape and the tab that was used to push the plugs out of the wells, are also contaminated and should be disinfected with 10 % bleach for at least 30 min if they will be washed and reused. PMID:25862053

  17. Protein Separation by Capillary Gel Electrophoresis: A Review

    PubMed Central

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

    2011-01-01

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

  18. Diffusion constant for the repton model of gel electrophoresis

    E-print Network

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

    1997-03-10

    The repton model is a simple model of the "reptation" motion by which DNA diffuses through a gel during electrophoresis. In this paper we show that the model can be mapped onto a system consisting of two types of particles with hard-sphere interactions diffusing on a one-dimensional lattice. Using this mapping we formulate an efficient Monte Carlo algorithm for the model which allows us to simulate systems more than twice the size of those studied before. Our results confirm scaling hypotheses which have previously been put forward for the model. We also show how the particle version of the model can be used to construct a transfer matrix which allows us to solve exactly for the diffusion constant of small repton systems. We give results for systems of up to 20 reptons.

  19. Monitoring Piscirickettsia salmonis by denaturant gel electrophoresis and competitive PCR.

    PubMed

    Heath, S; Pak, S; Marshall, S; Prager, E M; Orrego, C

    2000-05-25

    Reported strains of Piscirickettsia salmonis, a pathogen of salmonid fishes, were analyzed by amplifying part of the internal transcribed spacer (ITS) of the ribosomal RNA (rRNA) operon followed by denaturing gradient gel electrophoresis (DGGE) of the amplicons. All amplified fragments differing in sequence were distinguished by migration during DGGE. A simpler format, constant denaturant gel electrophoresis (CDGE), allowed the same diagnostic distinctions among strains. Sampling during 1997 and 1998 of salmonids from 5 different sites on and near Chilo Island in southern Chile displaying piscirickettsiosis revealed only P. salmonis resembling LF-89, the type strain first isolated in 1989. These observations are encouraging for control strategies, which might otherwise be compromised by unpredictable shifts of P. salmonis types in salmon farms. A competitive PCR assay offered insight about the power of PCR for quantification and about specific tissue invasiveness by this intracellular pathogen. This approach revealed that the PCR could amplify approximately 1 to 10 P. salmonis genome equivalents against a background of > 99.9% salmonid DNA. It also raised the possibility that the salmonid brain is an important site for P. salmonis survival, with its bacterial load in 1 individual having been about 100 times the loads observed in liver and kidney. Pathogen detection by competitive PCR in a surface seawater sample from a netpen in use indicated a density of about 3000 to 4000 P. salmonis cells (or their DNA remnants) 1(-1). Such quantitative estimates should aid decisions about disease prevention and management as, for example, choice of netpen sites following fallow periods and certification of ova, which are known conduits of infection. PMID:10907135

  20. Proteome mapping by two-dimensional polyacrylamide gel electrophoresis in combination with mass spectrometric protein sequence analysis

    Microsoft Academic Search

    Ettore Appella; David Arnott; Kazuyasu Sakaguchi; Peter J. Wirth

    \\u000a The high resolving power of two-dimensional polyacrylamide gel electrophoresis 2D-PAGE and its full analytical and preparative\\u000a potential have been described with special emphasis on reproducibility and standardization of protein spot patterns, enhanced\\u000a protein detection sensitivity, and computer analysis database development. New methodologies for peptide mass fingerprinting,\\u000a peptide, sequence, and fragmention tagging have been highlighted. Major challenges associated with 2D-PAGE\\/mass spectrometric

  1. An Iterative Algorithm for Segmenting Lanes in Gel Electrophoresis Images ALEXEI M. C. MACHADO1;3

    E-print Network

    An Iterative Algorithm for Segmenting Lanes in Gel Electrophoresis Images ALEXEI M. C. MACHADO1 detection on gel electrophoresis computer images. The problem can be posed as the determination fea- tures. The gel electrophoresis exam consists in breaking a molecule into many fragments

  2. Horizontal comparative fluorescence two-dimensional gel electrophoresis for improved spot coordinate detection.

    PubMed

    Hanneken, Marina; Knig, Simone

    2014-04-01

    Vertical comparative 2D fluorescence gel electrophoresis (CoFGE) has recently been shown to increase the reproducibility of coordinate assignment for protein spots, in particular in singular experiments, which cannot be investigated using DIGE. The method applies a standardized marker grid formed by a set of purified proteins to the sample proteome in a conglomerate of 1DE, 2DE, and DIGE. Here, improvements are demonstrated by transferring CoFGE to horizontal 2DE. These include the elimination of the protein modification by residual acrylamide monomer unavoidable in vertical CoFGE, reduced buffer volumes, and highly efficient laboratory procedures. Spot patterns are well defined and can be easily analyzed using commercially available warping algorithms. With horizontal CoFGE also a correction for changes in pI was introduced using a third fluorescent dye. Horizontal CoFGE holds high promises in comparative proteomics. PMID:24347295

  3. The state of the art in the analysis of two-dimensional gel electrophoresis images

    PubMed Central

    Berth, Matthias; Moser, Frank Michael; Kolbe, Markus

    2007-01-01

    Software-based image analysis is a crucial step in the biological interpretation of two-dimensional gel electrophoresis experiments. Recent significant advances in image processing methods combined with powerful computing hardware have enabled the routine analysis of large experiments. We cover the process starting with the imaging of 2-D gels, quantitation of spots, creation of expression profiles to statistical expression analysis followed by the presentation of results. Challenges for analysis software as well as good practices are highlighted. We emphasize image warping and related methods that are able to overcome the difficulties that are due to varying migration positions of spots between gels. Spot detection, quantitation, normalization, and the creation of expression profiles are described in detail. The recent development of consensus spot patterns and complete expression profiles enables one to take full advantage of statistical methods for expression analysis that are well established for the analysis of DNA microarray experiments. We close with an overview of visualization and presentation methods (proteome maps) and current challenges in the field. PMID:17713763

  4. Two-Dimensional Gel Electrophoresis: Discovering Biomolecules for Environmental Bioremediation

    NASA Astrophysics Data System (ADS)

    Singh, Om V.; Chandel, Anuj K.

    Environmental contamination has been viewed as an ecological malaise for which bioremediation can be prescribed as a perfect medicine. The solution to the problems with bioremediation lies in analyzing to what extent the microbes physiological machinery contributes to the degradation process and which biomolecules and their mechanisms are responsible for regulatory factors within the degradation system, such as protein, metabolite, and enzymatic chemical transformation. In the post-genomic era, recent advances in proteomics have allowed us to elucidate many complex biological mechanisms. Two-dimensional gel electrophoresis (2DE) in conjunction with mass spectrometry (MS) can be utilized to identify the biomolecules and their molecular mechanisms in bioremediation. A set of highly abundant global proteins over a pI range 4-7 was separated and compared by size fractionation (25-100 kDa) on 2DE. We identified a set of catabolic proteins, enzymes, and heat shock molecular chaperones associated with the regulatory network that was found to be overexpressed under phenol-stressed conditions. This chapter also offers optimized ideal directions for 2DE, followed by easy-to-follow directions for a protein identification strategy using MALDI-TOF and targeting novel proteins/enzymes for a universal set of experiments.

  5. Misincorporation during DNA synthesis, analyzed by gel electrophoresis.

    PubMed Central

    Hillebrand, G G; McCluskey, A H; Abbott, K A; Revich, G G; Beattie, K L

    1984-01-01

    A method has been developed for simultaneous comparison of the propensity of a DNA polymerase to misincorporate at different points on a natural template-primer. In this method elongation of a [5'-32P] primer, annealed to a bacteriophage template strand, is carried out in the presence of only three dNTPs (highly purified by HPLC). Under these conditions the rate of primer elongation (monitored by gel electrophoresis/autoradiography) is limited by the rate of misincorporation at template positions complementary to the missing dNTP. Variations in the rate of elongation (revealed by autoradiographic banding patterns) reflect variations in the propensity for misincorporation at different positions along the template. The effect on primer elongation produced by addition of a chemically modified dNTP to 'minus' reactions reveals the mispairing potential of the modified nucleotide during DNA synthesis. By use of this electrophoretic assay of misincorporation we have demonstrated that the fidelity of E. coli DNA polymerase I varies greatly at different positions along a natural template, and that BrdUTP and IodUTP can be incorporated in place of dCTP during chain elongation catalyzed by this enzyme. Images PMID:6326053

  6. Analysis of variations in band positions for normalization in across-gel denaturing gradient gel electrophoresis.

    PubMed

    Matsushita, Yuko; Yamamura, Kohji; Morimoto, Sho; Bao, Zhihua; Kurose, Daisuke; Sato, Ikuo; Yoshida, Shigenobu; Tsushima, Seiya

    2015-05-01

    Variation in band position between gels is a well-known problem in denaturing gradient gel electrophoresis (DGGE). However, few reports have evaluated the degree of variation in detail. In this study, we investigated the variation in band positions of DNA samples extracted from soil, normalized using reference positions within marker lanes for DGGE in three organismal (bacterial, fungal, and nematode) conditions. For sample lanes, marker DNA (as a control) and sample DNA were used. The test for normality of distribution showed that the position data of a large percentage of bands were normally distributed but not for certain bands. For the normally-distributed data, their variations [standard deviation of marker bands (SDM) and standard deviation of sample bands (SDS), respectively] were assessed. For all organismal conditions, the degree of within-gel variation were similar between SDMs and SDSs, while between-gel variations in SDSs were larger than those in SDMs. Due to the large effect of between-gel variations, the total variations in SDSs were more varied between sample bands, and the mean variations of all sample bands were higher than those in the markers. We found that the total variation in the fungal and nematode SDSs decreased when the intervals between marker bands were narrowed, suggesting that band interval is important for reducing total variation in normalized band positions. For the non-normally distributed data, the distribution was examined in detail. This study provided detailed information on the variation of band positions, which could help to optimize markers for reducing band position variation, and could aid in the accurate identification of bands in across-gel DGGE analyses. PMID:25725304

  7. Congruence between starch gel and polyacrylamide gel electrophoresis in detecting allozyme variation in pulmonate land slugs.

    PubMed

    Geenen, Sofie; Jordaens, Kurt; Castilho, Rita; Backeljau, Thierry

    2003-02-01

    The predominantly selfing slug species Arion (Carinarion) fasciatus, A. (C.) silvaticus and A. (C.) circumscriptus are native in Europe and have been introduced into North America, where each species consists of a single, homozygous multilocus genotype (strain), as defined by starch gel electrophoresis (SGE) of allozymes. In Europe, the "one strain per species" hypothesis does not hold since polyacrylamide gel electrophoresis (PAGE) of allozymes uncovered 46 strains divided over the three species. However, electrophoretic techniques may differ in their ability to detect allozyme variation. Therefore, several Carinarion populations from both continents were screened by applying the two techniques simultaneously on the same individual slugs and enzyme loci. SGE and PAGE yielded exactly the same results, so that the different degree of variation in North American and European populations cannot be attributed to differences in resolving power between SGE and PAGE. We found four A. (C.) silvaticus strains in North America indicating that in this region the "one strain per species" hypothesis also cannot be maintained. Hence, the discrepancies between previous electrophoretic studies on Carinarion are most likely due to sampling artefacts and possible founder effects. PMID:12601729

  8. technical manual protein electrophoresis

    E-print Network

    Kirschner, Marc W.

    . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Chapter 2 Polyacrylamide Gel Electrophoresis.2 Separating proteins on the basis of molecular weight: SDS gel electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 2.5 Native gel electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 2

  9. Photo-initiated cross-linked polyacrylamide gels for microdevice electrophoresis

    E-print Network

    Agrawal, Shilpa

    2005-08-29

    Photo-polymerized cross-linked polyacrylamide gels are becoming increasingly important for use in micro-fabricated DNA electrophoresis systems because they allow a concentrated sieving matrix to be precisely positioned at any location within a...

  10. Two dimensional gel electrophoresis analysis of DNA replication in Tetrahymena pyriformis

    E-print Network

    Ma, Yue

    1997-01-01

    TWO DIMENSIONAL GEL ELECTROPHORESIS ANALYSIS OF DNA REPLICATION IN TETRAHYMENA PYRIFORMJS A Thesis by YUE MA Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree... of MASTER OF SCIENCE December 1997 Major Subject: Medical Sciences TWO DIMENSIONAL GEL ELECTROPHORESIS ANALYSIS OF DNA REPLICATION IN TETRAHYMENA PYRIFORMIS A Thesis Submitted to Texas A&M University in partial fulfillment of the requirements...

  11. A versatile polyacrylamide gel electrophoresis based sulfotransferase assay

    PubMed Central

    2010-01-01

    Background Sulfotransferases are a large group of enzymes that regulate the biological activity or availability of a wide spectrum of substrates through sulfation with the sulfur donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS). These enzymes are known to be difficult to assay. A convenient assay is needed in order to better understand these enzymes. Results A universal sulfotransferase assay method based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is described. This assay has been successfully applied to substrates as small as ?-naphthol and as big as proteoglycans. As examples, we present the assays for recombinant human CHST4, TPST1, CHST3 and HS6ST1. In order to assess whether a small molecule can be applicable to this type of assay, a method to estimate the relative mobility of a molecule to PAPS is also presented. The estimated relative mobilities of various sulfated small molecules generated by SULT1A1, SULT1E1, SULT2A1 and CHST4 are in the range of 0.2 of the actual relative mobilities. Conclusion The versatility of the current method comes from the ability that SDS-PAGE can separate proteins and small molecules according to different parameters. While mobilities of proteins during SDS-PAGE are inversely related to their sizes, mobilities of small molecules are positively related to their charge/mass ratios. The predicted relative mobility of a product to PAPS is a good indicator of whether a sulfotransferase can be assayed with SDS-PAGE. Because phosphorylation is most similar to sulfation in chemistry, the method is likely to be applicable to kinases as well. PMID:20146816

  12. Sequence analyses of rat liver cytosolic proteins separated by gel electrophoresis using MALDI mass spectrometry

    E-print Network

    Ens, Werner

    was performed by in-gel tryptic digestion, MALDI mass spectrometry, and database search with peptide mass method by MALDI mass spectrometry also revealed that chemical modifications occurred at the residuesSequence analyses of rat liver cytosolic proteins separated by gel electrophoresis using MALDI mass

  13. Recovery of Native Proteins from Preparative Electrophoresis Gel Slices by Reverse Polarity ElutioN

    Microsoft Academic Search

    Aaron S. Abramovitz; Verrell Randolph; Aruna Mehra; Sylvia Chnstakos

    1984-01-01

    A technique for high yield recovery of native, biologically active proteins from preparative polyacrylamide gel slices by reverse polarity elution is described. No apparatus other than the standard slab gel electrophoresis system is required. Several proteins have been recovered in biologically active form at a 90% yield, in quantities ranging from 0.4 mg to 4.2 mg. The method is effective

  14. Agarose Gel Electrophoresis Reveals Structural Fluidity of a Phage T3 DNA Packaging Intermediate

    PubMed Central

    Serwer, Philip; Wright, Elena T.

    2012-01-01

    We find a new aspect of DNA packaging-associated structural fluidity for phage T3 capsids. The procedure is (1) glutaraldehyde cross-linking of in vivo DNA packaging intermediates for stabilization of structure and then (2) determining of effective radius by two-dimensional agarose gel electrophoresis (2d-AGE). The intermediates are capsids with incompletely packaged DNA (ipDNA) and without an external DNA segment; these intermediates are called ipDNA-capsids. We initially increase production of ipDNA-capsids by raising NaCl concentration during in vivo DNA packaging. By 2d-AGE, we find a new state of contracted shell for some particles of one previously identified ipDNA-capsid. The contracted shell-state is found when ipDNA length/mature DNA length (F) is above 0.17, but not at lower F. Some contracted-shell ipDNA-capsids have the phage tail; others do not. The contracted-shell ipDNA-capsids are explained by premature DNA maturation cleavage that makes accessible a contracted-shell intermediate of a cycle of the T3 DNA packaging motor. The analysis of ipDNA-capsids, rather than intermediates with uncleaved DNA, provides a simplifying strategy for a complete biochemical analysis of in vivo DNA packaging. PMID:22222979

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

    E-print Network

    Pal, Debnath

    Avoiding acidic region streaking in two-dimensional gel electrophoresis: Case study with two by two-dimensional gel electrophoresis. J. Commun. Dis. 38 255­262 Blokpoel MC, Smeulders MJ, Hubbard JA of IEF-program for 7 cm and 17 cm IPG strips. #12;Supplementary figure 6. 2DE gel images of E. coli WCPE

  16. Image processing methods and algorithms for accurate protein spot detection in 2-dimensional gel electrophoresis (2DGE)

    E-print Network

    Kouroupetroglou, Georgios

    -dimensional gel electrophoresis, denoising, image segmentation, spot detection, spot quantification, spot modeling electrophoresis (2DGE) and gives rise to protein spots of irregular shapes and sizes on the gel. Once proteinsImage processing methods and algorithms for accurate protein spot detection in 2-dimensional gel

  17. Sodium dodecyl sulfateagarose gel electrophoresis for the detection and isolation of amyloid curli fibers

    Microsoft Academic Search

    Chris Sitaras; Mahsa Naghavi; Muriel B. Herrington

    2011-01-01

    Curli are amyloid-like fibers on the surface of some strains of Escherichia coli and Salmonella enteritidis. We tested the use of horizontal sodium dodecyl sulfate (SDS)agarose gel electrophoresis to detect, isolate, and quantitate curli. Cell extracts fractionated in SDSagarose gels and stained with Coomassie blue exhibited a soluble fraction that entered the gel and an insoluble fraction that remained in

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

    PubMed

    Schrodt, Caroline; McHugh, Erin E; Gawinowicz, Mary Ann; Dupont, Herbert L; Brown, Eric L

    2013-01-01

    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) 90? 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. PMID:23922656

  19. Red wine proteins: two dimensional (2-D) electrophoresis and mass spectrometry analysis.

    PubMed

    Mainente, Federica; Zoccatelli, Gianni; Lorenzini, Marilinda; Cecconi, Daniela; Vincenzi, Simone; Rizzi, Corrado; Simonato, Barbara

    2014-12-01

    The aim of the present study was to optimize protein extraction from red wine (cv. Cabernet) in order to obtain a separation by two-dimensional electrophoresis (2-DE) compatible with mass spectrometry identification. Proteins were denatured by sodium dodecyl-sulphate (SDS) and precipitated as potassium salts. The potassium-DS (KDS) protein complexes obtained were treated with different solutions in order to remove the detergent. Proteins were solubilized with different buffers and separated by different electrophoretic approaches [native, urea, acid urea PAGEs and isoelectric focusing (IEF)] as the first-dimension (1-DE). The best 2D separation was achieved by using 10% saccharose in the DS removal step, and 6-cyclohexylhexyl ?-d-maltoside detergent in the solubilisation buffer combined with the IEF approach. Several well focalized protein spots were obtained and analyzed through mass-spectrometry. PMID:24996352

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

    PubMed

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

    2014-06-01

    Agarose gel electrophoresis of DNA and RNA is routinely performed using buffers containing either Tris, acetate, and EDTA (TAE) or Tris, borate, and EDTA (TBE). Gels are run at a low, constant voltage (?10 V/cm) to minimize current and asymmetric heating effects, which can induce band artifacts and poor resolution. In this study, alterations of gel structure and conductive media composition were analyzed to identify factors causing higher electrical currents during horizontal slab gel electrophoresis. Current was reduced when thinner gels and smaller chamber buffer volumes were used, but was not influenced by agarose concentration or the presence of ethidium bromide. Current was strongly dependent on the amount and type of EDTA used and on the concentrations of the major acid-base components of each buffer. Interestingly, resolution and the mobilities of circular versus linear plasmid DNAs were also affected by the chemical form and amount of EDTA. With appropriate modifications to gel structure and buffer constituents, electrophoresis could be performed at high voltages (20-25 V/cm), reducing run times by up to 3-fold. The most striking improvements were observed with small DNAs and RNAs (10-100 bp): high voltages and short run times produced sharper bands and higher resolution. PMID:24637158

  1. Separating DNA with different topologies by atomic force microscopy in comparison with gel electrophoresis

    PubMed Central

    Jiang, Yong; Rabbi, Mahir; Mieczkowski, Piotr A.; Marszalek, Piotr E.

    2010-01-01

    Atomic force microscopy, which is normally used for DNA imaging to gain qualitative results, can also be used for quantitative DNA research, at a single-molecular level. Here, we evaluate the performance of AFM imaging specifically for quantifying supercoiled and relaxed plasmid DNA fractions within a mixture, and compare the results with the bulk material analysis method - gel electrophoresis. The advantages and shortcomings of both methods are discussed in detail. Gel electrophoresis is a quick and well established quantification method. However, it requires a large amount of DNA, and needs to be carefully calibrated for even slightly different experimental conditions for accurate quantification. AFM imaging is accurate, in that single DNA molecules in different conformations can be seen and counted. When used carefully with necessary correction, both methods provide consistent results. Thus, AFM imaging can be used for DNA quantification, as an alternative to gel electrophoresis. PMID:20799746

  2. Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots

    DOEpatents

    Zhang, Jian-Shi (Shanghai, CN); Giometti, Carol S. (Glenview, IL); Tollaksen, Sandra L. (Montgomery, IL)

    1989-01-01

    After two-dimensional electrophoresis of proteins or the like, resulting in a polyacrylamide gel slab having a pattern of protein gel spots thereon, an individual protein gel spot is cored out from the slab, to form a gel spot core which is placed in an extraction tube, with a dialysis membrane across the lower end of the tube. Replicate gel spots can be cored out from replicate gel slabs and placed in the extraction tube. Molten agarose gel is poured into the extraction tube where the agarose gel hardens to form an immobilizing gel, covering the gel spot cores. The upper end portion of the extraction tube is filled with a volume of buffer solution, and the upper end is closed by another dialysis membrane. Upper and lower bodies of a buffer solution are brought into contact with the upper and lower membranes and are provided with electrodes connected to the positive and negative terminals of a DC power supply, thereby producing an electrical current which flows through the upper membrane, the volume of buffer solution, the agarose, the gel spot cores and the lower membrane. The current causes the proteins to be extracted electrophoretically from the gel spot cores, so that the extracted proteins accumulate and are contained in the space between the agarose gel and the upper membrane. A high percentage extraction of proteins is achieved. The extracted proteins can be removed and subjected to partial digestion by trypsin or the like, followed by two-dimensional electrophoresis, resulting in a gel slab having a pattern of peptide gel spots which can be cored out and subjected to electrophoretic extraction to extract individual peptides.

  3. Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots

    DOEpatents

    Zhang, Jian-Shi; Giometti, C.S.; Tollaksen, S.L.

    1987-09-04

    After two-dimensional electrophoresis of proteins or the like, resulting in a polyacrylamide gel slab having a pattern of protein gel spots thereon, an individual protein gel spot is cored out from the slab, to form a gel spot core which is placed in an extraction tube, with a dialysis membrane across the lower end of the tube. Replicate gel spots can be cored out from replicate gel slabs and placed in the extraction tube. Molten agarose gel is poured into the extraction tube where the agarose gel hardens to form an immobilizing gel, covering the gel spot cores. The upper end portion of the extraction tube is filled with a volume of buffer solution, and the upper end is closed by another dialysis membrane. Upper and lower bodies of a buffer solution are brought into contact with the upper and lower membranes and are provided with electrodes connected to the positive and negative terminals of a dc power supply, thereby producing an electrical current which flows through the upper membrane, the volume of buffer solution, the agarose, the gel spot cores and the lower membrane. The current causes the proteins to be extracted electrophoretically from the gel spot cores, so that the extracted proteins accumulate and are contained in the space between the agarose gel and the upper membrane. 8 figs.

  4. 2D capillary electrophoresis hyphenated with spectral detection for the determination of quinine in human urine.

    PubMed

    Mikus, Peter; Markov, Katarna; Veizerov, Lucia; Piestansky, Juraj; Galba, Jaroslav; Havrnek, Emil

    2012-01-01

    The possibilities of a column coupling two-dimensional capillary electrophoresis (2D CE) combined with fiber-based diode array detection (DAD) for the direct, highly reliable and ultrasensitive quantitative determination of quinine in real multicomponent ionic matrices (human urine) are demonstrated in this work. The capillary isotachophoresis (CITP) stage provided an on-line sample pretreatment (elimination of interfering matrix constituents, preseparation and preconcentration of the analyte) before the capillary zone electrophoresis (CZE) separation. Due to the large volume (30 L) sample injection and CITP sample preconcentration, a simple absorbance photometric detection was sufficient for obtaining very low concentration limits of detection (?8.6 ng/mL). The combination of the different separation mechanisms (CITP and CZE) resulted in enhanced separation selectivity. This enabled us to obtain a pure analyte zone in the directly injected real samples suitable for qualitative and quantitative evaluation. The spectral DAD allowed (i) characterization of the purity (i.e., spectral homogeneity) of the analyte zone; and (ii) preliminary indication of structurally related compounds (i.e., potential biodegradation products of quinine), via characteristic spectra recorded in intervals of 200-800 nm. The CITP-CZE-DAD method was characterized by favorable performance parameters that are suitable for its routine biomedical use. One of the primary benefits of the CITP-CZE-DAD method is the possibility of performing direct injections of real biological samples while avoiding external sample preparation procedures and, therefore, enhancing the reliability and applicability of analyses and the potential for method automatization and miniaturization. PMID:22677486

  5. Integrated protocol for reliable and fast quantification and documentation of electrophoresis gels.

    PubMed

    Rehbein, Peter; Schwalbe, Harald

    2015-06-01

    Quantitative analysis of electrophoresis gels is an important part in molecular cloning, as well as in protein expression and purification. Parallel quantifications in yield and purity can be most conveniently obtained from densitometric analysis. This communication reports a comprehensive, reliable and simple protocol for gel quantification and documentation, applicable for single samples and with special features for protein expression screens. As major component of the protocol, the fully annotated code of a proprietary open source computer program for semi-automatic densitometric quantification of digitized electrophoresis gels is disclosed. The program ("GelQuant") is implemented for the C-based macro-language of the widespread integrated development environment of IGOR Pro. PMID:25514201

  6. Use of denaturing gradient gel electrophoresis for analysis of the stool microbiota of hospitalized patients

    Microsoft Academic Search

    Curtis J. Donskey; Andrea M. Hujer; Sarbani M. Das; Nicole J. Pultz; Robert A. Bonomo; Louis B. Rice

    2003-01-01

    Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal RNA gene amplicons was used to study the stool microbiota of hospitalized patients and to examine the effect of antibiotic therapy. For one patient, 16 anaerobic species identified by random cloning and sequencing of PCR-amplified rRNA genes from stool were represented by bands on the DGGE gel. DGGE analysis and similarity index

  7. Kingella kingae KK247, an Atypical Pulsed-Field Gel Electrophoresis Clone A Strain

    PubMed Central

    Rouli, Laetitia; Robert, Catherine; Yagupsky, Pablo

    2014-01-01

    Kingella kingae strain KK247 was isolated from an adult Israeli patient with endocarditis. It belongs to pulsed-field gel electrophoresis clone A, has a 2,113,021-bp genome, a 15,507-bp plasmid that carries genes encoding ?-lactamases, and possesses 45 transposases, compared to the 5 detected in other K.kingae strains. PMID:25428974

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

    NASA Astrophysics Data System (ADS)

    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.

  9. Two-dimensional SDS-polyacrylamide gel electrophoresis of heat-modifiable outer-membrane proteins.

    PubMed

    Russell, R R

    1976-01-01

    An examination has been made of the effect which temperature of solubilization has upon the subsequent migration in SDS-polyacrylamide gel electrophoresis of proteins from the cell envelopes of Escherichia coli K12 and Neisseria sicca ATCC 9913. Conventional electrophoresis in tubes revealed substantial differences in the staining patterns of gels, depending upon whether the envelope samples were solubilized at 37 degrees C or 100 degrees C; in the case of N. sicca at least 6 of 13 discernible bands displayed heat-modifiable behavior. The relationship of the bands produced by each of the two temperatures was investigated by a two-dimensional electrophoresis procedure, in which a sample was solubilized at 37 degrees C and run in a usual cylindrical gel; the entire gel was then resolubilized at 100 degrees C, and laid along an acrylamide slab for electrophoresis in the second dimension. It was found that "free endotoxin" of both organisms examined contained the same major proteins as the total envelope fraction, and that these free endotoxin proteins showed the same heat-modifiable properties as when present in total envelopes. PMID:1252992

  10. Agar gel electrophoresis of proteolytic enzymes in gastric juice of patients with chronic gastritis

    Microsoft Academic Search

    Mona Agunod; George B. Jerzy Glass

    1972-01-01

    Agar gel electrophoresis with proteolytic digestion of albumin substrate and subsequent Chromoscan analysis of the agar slide, were used to study the proteolytic enzymes of gastric juice in 28 patients with atrophic gastritis with or without intestinal metaplasia, 7 patients with chronic superficial gastritis and 6 with a normal gastric mucosa. Five proteolytic enzyme spots, designated I through V in

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

    ERIC Educational Resources Information Center

    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

  12. Pulsed-Field Gel Electrophoresis and PCR Characterization of Environmental Vibrio parahaemolyticus Strains of Different Origins ?

    PubMed Central

    Suffredini, E.; Lopez-Joven, C.; Maddalena, L.; Croci, L.; Roque, A.

    2011-01-01

    The present study used pulsed-field gel electrophoresis (PFGE) characterization to examine the intraspecies variability and genetic relationships among environmental isolates of Vibrio parahaemolyticus from different European countries. This is first study performed on environmental V. parahaemolyticus that included more than one European country. PMID:21742917

  13. Single Cell Gel Electrophoresis (Comet) assay with plants: research on DNA repair and ecogenotoxicity testing.

    PubMed

    Ventura, Lorenzo; Giovannini, Annalisa; Savio, Monica; Don, Mattia; Macovei, Anca; Buttafava, Armando; Carbonera, Daniela; Balestrazzi, Alma

    2013-06-01

    Single Cell Gel Electrophoresis is currently used to investigate the cell response to genotoxic agents as well as to several biotic and abiotic stresses that lead to oxidative DNA damage. Different versions of Single Cell Gel Electrophoresis have been developed in order to expand the range of DNA lesions that can be detected and guidelines for their use in genetic toxicology have been provided. Applications of Single Cell Gel Electrophoresis in plants are still limited, compared to animal systems. This technique is now emerging as a useful tool in assessing the potential of higher plants as stable sensors in ecosystems and source of information on the genotoxic impact of dangerous pollutants. Another interesting application of Single Cell Gel Electrophoresis deals with Mutation Breeding or the combined use of irradiation and in vitro culture technique to enhance genetic variability in elite plant genotypes. SCGE, in combination with in situ detection of Reactive Oxygen Species (ROS) induced by ?-rays and expression analysis of both DNA repair and antioxidant genes, can be used to gather information on the radiosensitivity level of the target plant genotypes. PMID:23557725

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

    ERIC Educational Resources Information Center

    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

  15. Topological Patterns in Two-dimensional Gel Electrophoresis of DNA Knots

    E-print Network

    Michieletto, Davide; Orlandini, Enzo

    2015-01-01

    Gel electrophoresis is a powerful experimental method to probe the topology of DNA and other biopolymers. While there is a large body of experimental work which allows us to accurately separate different topoisomers of a molecule, a full theoretical understanding of these experiments has not yet been achieved. Here we show that the mobility of DNA knots depends crucially and subtly on the physical properties of the gel, and in particular on the presence of dangling ends. The topological interactions between these and DNA molecules can be described in terms of an "entanglement number", and yield a non-monotonic mobility at moderate fields. Consequently, in two-dimensional electrophoresis, gel bands display a characteristic arc pattern; this turns into a straight line when the density of dangling ends vanishes. We also provide a novel framework to accurately predict the shape of such arcs as a function of molecule length and topological complexity, which may be used to inform future experiments.

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

    PubMed Central

    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 and Alnus viridis subsp. crispa. Group C organisms were from A. incana subsp. rugosa and Comptonia peregrina nodules, and group D organisms were from A. incana subsp. rugosa, A. viridis subsp. cripsa, and Myrica pensylvanica root nodules. Isolates from each gel group were obtained at several widely separated geographical locations. The results indicate that two-dimensional polyacrylamide gel electrophoresis is useful for identifying Frankia isolates. Images PMID:16346488

  17. A new bond fluctuation method for a polymer undergoing gel electrophoresis

    E-print Network

    Ryuzo Azuma; Hajime Takayama

    1998-08-12

    We present a new computational methodology for the investigation of gel electrophoresis of polyelectrolytes. We have developed the method initially to incorporate sliding motion of tight parts of a polymer pulled by an electric field into the bond fluctuation method (BFM). Such motion due to tensile force over distances much larger than the persistent length is realized by non-local movement of a slack monomer at an either end of the tight part. The latter movement is introduced stochastically. This new BFM overcomes the well-known difficulty in the conventional BFM that polymers are trapped by gel fibers in relatively large fields. At the same time it also reproduces properly equilibrium properties of a polymer in a vanishing filed limit. The new BFM thus turns out an efficient computational method to study gel electrophoresis in a wide range of the electric field strength.

  18. Polymerase Chain Reaction (PCR) and Gel Electrophoresis Lab

    NSDL National Science Digital Library

    The HURI SURI project is developing a regional biotechnology workforce pipeline by expanding and supporting biotechnology research experiences for Jamestown Community College (JCC) undergraduates and disseminating these research experiences and materials to area high school teachers and students. This lab teaches students to "use PCR to amplify a plasmid form of DNA called pCDNA3.1(+)/GFP Plasmid DNA" and "run the DNA products formed during PCR on an agarose gel." The materials and procedure for this lab are described in detail.

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

    PubMed

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

    2004-08-01

    The metabolism of trace elements, in particular their binding to proteins in biological systems is of great importance in biochemical, toxicological, and pharmacological studies. As a result there has been a sustained interest over the last two decades in the speciation of protein-bound metals. Various analytical approaches have been employed, combining efficient separation of metalloproteins by liquid chromatography or electrophoresis with high-sensitivity elemental detection. Slab-gel electrophoresis (GE) is a key platform for high-resolution protein separation, and has been combined with autoradiography and various atomic spectrometric techniques for in-gel determination of protein-bound metals. Recently, the combination of GE with state-of-the-art inductively coupled plasma-mass spectrometry (ICP-MS), particularly when linked to laser ablation (LA) for direct gel interrogation, has opened up new opportunities for rapid characterization of metalloproteins. The use of GE and atomic spectrometry for the speciation of protein-bound trace elements is reviewed in this paper. Technical requirements for gel electrophoresis/atomic spectrometric measurement are considered in terms of method compatibilities, detection capability and potential usefulness. The literature is also surveyed to illustrate current status and future trends. PMID:15300764

  20. The whereabouts of 2D gels in quantitative proteomics Thierry Rabilloud

    E-print Network

    Paris-Sud XI, Universit de

    , completely dominated by tandem mass spectrometry (14, 15), 2D gel-based proteomics represents an exception in the sense that this is the only proteomics setup where i) protein quantification is not made in a mass that are then further characterized by mass spectrometry. These two cardinal features have put an enormous pressure

  1. Assessment of Pleiotropic Effects of a Gene Substitution in Pea by Two-Dimensional Polyacrylamide Gel Electrophoresis

    PubMed Central

    Gottlieb, L. D.; de Vienne, D.

    1988-01-01

    We examined, by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), near-isogenic lines of the r-gene in pea (Pisum sativum) which determines round (RR) vs. wrinkled (rr) seed. The study was undertaken to assess the number of protein changes resulting from a single gene substitution as a means of quantifying pleiotropic effects. A total of 636 to 770 resolvable polypeptides were identical in all respects between RR and rr for roots, shoots, leaflets, stipules, young ovaries, and young embryos. A single difference between the lines became evident about 21-23 days after anthesis in the embryos. Mature seeds of the two lines showed 62 spot differences in addition to differences in four clusters of spots, representing about 10% of the total number of spots visible on the gels. The protein differences are presumably involved in the many known physiological differences of the two seed types. 2-D PAGE analyses of near-isogenic lines are likely to be valuable in a number of quantitative and developmental genetic contexts. PMID:17246438

  2. Genetic distances in the tribe Bovini studied by gel electrophoresis

    E-print Network

    Butts, Kelley Elaine McRae

    1990-01-01

    volts 7 applications end load ice in tank DIA4 cello gel ENO1 cellulose acetate 0. 10 M Trizma base 0. 05 M Boric acid pH 8. 2 0. 10 M Trizma base 0, 10 M Maleic anhydride 0. 01 M MgC12. 6H20 pH 7. 4 (*membrane buffer- I:3 dilution) 2... hours 100 volts 2-5 applications middle load room temperature 45 minutes 150 volts 1-3 applications end load ice in tank ESD cellulose acetate 0. 90 M Trizma base 0. 019 M Na4EDTA 0. 50 M Boric acid pH 8. 6 (*membrane and tank buffers-1...

  3. Passivated gel electrophoresis of charged nanospheres by light-scattering video tracking.

    PubMed

    Zhu, Xiaoming; Mason, Thomas G

    2014-08-15

    Gel electrophoresis (gel-EP) has been used for decades to separate charged biopolymers, such as DNA, RNA, and proteins, yet propagation of other charged colloidal objects, such as nanoparticles, during gel-EP has been studied comparatively little. Simply introducing anionic nanoparticles, such as sulfate-stabilized polystyrene nanospheres, in standard large-pore agarose gels commonly used for biomolecules does not automatically ensure propagation or size-separation because attractive interactions can exist between the gel and the nanoparticles. Whereas altering the surfaces of the nanoparticles is a possible solution, here, by contrast, we show that treating a common type I-A low-electroendoosmosis agarose gel with a passivation agent, such as poly-(ethyleneglycol), enables charged nanoparticles to propagate through large-pore passivated gels in a highly reproducible manner. Moreover, by taking advantage of the significant optical scattering from the nanoparticles, which is not easily measurable for biopolymers, relative to scattering from the gel, we perform real-time, light-scattering, video-tracking gel-EP. Continuous optical measurements of the propagation of bands of uniformly sized nanospheres in passivated gels provides the propagation distance, L, and velocity, v, as a function of time for different sphere radii, electric field strengths, gel concentrations, and passivation agent concentrations. The steady-state particle velocities vary linearly with applied electric field strength, E, for small E, but these velocities become non-linear for larger E, suggesting that strongly driven nanoparticles can become elastically trapped in the smaller pores of the gel, which act like blind holes, in a manner that thermal fluctuations cannot overcome. Based on this assumption, we introduce a simple model that fits the measured v(E) in both linear and non-linear regimes over a relevant range of applied voltages. PMID:24910054

  4. Summary: You will be running gel electrophoresis to see if you got amplification of the 3 genes that you tried to amplify with pcr.

    E-print Network

    Sullivan, Matthew B.

    Summary: You will be running gel electrophoresis to see if you got amplification of the 3 genes pcr products from the ice bucket. Protocol Gel Electrophoresis of PCR Products Tucson High School that you tried to amplify with pcr. 1) Work in pairs to make your gel. 2) Add 0.5x TBE Buffer to your gel

  5. Microplate array diagonal gel electrophoresis for cohort studies of microsatellite loci.

    PubMed

    Chen, Xiao-he; O'Dell, Sandra D; Day, Ian N M

    2002-05-01

    After PCR amplification, we have achieved precise sizing of trinucleotide and tetranucleotide microsatellite alleles on 96-well open-faced polyacrylamide microplate array diagonal gel electrophoresis (MADGE) gels: two tetranucleotide repeats, HUMTHOI (five alleles 248-263 bp) and DYS390 (eight alleles 200-228 bp), and DYS392, a trinucleotide repeat (eight alleles 210-231 bp). A gel matrix of Duracryl, a high mechanical strength polyacrylamide derivative, and appropriate ionic conditions provide the 1.3%-1.5% band resolution required. No end-labeling of primers is needed, as the sensitive Vistra Green intercalating dye is used for the visualization of bands. Co-run markers bracketing the PCR fragments ensure accurate sizing without inter-lane variability. Electrophoresis of multiple gels in a thermostatically controlled tank allows up to 1000 samples to be run in 90 min. Gel images were analyzed using a Fluorlmager 595 fluorescent scanning system, and alleles were identified using Phoretix software for band migration measurement and Microsoft Excel to compute fragment sizes. Estimated sizes were interpolated precisely to achieve accurate binning. Microsatellite-MADGE represents a utilitarian methodfor high-throughput genotyping in cohort studies, using standard laboratory equipment. PMID:12019781

  6. Coupling isoelectric focusing gel electrophoresis to mass spectrometry by electrostatic spray ionization.

    PubMed

    Qiao, Liang; Tobolkina, Elena; Liu, Baohong; Girault, Hubert H

    2013-05-01

    Gel electrophoresis has been used for decades as a high-resolution separation technique for proteins and protein isomers but has been limited in the coupling with MS because of low throughput and poor automaticity compared with LC-MS. In this work, we have developed an ambient ionization strategy, electrostatic spray ionization, for in situ ionization of proteins or peptides inside a surfactant-free polyacrylamide gel. The samples can be first separated by isoelectric focusing in a gel and then quickly in situ detected by scanning the gel with the electrostatic spray ionization mass spectrometry. With this strategy, nanograms of proteins or peptides inside a band are enough to be ionized for MS detection. This method for protein/peptide spots visualization is sensitive, providing sample molecular weight information while avoiding spot staining and chemical extraction procedures that can introduce contaminants and sample loss. Proof-of-principle results have demonstrated that the electrostatic spray ionization can produce sample ions from a complex background, and with a spatial resolution matching the isoelectric focusing, it is therefore a good choice to couple directly isoelectric focusing gel electrophoresis with mass spectrometry. PMID:23510028

  7. New microsatellite markers for Anthyllis vulneraria (Fabaceae), analyzed with Spreadex gel electrophoresis1

    PubMed Central

    Kesselring, Halil; Hamann, Elena; Stcklin, Jrg; Armbruster, Georg F. J.

    2013-01-01

    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. PMID:25202507

  8. Detection of Mitochondrial DNA Mutations by Temporal Temperature Gradient Gel Electrophoresis

    Microsoft Academic Search

    Tian-Jian Chen; Richard G. Boles; Lee-Jun C. Wong

    Background: A unique requirement for the molecular diagnosis of mitochondrial DNA (mtDNA) disorders is the ability to detect heteroplasmic mtDNA mutations and to distinguish them from homoplasmic sequence variations before further testing (e.g., sequencing) is performed. We evaluated the potential utility of temporal temperature gradient gel electrophoresis (TTGE) for these purposes in patients with suspected mtDNA mutations. Methods: DNA samples

  9. Pulsed-Field Gel Electrophoresis as a Replacement for Bacteriophage Typing ofStaphylococcus aureus

    Microsoft Academic Search

    TAMMY L. BANNERMAN; GARY A. HANCOCK; FRED C. TENOVER; MICHAEL MILLER

    Bacteriophage typing (BT) (World Health Organization method) has been used at the Centers for Disease ControlandPreventionforover30yearstotypeisolatesofStaphylococcusaureus.Sincestudieshaveshownthat BT patterns have poor reproducibility and because BT fails to type a high percentage (15 to 20%) of isolates, the Centers for Disease Control and Prevention has converted from using BT to using pulsed-field gel electrophoresis (PFGE) for strain typingS. aureus. We compared the

  10. Isolation of Spiroplasma citri membranes and characterization of membrane proteins by two-dimensional gel electrophoresis

    Microsoft Academic Search

    Philippe Simoneau; Jacques Labarre

    1988-01-01

    This paper describes a method for isolating plasma membranes fromSpiroplasma citri and for comparing membrane and cytoplasmic proteins by two-dimensional gel electrophoresis. Plasma membranes ofS. citri were stabilized against fragmentation by coating cell with Concanavalin A just prior to lysis. After lysis of the cells by ultrasonic irradiation, membranes were purified by differential centrifugation and step gradients. The purified fraction,

  11. Differentiation of Mycoplasma Species by 16S Ribosomal DNA PCR and Denaturing Gradient Gel Electrophoresis Fingerprinting

    PubMed Central

    McAuliffe, Laura; Ellis, Richard J.; Ayling, Roger D.; Nicholas, Robin A. J.

    2003-01-01

    Denaturing gradient gel electrophoresis (DGGE) of a 16S ribosomal DNA PCR product was used to differentiate 32 mycoplasma species of veterinary significance. Twenty-seven (85%) species could be differentiated by DGGE. This method could enable the rapid identification of many mycoplasma species for which there is no specific PCR available and which are currently identified by using culture and serological tests. PMID:14532239

  12. Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology

    Microsoft Academic Search

    Steven P. Gygi; Garry L. Corthals; Yanni Zhang; Yvan Rochon; Ruedi Aebersold

    2000-01-01

    Proteome analysis is most commonly accomplished by a combination of two-dimensional gel electrophoresis (2DE) to separate and visualize proteins and mass spectrometry (MS) for protein identification. Although this technique is powerful, mature, and sensitive, questions remain concerning its ability to characterize all of the elements of a proteome. In the current study, more than 1,500 features were visualized by silver

  13. Rapid Preparation of Bacterial DNA for Pulsed-Field Gel Electrophoresis

    Microsoft Academic Search

    MARIAN G. MATUSHEK; MARC J. M. BONTEN; ANDMARY K. HAYDEN

    1996-01-01

    Pulsed-field gel electrophoresis (PFGE) is a highly repro- ducible and discriminating tool for the molecular typing of bacteria. It has been successfully applied to a broad range of different gram-negative and gram-positive organisms and my- cobacterial species in epidemiological studies of populations wherediseasesareendemic,aswellasinoutbreaks(2,4,5).Its major disadvantage is that it requires up to 6 days from isola- tion of a pure colony

  14. Bifidobacterial Diversity in Human Feces Detected by Genus-Specific PCR and Denaturing Gradient Gel Electrophoresis

    Microsoft Academic Search

    REETTA M. SATOKARI; ELAINE E. VAUGHAN; ANTOON D. L. AKKERMANS; MARIA SAARELA; WILLEM M. DE VOS

    2001-01-01

    We describe the development and validation of a method for the qualitative analysis of complex bifidobac- terial communities based on PCR and denaturing gradient gel electrophoresis (DGGE). Bifidobacterium genus-specific primers were used to amplify an approximately 520-bp fragment from the 16S ribosomal DNA (rDNA), and the fragments were separated in a sequence-specific manner in DGGE. PCR products of the same

  15. Method for quantitating cholesterol in subfractions of serum lipoproteins separated by gradient gel electrophoresis

    Microsoft Academic Search

    M.-L. Cheng; C. M. Kammerer; W. F. Lowe; B. Dyke; J. L. VandeBerg

    1988-01-01

    Extensive heterogeneity in particle size distribution of serum lipoproteins of baboons was resolved by a procedure that combined\\u000a Sudan black B prestaining, polyacrylamide gradient gel electrophoresis (GGE), and quantitative densitometry. Each densitometric\\u000a scan represented a continuous distribution of the relative amount of cholesterol in a serum sample, as a function of the lipoprotein\\u000a particle size. For analytical purposes, each scan

  16. Detection of metals in proteins by means of polyacrylamide gel electrophoresis and laser ablation-inductively coupled plasma-mass spectrometry: application to selenium.

    PubMed

    Chry, Cyrille C; Gnther, Detlef; Cornelis, Rita; Vanhaecke, Frank; Moens, Luc

    2003-10-01

    The capabilities of laser ablation-inductively coupled plasma-mass spectrometry for the detection of trace elements in a gel after gel electrophoresis were systematically studied. Figures of merit, such as limit of detection, linearity, and repeatability, were evaluated for various elements (Li, V, Cr, Mn, Ni, Cu, Zn, As, Se, Mo, Pd, Ag, Cd, Pt, Tl, Pb). Two ablation strategies were followed: single hole drilling, relevant for ablation of spots after two-dimensional (2-D) separations, and ablation with translation, i.e., on a line, relevant for one-dimensional (1-D) separations. This technique was applied to the detection of selenoproteins in red blood cells extracts after a 1-D separation (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and the detection of selenium-containing proteins in yeast after 2-D electrophoresis (2-DE). The detection procedure was further improved by using the dynamic reaction cell technology, which allowed the removal of the Ar_2(+) interference and hence the use of the most abundant Se isotope, (80)Se. Reaction gases were compared (methane, carbon monoxide, ammonia, oxygen and the combination of argon (collision gas) and hydrogen (reaction gas)). In each instance, the reaction cell parameters were optimized in order to obtain the lowest detection limit for Se (as (80)Se(+), (82)Se(+) or (77)Se(+); and as (80)Se(16)O(+), (82)Se(16)O(+) or (77)Se(16)O(+) with O(2) as the reaction gas). Carbon monoxide was found to offer the best performance. The detection limit with the use of DRC and He as transport gas was 0.07 microg Se g(-1) gel with single hole drilling and 0.15 microg Se g(-1) gel for ablation with translation. PMID:14595676

  17. Development of an integrated approach for evaluation of 2-D gel image analysis: Impact of multiple proteins in single spots on comparative proteomics in conventional 2-D gel/MALDI workflow

    Technology Transfer Automated Retrieval System (TEKTRAN)

    With 2-D gel mapping, it is often observed that essentially identical proteins migrate to different positions in the gel, while some seemingly well-resolved protein spots consist of multiple proteins. These observations can undermine the validity of gel-based comparative proteomic studies. Through...

  18. Optimization of separation and detection schemes for DNA with pulsed field slab gel and capillary electrophoresis

    SciTech Connect

    McGregor, D.A.

    1993-07-01

    The purpose of the Human Genome Project is outlined followed by a discussion of electrophoresis in slab gels and capillaries and its application to deoxyribonucleic acid (DNA). Techniques used to modify electroosmotic flow in capillaries are addressed. Several separation and detection schemes for DNA via gel and capillary electrophoresis are described. Emphasis is placed on the elucidation of DNA fragment size in real time and shortening separation times to approximate real time monitoring. The migration of DNA fragment bands through a slab gel can be monitored by UV absorption at 254 nm and imaged by a charge coupled device (CCD) camera. Background correction and immediate viewing of band positions to interactively change the field program in pulsed-field gel electrophoresis are possible throughout the separation. The use of absorption removes the need for staining or radioisotope labeling thereby simplifying sample preparation and reducing hazardous waste generation. This leaves the DNA in its native state and further analysis can be performed without de-staining. The optimization of several parameters considerably reduces total analysis time. DNA from 2 kb to 850 kb can be separated in 3 hours on a 7 cm gel with interactive control of the pulse time, which is 10 times faster than the use of a constant field program. The separation of {Phi}X174RF DNA-HaeIII fragments is studied in a 0.5% methyl cellulose polymer solution as a function of temperature and applied voltage. The migration times decreased with both increasing temperature and increasing field strength, as expected. The relative migration rates of the fragments do not change with temperature but are affected by the applied field. Conditions were established for the separation of the 271/281 bp fragments, even without the addition of intercalating agents. At 700 V/cm and 20{degrees}C, all fragments are separated in less than 4 minutes with an average plate number of 2.5 million per meter.

  19. Application of MALDI-TOF-mass spectrometry to proteome analysis using stain-free gel electrophoresis.

    PubMed

    Susnea, Iuliana; Bernevic, Bogdan; Wicke, Michael; Ma, Li; Liu, Shuying; Schellander, Karl; Przybylski, Michael

    2013-01-01

    The combination of MALDI-TOF-mass spectrometry with gel electrophoretic separation using protein visualization by staining procedures involving such as Coomassie Brilliant Blue has been established as a widely used approach in proteomics. Although this approach has been shown to present high detection sensitivity, drawbacks and limitations frequently arise from the significant background in the mass spectrometric analysis. In this chapter we describe an approach for the application of MALDI-MS to the mass spectrometric identification of proteins from one-dimensional (1D) and two-dimensional (2D) gel electrophoretic separation, using stain-free detection and visualization based on native protein fluorescence. Using the native fluorescence of aromatic protein amino acids with UV transmission at 343 nm as a fast gel imaging system, unstained protein spots are localized and, upon excision from gels, can be proteolytically digested and analyzed by MALDI-MS. Following the initial development and testing with standard proteins, applications of the stain-free gel electrophoretic detection approach to mass spectrometric identification of biological proteins from 2D-gel separations clearly show the feasibility and efficiency of this combination, as illustrated by a proteomics study of porcine skeleton muscle proteins. Major advantages of the stain-free gel detection approach with MALDI-MS analysis are (1) rapid analysis of proteins from 1D- and 2D-gel separation without destaining required prior to proteolytic digestion, (2) the low detection limits of proteins attained, and (3) low background in the MALDI-MS analysis. PMID:22547356

  20. Identification of isolate-specific sporozoite proteins of Cryptosporidium parvum by two-dimensional gel electrophoresis.

    PubMed Central

    Mead, J R; Humphreys, R C; Sammons, D W; Sterling, C R

    1990-01-01

    Five isolates of Cryptosporidium parvum collected from human, horse, and calf sources were compared for differences in sporozoite protein patterns by using two-dimensional gel electrophoresis. Silver-stained two-dimensional gels contained over 300 protein spots from detergent-solubilized sporozoites. A distinguishing 106-kilodalton peptide that shifted in isoelectric point was detected in four of the five isolates. Computerized two-dimensional gel analysis was performed to obtain objective quantitation of the pI shift. Three of these four isolates could be differentiated from one other by the pI shift in this peptide. The fifth isolate was distinguished by the absence of the 106-kilodalton peptide and the presence of a 40-kilodalton peptide that was not observed in any other isolate. Images PMID:2365452

  1. Single universal primer multiplex ligation-dependent probe amplification with sequencing gel electrophoresis analysis.

    PubMed

    Shang, Ying; Zhu, Pengyu; Xu, Wentao; Guo, Tianxiao; Tian, Wenying; Luo, Yunbo; Huang, Kunlun

    2013-12-15

    In this study, a novel single universal primer multiplex ligation-dependent probe amplification (SUP-MLPA) technique that uses only one universal primer to perform multiplex polymerase chain reaction (PCR) was developed. Two reversely complementary common sequences were designed on the 5' or 3' end of the ligation probes (LPs), which allowed the ligation products to be amplified through only a single universal primer (SUP). SUP-MLPA products were analyzed on sequencing gel electrophoresis with extraordinary resolution. This method avoided the high expenses associated with capillary electrophoresis, which was the commonly used detection instrument. In comparison with conventional multiplex PCR, which suffers from low sensitivity, nonspecificity, and amplification disparity, SUP-MLPA had higher specificity and sensitivity and a low detection limit of 0.1 ng for detecting single crop species when screening the presence of genetically modified crops. We also studied the effect of different lengths of stuffer sequences on the probes for the first time. Through comparing the results of quantitative PCR, the LPs with different stuffer sequences did not affect the ligation efficiency, which further increased the multiplicity of this assay. The improved SUP-MLPA and sequencing gel electrophoresis method will be useful for food and animal feed identification, bacterial detection, and verification of genetic modification status of crops. PMID:24050969

  2. Mutations and a polymorphism in the factor VIII gene discovered by denaturing gradient gel electrophoresis

    SciTech Connect

    Kogan, S.; Gitschier, J. (Univ. of California, San Francisco (USA))

    1990-03-01

    Hemophilia A results from mutations in the gene coding for coagulation factor VIII. The authors gradient gel electrophoresis to screen for mutations in the region of the factor VIII gene coding for the first acidic domain. Amplification primers were designed employing the MELTMAP computer program to optimize the ability to detect mutations. Screening of amplified DNA from 228 unselected hemophilia A patients revealed two mutations and one polymorphism. Rescreening the same population by making heteroduplexes between amplified patient and control samples prior to electrophoresis revealed one additional mutation. The mutations include two missense and one 4-base-pair deletion, and each mutation was found in patients with severe hemophilia. The polymorphism, located adjacent to the adenine branch site in intron 7, is useful for genetic prediction in some cases where the Bcl I and Xba I polymorphisms are uninformative. These results suggest that DNA amplification and denaturing gradient gel electrophoresis should be an excellent strategy for identifying mutations and polymorphisms in defined regions of the factor VIII gene and other large genes.

  3. Detection of genotoxic insult as DNA strand breaks in fish blood cells by agarose gel electrophoresis

    SciTech Connect

    Theodorakis, C.W. (Univ. of Tennessee, Knoxville, TN (United States)); D'Surney, S.J. (Univ. of Mississippi, University, MS (United States). Dept. of Biology); Shugart, L.R. (Oak Ridge National Lab., TN (United States). Environmental Sciences Division)

    1994-07-01

    DNA, isolated from the blood cells of bluegill sunfish (Lepomis macrochirus) exposed in the lab to bedded sediment collected from a site contaminated with genotoxic compounds (i.e., PAHs, PCBs, and heavy metals), was examined for strand breakage by agarose gel electrophoresis. Before electrophoresis the blood cells were embedded in agarose plugs and incubated with proteinase. After electrophoresis under both neutral (pH 7) or alkaline (pH 12) conditions, the median molecular length (MML) of the DNA distributed in the gel was determined. These quantitative measures were used to estimate the difference in the number of double- and single-strand breaks between DNA preparations. Both types of strand breakage were found to be greater in fish exposed to sediment contaminated with genotoxic compounds as compared to nonexposed fish. A statistically significant correlation was demonstrated between the MML value obtained by the electrophoretic assay reported here and the F value (measure of DNA double-strandedness) obtained by the alkaline unwinding assay.

  4. Quantitative analysis of two-dimensional gel electrophoresis protein patterns: a method for studying genetic relationships among Globodera pallida populations.

    PubMed

    Fullaondo, A; Vicario, A; Aguirre, A; Barrena, I; Salazar, A

    2001-09-01

    A method based in two-dimensional protein gel electrophoresis has been developed in order to improve the analysis of genetic relationships among populations of Globodera. It has been used to estimate genetic divergence among nine Globodera pallida nematode populations. Sixty-one anonymous polypeptide spots were resolved using silver-stained high-resolution 2D gels and they were quantified in each population to establish genetic variation among G. pallida populations. The results of this analysis were compared with those obtained after a study of allelic frequency variation, which was carried out using seven previously described loci. Genetic distances among populations were calculated by means of both studies, the quantitative analysis and the allelic frequency variation, and phylogenetic trees were constructed for each type of analysis. A correlation analysis between the two distance matrices was carried out and a bootstrap analysis was performed to determine the strength of the clusters obtained with each method. The results obtained support the idea that quantitative protein analysis can be successfully applied to phylogenetic analysis of G. pallida populations. PMID:11737273

  5. Age-associated changes in synaptic lipid raft proteins revealed by two-dimensional fluorescence difference gel electrophoresis

    PubMed Central

    Jiang, Lei; Fang, Jianwen; Moore, David S.; Gogichaeva, Natalia V.; Galeva, Nadezhda A.; Michaelis, Mary L.; Zaidi, Asma

    2009-01-01

    Brain aging is associated with a progressive decline in cognitive function though the molecular mechanisms remain unknown. Functional changes in brain neurons could be due to age-related alterations in levels of specific proteins critical for information processing. Specialized membrane microdomains known as lipid rafts contain protein complexes involved in many signal transduction processes. This study was undertaken to determine if two-dimensional fluorescence difference gel electrophoresis (2D DIGE) analysis of proteins in synaptic membrane lipid rafts revealed age-dependent alterations in levels of raft proteins. Five pairs of young and aged rat synaptic membrane rafts were subjected to DIGE separation, followed by image analysis and identification of significantly altered proteins. Of 1046 matched spots on DIGE gels, 94 showed statistically significant differences in levels between old and young rafts, and 87 of these were decreased in aged rafts. The 41 most significantly altered (p < 0.03) proteins included several synaptic proteins involved in energy metabolism, redox homeostasis, and cytoskeletal structure. This may indicate a disruption in bioenergetic balance and redox homeostasis in synaptic rafts with brain aging. Differential levels of representative identified proteins were confirmed by immunoblot analysis. Our findings provide novel pathways in investigations of mechanisms that may contribute to altered neuronal function in aging brain. PMID:19118924

  6. CCMR: Screening of Point Mutation in DNA Using Constant Denaturant Gel Electrophoresis

    NSDL National Science Digital Library

    Hsu, Tien Theresa

    2005-08-17

    Point mutations, the mismatch of one nucleotide base pair, are the most common type of mutations. These point mutations, if found on several important parts of a gene, may have a serious impact. For example, the growth of human tumors have been associated with point mutations in the p53 gene (1). Past surveys have used loss of heterozygosity as an indirect essay for inactivation of genes (2). However, although this method gives rapid results, it cannot detect point mutations. The most sensitive screening is to sequence the actual genome, but this method is requires intensive time and labor. More recently, there has been several nucleic acid-based screening methods that can detect mutations within short fragments of DNA, including denaturing gradient gel electrophoresis (DGGE) (3). Constant denaturant gel electrophoresis (CDGE) (4) is a modification of DGGE. The separation principle of CDGE is based on the melting behavior of double-stranded DNA. DNA strands with mismatches are destabilized by the mismatch, since the hydrogen bonds between mismatched base pairs are weaker, and therefore is unstranded (or melts) at a lower temperature than strands without mismatches. Strand separation can be detected as a reduction in the mobility of the fragment as it moves through an acrylamide gel containing a chemical denaturant. In this study, the effectiveness of CDGE is investigated with DNA fragments of known sequences.

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

    PubMed Central

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

    2012-01-01

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

  8. Single nucleus versus single-cell gel electrophoresis: Kinetics of DNA track formation.

    PubMed

    Afanasieva, Katerina; Chopei, Marianna; Sivolob, Andrei

    2015-04-01

    Single-cell gel electrophoresis, or the comet assay, is usually performed with nucleoids prepared after a lysis of either whole cells (more often) or isolated cell nuclei (rarely). Electrophoretic properties of the second type of nucleoids have never been investigated carefully. We measured the kinetics of the DNA exit from nuclei-derived nucleoids in comparison with cell-derived nucleoids. The results show that general organization of the nuclei-derived nucleoids is not changed very much in comparison with nucleoids commonly obtained from whole cells. At the same time, in contrast to the cell-derived nucleoids, for which the exit is stepwise and cooperative, the DNA exit from the nuclei-derived nucleoids can be described by a simple monomolecular kinetics. This difference is probably due to agarose penetration into nuclei (but not into cells) before polymerization of the agarose gel. We suggest that single-nucleus gel electrophoresis may be a way for the comet assay standardization. PMID:25631953

  9. Application of the commercial gel electrophoresis apparatus with intermittent fluorescence scanning to a nonfluorescing protein.

    PubMed

    Chen, N; Chrambach, A

    1996-11-01

    Gel electrophoretic instrumentation has taken a quantum jump forward with the commercial introduction of an apparatus which, after loading of the sample and initiation of electrophoresis, provides real-time gel patterns at desired time intervals, with a computer printout of mobility values characterizing each band and the means to isolate each desired band with known and maximizeable recovery. However, a major limitation of that apparatus has been that it employs fluorescence detection and therefore requires the fluorescent labeling of the macromolecules of interest. That limitation was first overcome by E. Gombocz and E. Cortez (Application Note 8, 1994, LabIntelligence, Belmont, CA) in the detection of nonfluorescing carrier ampholytes. In that application, fluorescent, immobile (uncharged) umbelliferone was added to the gel to provide a uniform background of fluorescence upon excitation at 280-360 nm. The isoelectric carrier ampholyte zones could be detected as inverted peaks due to their reduction of the fluorescence intensity of umbelliferone. A similar approach was applied to a representative SDS-protein, conalbumin-SDS, in the present study, replacing umbelliferone in the gel by a fluorescing paper sheet in contact with the lower external surface of the electrophoresis cell. Passage of the proteins reduced the intensity of the light excitation incident on the fluorescent paper so as to decrease the emitted fluorescence signal and allow for the detection of the proteins as "inverted peaks." Presumably, the reduction of background fluorescence is due to the absorbance at 280 nm of the protein passing through the gel, and reduction of the incident light intensity by that absorbance. The resulting detection of the representative unlabeled SDS-protein by "fluorescence reduction" was found to be less sensitive by a factor of 10-20 than detection of the fluorescently labeled protein (at a molar ratio of fluorescein carboxylate to conalbumin of 1/1). The area of the inverted bands of conalbumin-SDS was found to be independent of migration distance. PMID:8923965

  10. Sodium dodecyl sulphate-polyacrylamide denaturing gel electrophoresis and Western blotting techniques.

    PubMed

    Blancher, Christine; Cormick, Rob Mc

    2012-01-01

    The study of proteins, their expression and post-translational modification, is a key process in molecular biology. Immunoblotting is a well-established and powerful tool for the study of proteins, which continues to evolve as new reagents and apparatus are developed. This chapter describes in detail the process by which proteins are extracted from cells, quantified, fractionated using poly-acrylamide gel electrophoresis, transferred to a membrane, and assessed by immunoblotting. Variations in experimental technique, and new technologies available to the researcher, are also discussed. PMID:22674128

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

    SciTech Connect

    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.

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

    NASA Technical Reports Server (NTRS)

    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.

  13. Nanoparticle size distributions measured by optical adaptive-deconvolution passivated-gel electrophoresis.

    PubMed

    Zhu, Xiaoming; Mason, Thomas G

    2014-12-01

    We image visible light scattered from dispersions of charged spherical nanoparticles propagating through a passivated agarose gel during electrophoresis. By analyzing one-dimensional light intensities along different lanes, we measure the mobility distributions of the nanoparticles and thereby infer their size distributions, which become time-independent after adequate propagation and separation have occurred. For a given large-pore passivated agarose gel, experiments using monodisperse, surfactant-free, sulfate-stabilized, polystyrene nanopheres establish the propagation distance as a function of time for a range of different sphere radii having known surface charges. As bands of monodisperse nanospheres propagate through the gel, the bands become smeared, developing asymmetric tails as some nanospheres experience additional delays compared to others of the same size. After background subtraction, these bands, including their tails, can be fit well using a modified log-normal distribution, yielding deconvolution parameters that vary with propagation distance and transit time. To demonstrate the approach for complex nanosphere dispersions, such as a multi-modal mixture or a broadly polydisperse nanoemulsion, we measure scattered light intensities as a function of propagation distance and time during gel-EP. Iterative deconvolution using a modified log-normal point-spread function, which changes shape according to propagation distance and time, directly yields unsmeared, high-resolution electrophoretic mobility distributions, from which detailed particle size distributions are inferred. PMID:25218049

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

    NASA Astrophysics Data System (ADS)

    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.

  15. A Novel Universal Primer-Multiplex-PCR Method with Sequencing Gel Electrophoresis Analysis

    PubMed Central

    Huang, Kunlun; Zhang, Nan; Yuan, Yanfang; Shang, Ying; Luo, Yunbo

    2012-01-01

    In this study, a novel universal primer-multiplex-PCR (UP-M-PCR) method adding a universal primer (UP) in the multiplex PCR reaction system was described. A universal adapter was designed in the 5?-end of each specific primer pairs which matched with the specific DNA sequences for each template and also used as the universal primer (UP). PCR products were analyzed on sequencing gel electrophoresis (SGE) which had the advantage of exhibiting extraordinary resolution. This method overcame the disadvantages rooted deeply in conventional multiplex PCR such as complex manipulation, lower sensitivity, self-inhibition and amplification disparity resulting from different primers, and it got a high specificity and had a low detection limit of 0.1 ng for single kind of crops when screening the presence of genetically modified (GM) crops in mixture samples. The novel developed multiplex PCR assay with sequencing gel electrophoresis analysis will be useful in many fields, such as verifying the GM status of a sample irrespective of the crop and GM trait and so on. PMID:22272223

  16. Investigating DNA Migration in Pulsed Fields Using a Miniaturized Field Inversion Gel Electrophoresis System

    PubMed Central

    Chen, Xiaojia; Ugaz, Victor M.

    2010-01-01

    Pulsed field gel electrophoresis (PFGE) is a well-established technique for fractionation of DNA fragments ranging from kilobases to megabases in length. But many of these separations require an undesirable combination of long experiment times (often approaching tens of hours) and application of high voltages (often approaching tens of kV). Here we present a simple miniaturized field inversion gel electrophoresis (FIGE) apparatus capable of separating DNA fragments up to 32.5 kb in length within 3 hours using a modest applied potential of 20 V. The device is small enough to be imaged under a fluorescence microscope, permitting the migrating DNA bands to be observed during the course of the separation run. We use this capability to investigate how separation performance is affected by parameters including the ratio of forward and backward voltage, pulse time, and temperature. We also characterize the dependence of DNA mobility on fragment size N, and observe a scaling in the vicinity of N?0.5 over the size range investigated. The high speed, low power consumption, and simple design of this system may help enable future studies of DNA migration in PFGE to be performed quickly and inexpensively. PMID:19053074

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

    PubMed

    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

  18. Comparison of field inversion gel electrophoresis with contour-clamped homogeneous electric field electrophoresis as a typing method for Enterococcus faecium.

    PubMed Central

    Green, M; Barbadora, K; Donabedian, S; Zervos, M J

    1995-01-01

    Direct comparisons between contour-clamped homogeneous electric field (CHEF) electrophoresis and field inversion gel electrophoresis (FIGE) to determine the epidemiology of antibiotic-resistant enterococci have not been previously published. Fifty non-beta-lactamase-producing, ampicillin-resistant Enterococcus faecium isolates and 10 vancomycin-resistant E. faecium strains collected from multiple centers were analyzed in a blinded fashion by CHEF electrophoresis and FIGE after digestion with SmaI. Isolates were considered clonally related if there was a difference of three of fewer bands between electrophoretic patterns. Agreement between CHEF electrophoresis and FIGE was seen for 12 of 13 identified groups of ampicillin-resistant E. faecium and 7 of 7 groups of vancomycin-resistant E. faecium. The lone discordance was accounted for by a fourth band difference between two strains recognized near 350 kb by CHEF electrophoresis but not by FIGE, placing them into different clonal groups. Better band separation was noted in the 50- to 200-kb range for FIGE, while CHEF electrophoresis revealed better resolution over 250 kb more reliably, including detection of some bands not seen on FIGE. Molecular epidemiologic investigations of E. faecium by either technique should provide comparable results. PMID:7650185

  19. The development of simple and sensitive small-molecule fluorescent probes for the detection of serum proteins after native polyacrylamide gel electrophoresis.

    PubMed

    Wang, Fangfang; Huang, Lingyun; Na, Na; He, Dacheng; Sun, Dezhi; Ouyang, Jin

    2012-05-21

    In this paper, a simple and sensitive small-molecule fluorescent probe, 2,5-dihydroxy-4'-dimethylaminochalcone (DHDMAC), was designed and synthesized for the detection of human serum proteins via hydrophobic interactions after polyacrylamide gel electrophoresis (PAGE). This probe produced lower fluorescence emission in the absence of proteins, and the emission intensity was significantly increased after the interaction with serum proteins. To demonstrate the imaging performance of this probe as a fluorescent dye, a series of experiments was conducted that included sensitivity comparison and 2D-PAGE. The results indicated that the sensitivity of DHDMAC staining is comparable to that of the most widely used fluorescent dye, SYPRO Ruby, and more protein spots (including thyroxine-binding globulin, angiotensinogen, afamin, zinc-?-2-glycoprotein and ?-1-antichymotrypsin) were detected after 2D-PAGE. Therefore, DHDMAC is a good protein reporter due to its fast staining procedure, low detection limits and high resolution. PMID:22475746

  20. Rearrangements, Vibrations and Microscopic Glassy Dynamics and Structure in Quasi-2D Dense Colloidal Gels

    NASA Astrophysics Data System (ADS)

    Lohr, Matthew; Yodh, Arjun

    2012-02-01

    In this work, we investigate the microscopic dynamics of quasi-2D dense attractive colloidal systems. We confine bidisperse polystyrene spheres between glass coverslips in a suspension of water and 2,6-lutidine; as we increase the temperature of the sample into a critical regime, lutidine wets the colloids, creating a strong attractive interaction (> 4kT). We specifically study suspensions in the ``dense gel'' regime, i.e., at a volume fraction high enough that the attractive particles form a spanning cluster, yet just low enough that there exists some structural heterogeneity larger than the individual particle size. We track the particle locations via bright-field video microscopy and analyze the dynamics of both lower-volume-fraction gel states and higher-volume-fraction glassy states. Specifically, we make correlations between local structure, rearrangement-prone regions, and low-frequency vibrational modes. In doing so, we not only characterize the structural and dynamical differences and similarities between colloidal gels and glasses, but we also gain further insight into the origins of dynamic heterogeneity in glassy systems.

  1. Identification of cellular proteome using two-dimensional difference gel electrophoresis in ST cells infected with transmissible gastroenteritis coronavirus

    PubMed Central

    2013-01-01

    Background Transmissible gastroenteritis coronavirus (TGEV) is an enteropathogenic coronavirus that causes diarrhea in pigs, which is correlated with high morbidity and mortality in suckling piglets. Information remains limited about the comparative protein expression of host cells in response to TGEV infection. In this study, cellular protein response to TGEV infection in swine testes (ST) cells was analyzed, using the proteomic method of two-dimensional difference gel electrophoresis (2D DIGE) coupled with MALDI-TOF-TOF/MS identification. Results 33 differentially expressed protein spots, of which 23 were up-regulated and 10 were down-regulated were identified. All the protein spots were successfully identified. The identified proteins were involved in the regulation of essential processes such as cellular structure and integrity, RNA processing, protein biosynthesis and modification, vesicle transport, signal transduction, and the mitochondrial pathway. Western blot analysis was used to validate the changes of alpha tubulin, keratin 19, and prohibitin during TGEV infection. Conclusions To our knowledge, we have performed the first analysis of the proteomic changes in host cell during TGEV infection. 17 altered cellular proteins that differentially expressed in TGEV infection were identified. The present study provides protein-related information that should be useful for understanding the host cell response to TGEV infection and the underlying mechanism of TGEV replication and pathogenicity. PMID:23855489

  2. Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

    PubMed Central

    Fiala, Gina J.

    2011-01-01

    Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Schgger and von Jagow 1991); (Schgger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes. PMID:21403626

  3. Detection and analyses by gel electrophoresis of cisplatin-mediated DNA damage.

    PubMed

    Zhang, B; Seki, S; Akiyama, K; Tsutsui, K; Li, T; Nagao, K

    1992-12-01

    DNA damage induced by cis-diamminedichloroplatinum (II) (cisplatin: cis-DDP), an anticancer drug, was studied in vitro by monitoring the drug-induced conformational change of pUC18 plasmid DNA, the sensitivity to some restriction enzymes of the damaged DNA and the sequence-dependent termination of DNA synthesis caused by cisplatin. Closed circular, superhelical pUC18 DNA was treated at 37 degrees C for 16 h with various concentrations of cisplatin. Cisplatin-dose-dependent conformational change due to unwinding of the treated DNA was detected by agarose gel electrophoresis. To analyze the base-specificity of the cisplatin damage, the measurement for sensitivity of cisplatin-treated DNA to various types of restriction enzyme and sequence gel analysis of the treated DNA were conducted. The results suggested that cisplatin attacked preferentially the sequence of GG > AG > GNG in the order. In the present assay condition, the cisplatin/DNA nucleotide ratios required for the DNA damage detection were roughly 0.025 for the conformational analysis, 0.001 or more for the restriction enzyme analysis, and less than 0.001 for the sequence gel analysis. By using the present method, it was demonstrated that the cisplatin-mediated DNA damage was inhibited by NaCl, KCl, CaCl2 or MgCl2 at their nearly physiological concentrations, and by reducing agents such as thiourea and 2-mercaptoethanol in the reaction mixture. PMID:1336637

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

    PubMed

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

    2014-12-10

    Abundant isoforms and dynamic posttranslational modifications cause the separation and identification of histone variants to be experimentally challenging. To meet this need, we employ two-dimensional electrophoretic gel separation followed by mass spectrometric detection which takes advantage of the chelation of Cu(2+) with amino acid residues exposed on the surfaces of the histone proteins. Acid-extracted rat liver histones were first mixed with CuSO4 solution and then separated in one dimension with triton-acid-urea (TAU) gel electrophoresis and in a second dimension using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separations result from both the changes in charge and mass upon Cu(2+) chelation. Identities of each separated gel bands were obtained by using matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). It was found that the migration of H3 histone isoforms of rat liver is markedly affected by the use of Cu(2+) ions. PMID:25441888

  5. Colorful Electrophoresis

    NSDL National Science Digital Library

    2012-06-26

    In this activity, learners follow step-by-step instructions to build a gel electrophoresis chamber using inexpensive materials from local hardware and electronic stores. Then, learners follow instructions to simulate DNA electrophoresis using food colors from the kitchen pantry.

  6. Phylogenetic Compositions of Bacterioplankton from Two California Estuaries Compared by Denaturing Gradient Gel Electrophoresis of 16S rDNA Fragments

    Microsoft Academic Search

    ALISON E. MURRAY; JAMES T. HOLLIBAUGH; ANDCRISTIAN ORREGO

    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

  7. Analytical techniques for cell fractions. XXII. Two-dimensional analysis of serum and tissue proteins: multiple gradient-slab gel electrophoresis

    Microsoft Academic Search

    N. L. Anderson; N. G. Anderson

    1978-01-01

    Two-dimensional electrophoresis of proteins and protein subunits, employing isoelectric focusing in the first dimension and electrophoresis in the presence of sodium dodecyl sulfate in the second, yields the highest resolutions currently available. In this paper separations in the second dimension are considered (the socalled DALT system). Methods for multiple-parallel casting of gradient gels in slab gel holders are described. The

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

    NASA Technical Reports Server (NTRS)

    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.

  9. Recovery of functional DNA inserts by electroendosmotic elution during gel electrophoresis.

    PubMed Central

    Tan, H V; Kitzis, A; Berthollet, T; Hamard, G; Beldjord, C; Benarous, R

    1988-01-01

    In contrast to all previous preparative electrophoresis apparatus which used a pump, electroendosmotic elution uses bound electrical charges at the end of the separating gel to generate a buffer flow. The electroendosmotic flow increased with increasing currents and decreasing buffer concentrations: its exact characteristics for the built apparatus were determined. The electroendosmotic device was able to separate two DNA fragments differing in size by only 5% with a recovery over 95%. As demonstrated in practical examples of recovery and uses of DNA inserts, up to 10 micrograms of DNA per band can be loaded at a time. The recovered DNA can be used directly for nick-translation, ligation... without further treatment. The performances of the method are expected to improve still further if the charge density and pores of the electroendosmotic medium can be "made-to-order" to provide a better flow profile of the eluting buffer. Images PMID:2833722

  10. Polyacrylamide gel electrophoresis (PAGE) of whole-cell proteins of cutaneous Propionibacterium species.

    PubMed

    Nordstrom, K M

    1985-02-01

    Polyacrylamide gel electrophoresis (PAGE) was applied to the study of whole-cell proteins of cutaneous propionibacteria in an attempt to characterise possible protein patterns that may be typical for strains isolated from acne skin. Isolates were obtained from the faces of 33 individuals aged 7-16 years. Some of these subjects had apparently normal healthy skin, whereas others had acne vulgaris of varying severity. Twenty-five facial isolates of Propionibacterium acnes and eight of P. granulosum were studied. A further seven axillary strains of P. avidum were included for purely taxonomic interest. No particular protein pattern was characteristic of an isolate from acne skin; in fact the P. acnes strains from all sources appeared to be identical. PMID:3155801

  11. Derivation of clones close to met by preparative field inversion gel electrophoresis

    SciTech Connect

    Michiels, F.; Burmeister, M.; Lehrach, H.

    1987-06-05

    The molecular analysis of genes identified by mutations is a major problems in mammalian genetics. As a step toward this goal, preparative field inversion gel electrophoresis (FIGE) was used to selectively isolate clones from the environment of genetically linked markers, and to select a subset of these clones containing sequences next to specific restriction sites rare in mammalian DNA. This approach has been used to generate a library highly enriched in sequences closely linked to the cystic fibrosis marker met. One clone derived from the end of a Not I restriction fragment containing the met sequence was analyzed in detail and localized within a long range map to a position of 300 kilobase pairs 5' of the metD sequence.

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

    PubMed Central

    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 in each digest yielded estimates for the size of the H. influenzae chromosome ranging from 1,834 kb. Images PMID:2842319

  13. Using microchip gel electrophoresis to probe DNA-drug binding interactions.

    PubMed

    Shi, Nan; Ugaz, Victor M

    2014-01-01

    Binding of small molecules with DNA plays an important role in many biological functions such as DNA replication, repair, and transcription. These interactions also offer enormous potential as targets for diagnostics and therapeutics, leading to intense interest in development of methods to probe the underlying binding events. In this chapter, we present a new approach to investigate the structural changes that accompany binding of DNA and small molecules. Instead of relying on conventional yet delicate single-molecule imaging methods, we show how a single microchip gel electrophoresis experiment incorporating both constant electric field and on-off actuation over a specific frequency range enables fundamental structural parameters (e.g., contour and persistence lengths) to be simultaneously determined. The microchip format offers an attractive combination of simplicity and scale-up potential that makes it amenable for high-throughput screening. PMID:24162976

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

    E-print Network

    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...

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

    SciTech Connect

    Cavinato, A.G.; Macleod, R.M.; Ahmed, M.S.

    1988-01-01

    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 /sup 3/H-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.

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

    PubMed Central

    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.; Franco, C.; 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. monocytogenes isolates from clinical samples and feedstuffs consumed by the diseased animals were analyzed in 21 flocks. In most cases, clinical strains from different animals of the same flock had identical pulsotypes, confirming the existence of a listeriosis outbreak. L. monocytogenes strains with pulsotypes identical to those of clinical strains were isolated from silage, potatoes, and maize stalks. This is the first study wherein potatoes and maize stalks are epidemiologically linked with clinical listeriosis. PMID:11722943

  17. [Characterization of Amycolatopsis meditteranei U32 chromosome by pulse-field gel electrophoresis].

    PubMed

    Ma, Wei; Yao, Yufeng; Jiang, Weihong; Jiao, Ruishen; Zhao, Guoping

    2003-12-01

    Amycolatopsis meditteranei U32 is an industrial strain with high yield of rifamycin SV. Its chromosome structure was characterized by pulse-field gel electrophoresis. The topological structure of U32 chromosome was linear and its genome size was about 10 Mb. There was no internal plasmid found. Gene encoding several enzymes involved in rifamycin bio-synthesis and relevant primary metabolism as well as protein related to metabolic regulation were localized by Southern blot. The results revealed that there were at least five copies of rrn operon in U32. The rifamycin bio-synthesis gene cluster was located on one about 700 kb PshBI fragement where several other important genes related to primary and secondary metabolism were also situated. They were the nasD gene, the mcm gene encoding methylmalonyl CoA mutase, the accA gene encoding acethyl CoA carboxylase biotin carrier protein and one copy of the rrn operons. PMID:16276900

  18. Molecular typing by pulsed-field gel electrophoresis of Bacillus thuringiensis from root voles.

    PubMed

    Swiecicka, Izabela

    2003-04-01

    The root voles intestinal strains of Bacillus thuringiensis, were characterised by pulsed-field gel electrophoresis (PFGE). For 14 isolates, three pulsotypes were found, with the use of SmaI or NotI as restriction enzymes. Strains in each pulsotypes presented identical DNA patterns, indicating that the population structure of B. thuringiensis from root voles is clonal. The similarities in banding patterns were estimated at 56% and 33% for SmaI and NotI digests, respectively. The strains under study differed significantly in the size of their entire genome, which varied between 2.4 and 4.2 Mb. No significant differences were detected among the isolates subjected to biochemical properties determined by API tests. Present study showed that genomic diversity is a common feature of B. thuringiensis originating from one ecological niche. PFGE appears to be a useful technique for use in studies on the spread of B. thuringiensis in the environment. PMID:12732973

  19. Molecular basis of apolipoprotein (a) isoform size heterogeneity as revealed by pulsed-field gel electrophoresis.

    PubMed Central

    Lackner, C; Boerwinkle, E; Leffert, C C; Rahmig, T; Hobbs, H H

    1991-01-01

    Lipoprotein(a) [Lp(a)] is a cholesterol-rich lipoprotein that is distinguished by its content of a glycoprotein called apolipoprotein(a) [apo(a)]. Apo(a) varies in size among individuals owing to different numbers of cysteine-rich sequences that are homologous to kringle 4 of plasminogen. The genetic basis for this variation is not understood at the genomic level. In this study we used pulsed-field gel electrophoresis and genomic blotting to identify a highly polymorphic restriction fragment from the apo(a) gene. The fragment contains multiple tandem repeats of a kringle 4-encoding sequence and varies in length from 48 to 190 kb depending on the number of kringle 4-encoding sequences. A total of 19 different alleles were identified among 102 unrelated Caucasian Americans. 94% of individuals studied had two different alleles which could be distinguished by size on pulsed-field gel electrophoresis. The degree of size heterogeneity was much greater than had been previously appreciated based on the analysis of the apparent molecular mass of the protein. The size of the apo(a) gene correlated directly with the size of the apo(a) protein, and inversely with the concentration of Lp(a) in plasma. Segregation analysis of the apo(a) gene was performed in families; siblings with identical apo(a) genotypes had similar plasma levels of Lp(a). These results suggest that in the normal population, the level of plasma Lp(a) is largely determined by alleles at the apo(a) locus. Images PMID:1645755

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

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

    Microsoft Academic Search

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

    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 propa- gated 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

  2. Multilocus Sequence Typing Lacks the Discriminatory Ability of Pulsed-Field Gel Electrophoresis for Typing Salmonella enterica Serovar Typhimurium

    Microsoft Academic Search

    Mohamed K. Fakhr; Lisa K. Nolan; Catherine M. Logue

    2005-01-01

    Nontyphoidal salmonellae are among the leading causes of food-borne disease in the United States. Because of the importance of Salmonella enterica in food-borne disease, numerous typing methodologies have been developed. Among the several molecular typing methods, pulsed-field gel electrophoresis (PFGE) is currently considered the \\

  3. Pulsed-field gel electrophoresis analysis of Bordetella pertussis populations in various European countries with different vaccine policies

    Microsoft Academic Search

    Valrie Caro; Elisabeth Njamkepo; Shirley C. M. Van Amersfoorth; Frits R. Mooi; Abdolreza Advani; Hans O. Hallander; Qiushui He; Jussi Mertsola; Marion Riffelmann; Carola Vahrenholz; Carl H. W. Von Knig; Nicole Guiso

    2005-01-01

    The increasing incidence of pertussis in a number of countries, despite good vaccination coverage, is a cause for concern. We used pulsed-field gel electrophoresis (PFGE) typing to examine the genetic diversity of 101 clinical isolates of Bordetella pertussis, recovered during 19992001, and circulating in five different European countries to evaluate temporal and geographical distribution. This DNA fingerprinting approach seems to

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  5. Detection of rpoB Mutations Associated with Rifampin Resistance in Mycobacterium tuberculosis Using Denaturing Gradient Gel Electrophoresis

    Microsoft Academic Search

    Mark T. McCammon; John S. Gillette; Derek P. Thomas; Srinivas V. Ramaswamy; Edward A. Graviss; Barry N. Kreiswirth; Jan Vijg; Teresa N. Quitugua

    2005-01-01

    Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with rifampin (RIF) resistance in the rpoB gene of Mycobacterium tuberculosis. DGGE scans for mutations across large regions of DNA and is comparable to DNA sequencing in detecting DNA alterations. Specific mutations are often recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint. Five DGGE

  6. Electrophoresis of /sup 35/S-labeled proteoglycans of polyacrylamide-agarose composite gels and their visualization by fluorography

    SciTech Connect

    Carney, S.L.; Bayliss, M.T.; Collier, J.M.; Muir, H.

    1986-01-01

    Techniques for the electrophoresis of /sup 35/S-labeled proteoglycans on polyacrylamide-agarose gel slabs and subsequent fixation, impregnation, and fluorography of such electrophoretograms have been developed. The procedure permits the examination of newly synthesized proteoglycan subspecies using a rapid technique, previously unavailable for these labeled molecules.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    PubMed Central

    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. PMID:16672538

  9. DNA Replication of Bacteriophage T5. 1. Fractionation of Intracellular T5 DNA by Agarose Gel Electrophoresis

    Microsoft Academic Search

    ROGER D. EVERETT; MARY R. LUNT

    1980-01-01

    SUMMARY Two forms of DNA may be isolated from gently lysed T5-infected bacteria. In neutral sucrose gradients a fast sedimenting form (fsf) is distinguishable from a slow sedimenting form (ssf) which moves at a rate similar to that of DNA ex- tracted from mature T 5 virus particles. A method of agarose gel electrophoresis is described which gives complete separation

  10. Determination of the enantiomeric purity of (?) terbutaline by capillary electrophoresis using cyclodextrins as chiral selectors in a polyethylene glycol gel

    Microsoft Academic Search

    Theo de Boer; Kees Ensing

    1998-01-01

    A method was developed for determination of the enantiomeric purity of the therapeutic-pharmacological active (?)-enantiomer of terbutaline using cyclodextrins as a chiral selector dissolved in a removable liquid polyethylene glycol gel by use of capillary electrophoresis. The effect of temperature, type and concentration of polyethylene glycol and cyclodextrins was studied on the resolution between the two enantiomers. Best results were

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  12. Serum protein electrophoresis by using high-resolution agarose gel in clinically healthy and Aspergillus species-infected falcons.

    PubMed

    Kummrow, Maya; Silvanose, Christudas; Di Somma, Antonio; Bailey, Thomas A; Vorbrggen, Susanne

    2012-12-01

    Serum protein electrophoresis has gained importance in avian medicine during the past decade. Interpretation of electrophoretic patterns should be based on species-specific reference intervals and the electrophoresis gel system. In this study, serum protein electrophoresis by using high-resolution agarose gels was performed on blood samples collected from 105 falcons, including peregrine falcons (Falco peregrinus), gyrfalcons (Falco rusticolus), saker falcons (Falco cherrug), red-naped shaheens (Falco pelegrinoides babylonicus), and hybrid falcons, that were submitted to the Dubai Falcon Hospital (Dubai, United Arab Emirates) between 2003 and 2006. Reference values were established in clinically healthy birds and compared with values from falcons infected with Aspergillus species (n = 32). Falcons with confirmed aspergillosis showed significantly lower prealbumin values, which is a novel finding. Prealbumin has been documented in many avian species, but further investigation is required to illuminate the diagnostic significance of this negative acute-phase protein. PMID:23409432

  13. Immunoreactivity and two-dimensional gel-electrophoresis characterization of Egyptian cobra venom proteome.

    PubMed

    Almehdar, Hussein Abduelrahman; Adel-Sadek, Mahmoud Abass; Redwan, Elrashdy Moustafa

    2015-01-01

    The first and second (two) dimensional gel electrophoresis has a broad protein resolution power. It was used to separate and identify cobra venom proteome. The importance of characterizing venom proteins contents from the Egyptian elapidae, specifically neurotoxins, is based on the need to produce effective anti-venom. About 30-55distinct protein spots were identified on silver stained two-dimensional gels. Around two-thirds of the venom proteins displayed low a molecular weight and a migration into hydrophobic side. The venoms from Naja haja and Naja nigricollus showed 45-55 spots, while Walternnesia aegyptia had less (31-37) spots. The commercial prepared polyclonal antivenom had a strong signal for anionic and cationic venom protein spots with molecular weight 20-115 kDa. However, it showed weak or non immunoreactivity toward anionic low molecular weight spots (2.5-15 kDa). These results suggest the need to change the immunization schedule to include low molecular weight toxin-proteomes as separate dose or sequester injection. PMID:25553707

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

    PubMed

    Singh, Sanjay Kumar; Ramaiah, Nagappa

    2011-05-01

    Denaturing gradient gel electrophoresis (DGGE) was used to elucidate spatial and temporal variations in bacterial community composition (BCC) from four locations along the central west coast of India. DNA extracts from 36 water samples collected from surface, mid-depth (-10 m) and dose to bottom (-20 m) during premonsoon, postmonsoon, monsoon were analyzed by PCRfor amplifying variable region of 16S rRNAgene and subsequently through DGGE. Prominent bands were excised, cloned 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 in the BCC appear to be influenced by monsoonal processes. The canonical correspondence analyses suggest that the concentration of chlorophyll a and nitrate are two important environmental factors for both spatial and temporal variations in BCC. Chlorophyll a seems to be impart a top-down control of BCC while nitrate, the bottom-up control. Our results also suggest that BCC can vary over a small geographic distance in highly dynamic, seasonally predisposed tropical coastal waters. PMID:22167947

  15. Urine proteins identified by two-dimensional differential gel electrophoresis facilitate the differential diagnoses of scrapie.

    PubMed

    Lamoureux, Lise; Simon, Sharon L R; Plews, Margot; Ruddat, Viola; Brunet, Simone; Graham, Catherine; Czub, Stefanie; Knox, J David

    2013-01-01

    The difficulty in developing a diagnostic assay for Creutzfeldt - Jakob disease (CJD) and other transmissible spongiform encephalopathies (TSEs) stems in part from the fact that the infectious agent is an aberrantly folded form of an endogenous cellular protein. This precludes the use of the powerful gene based technologies currently applied to the direct detection of other infectious agents. To circumvent this problem our research objective has been to identify a set of proteins exhibiting characteristic differential abundance in response to TSE infection. The objective of the present study was to assess the disease specificity of differentially abundant urine proteins able to identify scrapie infected mice. Two-dimensional differential gel electrophoresis was used to analyze longitudinal collections of urine samples from both prion-infected mice and a transgenic mouse model of Alzheimer's disease. The introduction of fluorescent dyes, that allow multiple samples to be co-resolved and visualized on one two dimensional gel, have increased the accuracy of this methodology for the discovery of robust protein biomarkers for disease. The accuracy of a small panel of differentially abundant proteins to correctly classify an independent nave sample set was determined. The results demonstrated that at the time of clinical presentation the differential abundance of urine proteins were capable of identifying the prion infected mice with 87% sensitivity and 93% specificity. The identity of the diagnostic differentially abundant proteins was investigated by mass spectrometry. PMID:23704971

  16. Urine Proteins Identified by Two-Dimensional Differential Gel Electrophoresis Facilitate the Differential Diagnoses of Scrapie

    PubMed Central

    Lamoureux, Lise; Simon, Sharon L. R.; Plews, Margot; Ruddat, Viola; Brunet, Simone; Graham, Catherine; Czub, Stefanie; Knox, J. David

    2013-01-01

    The difficulty in developing a diagnostic assay for Creutzfeldt - Jakob disease (CJD) and other transmissible spongiform encephalopathies (TSEs) stems in part from the fact that the infectious agent is an aberrantly folded form of an endogenous cellular protein. This precludes the use of the powerful gene based technologies currently applied to the direct detection of other infectious agents. To circumvent this problem our research objective has been to identify a set of proteins exhibiting characteristic differential abundance in response to TSE infection. The objective of the present study was to assess the disease specificity of differentially abundant urine proteins able to identify scrapie infected mice. Two-dimensional differential gel electrophoresis was used to analyze longitudinal collections of urine samples from both prion-infected mice and a transgenic mouse model of Alzheimer's disease. The introduction of fluorescent dyes, that allow multiple samples to be co-resolved and visualized on one two dimensional gel, have increased the accuracy of this methodology for the discovery of robust protein biomarkers for disease. The accuracy of a small panel of differentially abundant proteins to correctly classify an independent nave sample set was determined. The results demonstrated that at the time of clinical presentation the differential abundance of urine proteins were capable of identifying the prion infected mice with 87% sensitivity and 93% specificity. The identity of the diagnostic differentially abundant proteins was investigated by mass spectrometry. PMID:23704971

  17. Fluorescent monitoring of proteins during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting.

    PubMed

    Falk, B W; Elliott, C

    1985-02-01

    Fluorescent labeling of proteins was found to be a very sensitive and reliable alternative to conventional methods for monitoring proteins on Western blots. Proteins were labeled with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) before SDS-PAGE. After electrophoresis and subsequent electro-blotting the fluorescent-labeled proteins were visible upon ultraviolet illumination of the nitrocellulose membranes, and could be photographed to yield an accurate record of the blots before subsequent serological analysis. The sensitivity for detecting MDPF-labeled proteins on nitrocellulose was 100-200 ng, 50 to 100 fold less sensitive than on gels. Fluorescent-labeled TMV and MStpV capsid proteins that were blotted onto nitrocellulose still reacted in serological tests and were detected when present in quantities as low as 100 pg. Fluorescent labeling allows accurate photographic records of the SDS-gel, blot and probed blot using only one sample, and no subsequent staining steps are required. PMID:3887982

  18. An Iterative Algorithm for Segmenting Lanes in Gel Electrophoresis Images ALEXEI M. C. MACHADO 1;3 , MARIO F. M. CAMPOS 1 , ARI M. SIQUEIRA 2 , OSVALDO S. F. DE CARVALHO 1

    E-print Network

    An Iterative Algorithm for Segmenting Lanes in Gel Electrophoresis Images ALEXEI M. C. MACHADO 1 detection on gel electrophoresis computer images. The problem can be posed as the determination fea­ tures. The gel electrophoresis exam consists in breaking a molecule into many fragments

  19. Phosphopeptide quantitation using amine-reactive isobaric tagging reagents and tandem mass spectrometry: application to proteins isolated by gel electrophoresis.

    PubMed

    Sachon, E; Mohammed, S; Bache, N; Jensen, O N

    2006-01-01

    Polyacrylamide gel electrophoresis is widely used for protein separation and it is frequently the final step in protein purification in biochemistry and proteomics. Using a commercially available amine-reactive isobaric tagging reagent (iTRAQ) and mass spectrometry we obtained reproducible, quantitative data from peptides derived by tryptic in-gel digestion of proteins and phosphoproteins. The protocol combines optimized reaction conditions, miniaturized peptide handling techniques and tandem mass spectrometry to quantify low- to sub-picomole amounts of (phospho)proteins that were isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immobilized metal affinity chromatography (FeIII-IMAC) was efficient for removal of excess reagents and for enrichment of derivatized phosphopeptides prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. Phosphopeptide abundance was determined by liquid chromatography/tandem mass (LC/MS/MS) using either MALDI time-of-flight/time-of-flight (TOF/TOF) MS/MS or electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS/MS instruments. Chemically labeled isobaric phosphopeptides, differing only by the position of the phosphate group, were distinguished and characterized by LC/MS/MS based on their LC elution profile and distinct MS/MS spectra. We expect this quantitative mass spectrometry method to be suitable for systematic, comparative analysis of molecular variants of proteins isolated by gel electrophoresis. PMID:16521170

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

    SciTech Connect

    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.

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

    SciTech Connect

    McGrew, B.R.; Green, D.M. (Univ. of New Hampshire, Durham (USA))

    1990-08-15

    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.

  2. Characterization of lipopolysaccharide heterogeneity in Salmonella enteritidis by an improved gel electrophoresis method.

    PubMed Central

    Guard-Petter, J; Lakshmi, B; Carlson, R; Ingram, K

    1995-01-01

    Salmonella enteritidis field isolates of different phage types and pathogenicities were assessed for changes in lipopolysaccharide (LPS) structure, using an improved method of polyacrylamide gel electrophoresis (PAGE) that revealed the same degree of structural detail as mass spectroscopy. The method allowed characterization of an LPS chemotype that may be associated, regardless of phage type, with increased virulence of S. enteritidis. The virulent variant SE6-E21, which efficiently contaminates eggs and yields high numbers of organisms from chick spleens, had an O-antigen/core ratio of 2.8, as determined from gels by densitometry, and 1.67 micrograms of mannose per microgram of 2-keto-3-deoxy-octulosonic acid (KDO), while the avirulent variant SE6-E5 had O-antigen/core ratios of 1.2 and 1.00. The association between O antigen and virulence was also seen on analysis of five new field isolates. One of the new field isolates generated a mixed population of smooth and semismooth variants in agreement with its mixed virulence in chicks. When LPS was purified from large-volume cultures, only the most virulent isolate yielded high amounts of O antigen (1.6 micrograms of mannose per microgram of KDO), while the other isolates had ratios characteristic of semismooth variants (< or = 1.0 microgram of mannose per microgram of KDO), including the isolate of mixed virulence. These results indicate that the improved PAGE method might provide a rapid, sensitive, in vitro assessment of field isolate virulence prior to the performance of definitive infectivity trials. PMID:7487016

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

    SciTech Connect

    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.

  4. Determination of Wolbachia Genome Size by Pulsed-Field Gel Electrophoresis

    PubMed Central

    Sun, Ling V.; Foster, Jeremy M.; Tzertzinis, George; Ono, Midori; Bandi, Claudio; Slatko, Barton E.; O'Neill, Scott L.

    2001-01-01

    Genome sizes of six different Wolbachia strains from insect and nematode hosts have been determined by pulsed-field gel electrophoresis of purified DNA both before and after digestion with rare-cutting restriction endonucleases. Enzymes SmaI, ApaI, AscI, and FseI cleaved the studied Wolbachia strains at a small number of sites and were used for the determination of the genome sizes of wMelPop, wMel, and wMelCS (each 1.36 Mb), wRi (1.66 Mb), wBma (1.1 Mb), and wDim (0.95 Mb). The Wolbachia genomes studied were all much smaller than the genomes of free-living bacteria such as Escherichia coli (4.7 Mb), as is typical for obligate intracellular bacteria. There was considerable genome size variability among Wolbachia strains, especially between the more parasitic A group Wolbachia infections of insects and the mutualistic C and D group infections of nematodes. The studies described here found no evidence for extrachromosomal plasmid DNA in any of the strains examined. They also indicated that the Wolbachia genome is circular. PMID:11244060

  5. High resolution melt analysis (HRMA); a viable alternative to agarose gel electrophoresis for mouse genotyping.

    PubMed

    Thomsen, Nicole; Ali, Radiya G; Ahmed, Jehangir N; Arkell, Ruth M

    2012-01-01

    Most mouse genetics laboratories maintain mouse strains that require genotyping in order to identify the genetically modified animals. The plethora of mutagenesis strategies and publicly available mouse alleles means that any one laboratory may maintain alleles with random or targeted insertions of orthologous or unrelated sequences as well as random or targeted deletions and point mutants. Many experiments require that different strains be cross bred conferring the need to genotype progeny at more than one locus. In contrast to the range of new technologies for mouse mutagenesis, genotyping methods have remained relatively static with alleles typically discriminated by agarose gel electrophoresis of PCR products. This requires a large amount of researcher time. Additionally it is susceptible to contamination of future genotyping experiments because it requires that tubes containing PCR products be opened for analysis. Progress has been made with the genotyping of mouse point mutants because a range of new high-throughput techniques have been developed for the detection of Single Nucleotide Polymorphisms. Some of these techniques are suitable for genotyping point mutants but do not detect insertion or deletion alleles. Ideally, mouse genetics laboratories would use a single, high-throughput platform that enables closed-tube analysis to genotype the entire range of possible insertion and deletion alleles and point mutants. Here we show that High Resolution Melt Analysis meets these criteria, it is suitable for closed-tube genotyping of all allele types and current genotyping assays can be converted to this technology with little or no effort. PMID:23028882

  6. Bacterial community dynamics of salted and fermented shrimp based on denaturing gradient gel electrophoresis.

    PubMed

    Han, Kook-Il; Kim, Yong Hyun; Hwang, Seon Gu; Jung, Eui-Gil; Patnaik, Bharat Bhusan; Han, Yeon Soo; Nam, Kung-Woo; Kim, Wan-Jong; Han, Man-Deuk

    2014-12-01

    The Korean traditional seafood jeotgal is consumed directly or as an additive in other foods to improve flavor or fermentation efficiency. Saeujot, made from salted and fermented tiny shrimp (SFS; Acetes japonicus), is the best-selling jeotgal in Korea. In this study, we reveal the microbial diversity and dynamics in naturally fermented shrimp by denaturing gradient gel electrophoresis (DGGE). The population fingerprints of the predominant microbiota and its succession were generated by DGGE analysis of universal V3 16S rDNA polymerase chain reaction (PCR) amplicons. Overall, 17 strains were identified from sequencing of 30 DGGE bands. The DGGE profiles showed diverse bacterial populations in the sample, throughout the fermentation of SFS. Staphylococcus equorum, Halanaerobium saccharolyticum, Salimicrobium luteum, and Halomonas jeotgali were the dominant bacteria, and their levels steadily increased during the fermentation process. Certain other bacteria, such as Psychrobacter jeotgali and Halomonas alimentaria appeared during the early-fermentation process, while Alkalibacterium putridalgicola, Tetragenococcus muriaticus, and Salinicoccus jeotgali appeared during the late-fermentation process. The members of the order Bacillales were found to be predominant during the fermentation of SFS. Furthermore, S. equorum was identified as the dominant bacterial isolate by the traditional method of culturing under aerobic and facultative anaerobic conditions. We expect that this information will facilitate the design of autochthonous starter cultures for the production of SFS with desired characteristic sensory profiles and shorter ripening times. PMID:25393163

  7. Brownian Dynamics Studies on DNA Gel Electrophoresis. II. `Defect' Dynamics in the Elongation-Contraction Motion

    E-print Network

    Ryuzo Azuma

    2001-11-07

    By means of the Brownian dynamics (BD) method of simulations we have developed, we study dynamics of individual DNA undergoing constant field gel electrophoresis (CFGE), focusing on the relevance of the `defect' concept due to de Gennes in CFGE. The corresponding embodiment, which we call {\\it slack beads} (s-beads) is explicitly introduced in our BD model. In equilibrium under a vanishing field the distance between s-beads and their hopping range are found to be randomly distributed following a Poisson distribution. In the strong field range, where a chain undergoes the elongation-contraction motion, s-beads are observed to be alternatively annihilated in elongation and created in contraction of the chain. On the other hand, the distribution of hopping range of s-beads does not differ much from that in equilibrium. The results indicate that the motion of the chain elongated consists of a huge number of random movements of s-beads. We have also confirmed that these features of s-beads agree qualitatively with those of s-monomers in the extended bond fluctuation model (EBFM) which we recently proposed. The coincidence of the two simulations strongly supports the stochastic semi-local movement of s-monomers which we {\\it a priori} introduced into the EBFM.

  8. Differentiation of Erwinia amylovora strains by pulsed-field gel electrophoresis.

    PubMed Central

    Zhang, Y; Geider, K

    1997-01-01

    Erwinia amylovora strains, isolated from several host plants in various geographic regions during different years, were analyzed by pulsed-field gel electrophoresis (PFGE) after digestion of the DNA from lysed, agar-embedded cells with rare-cutting restriction enzymes. The banding patterns obtained with enzyme XbaI digests revealed significant differences among strains from different areas. North American strains E9 and Ea-Rb, a Rubus strain, were highly divergent from other E. amylovora strains. French strains were different from central European and English strains. E. amylovora strains from central Europe and New Zealand had identical PFGE patters, as had strains from Egypt, Greece, and Turkey. PFGE of genomic DNA from American and English strains gave rise to dissimilar patterns. Patterns of some American strains resembled those from strains isolated in other parts of the world. The restriction fragment length polymorphisms observed by PFGE analysis can be used to group strains and may give hints about the course of distribution of the plant disease. From the sizes of the restriction fragments obtained, a molecular mass of approximately 4.5 Mb was calculated for the genome of E. amylovora. PMID:9361429

  9. Application of denaturing gradient gel electrophoresis (DGGE) for assessing fecal pollution sources at a recreational beach.

    PubMed

    Esseili, M A; Kassem, I I; Lis, J; Sigler, V

    2014-12-01

    We investigated the effectiveness of Escherichia coli community fingerprinting for identifying fecal pollution sources impacting a recreational beach. E. coli in water collected from the beach, nearby creek and storm sewer outfall were enumerated using membrane filtration, while E. coli communities were characterized following polymerase chain reaction analysis and denaturing gradient gel electrophoresis (DGGE) fingerprinting. Analysis of E. coli densities to determine the contributions of the creek and storm sewer during dry weather was inconclusive. However, DGGE fingerprinting indicated that the creek E. coli communities had a greater impact on the beach community composition (80-95% similarity), than on storm sewer communities (41-64%). Following rainfall events, E. coli communities in the creek were at least 93% similar to those at the beach, while the similarity of the outfall and beach communities varied from 72 to 96%. Furthermore, E. coli communities at the beach were more similar to creek communities than to storm sewer communities after the first 2 h and 48 h following the onset of rainfall, and of comparable similarity following 24 h of rainfall, suggesting transient contributions from the storm sewer. DGGE analysis of E. coli communities provided evidence that the creek was a consistent source of E. coli to the beach, while the storm sewer was a transient source. PMID:25473994

  10. Pulsed-field gel electrophoresis as a replacement for bacteriophage typing of Staphylococcus aureus.

    PubMed Central

    Bannerman, T L; Hancock, G A; Tenover, F C; Miller, J M

    1995-01-01

    Bacteriophage typing (BT) (World Health Organization method) has been used at the Centers for Disease Control and Prevention for over 30 years to type isolates of Staphylococcus aureus. Since studies have shown that BT patterns have poor reproducibility and because BT fails to type a high percentage (15 to 20%) of isolates, the Centers for Disease Control and Prevention has converted from using BT to using pulsed-field gel electrophoresis (PFGE) for strain typing S. aureus. We compared the results of BT with results of PFGE for typing 300 isolates of S. aureus, including strains from several well-characterized outbreaks. Ninety-six isolates were BT group I, 19 were group II, 82 were group III, 7 were group V, and 96 were nontypeable. PFGE identified subgroups within each phage group and thus was more discriminating than BT, which identified no subgroups. PFGE was able to type all isolates and distinguish related from unrelated strains of S. aureus. Our modified, standardized PFGE methodology should enable typing laboratories to obtain rapid, reliable results in 3 to 4 days when starting with an isolated colony on agar media. PMID:7751356

  11. Comparative denaturing gradient gel electrophoresis analysis of fungal communities associated with whole plant corn silage.

    PubMed

    May, L A; Smiley, B; Schmidt, M G

    2001-09-01

    Significant portions of grain produced for livestock consumption are convened into ensiled forage. Silage producers have long recognized the positive effects of using an inoculant to insure the proper transformation of forage into a palatable and digestible feedstuff. When silage is fed from a storage structure, exposure to air stimulates the growth of epiphytic aerobes that may result in the loss of up to 50% of the dry matter. Moreover, fungi have been found to be associated with ensiled forage, but their growth is normally suppressed by the anaerobic conditions. However, the introduction of oxygen results in a fungal bloom, and the fungi and the associated metabolites may result in lost productivity in the livestock consuming the contaminated forage. In this study, we report on the diversity of the fungal community associated with whole plant corn silage during the ensiling process, and the effect of two different bacterial inoculants as compared with the uninoculated natural epiphytic fermentation on the distribution of the fungi associated with the silage. The fungal community from duplicate mini-silo packages of the same treatment was analyzed by denaturing gradient gel electrophoresis and direct sequencing of the resulting operational taxonomic units. This method proved useful in analyzing the complex microbial communities associated with the forage in that it was possible to determine that one inoculant dramatically influenced the fungal community associated with whole plant corn silage. PMID:11683465

  12. First clinical isolates of Cronobacter spp. (Enterobacter sakazakii) in Argentina: characterization and subtyping by pulsed-field gel electrophoresis.

    PubMed

    Asato, Valeria C; Vilches, Viviana E; Pineda, Mara G; Casanueva, Enrique; Cane, Alejandro; Moroni, Mirian P; Brengi, Silvina P; Pichel, Mariana G

    2013-01-01

    Cronobacter species are opportunistic pathogens associated with severe infections in neonates and immunocompromised infants. From January 2009 through September 2010, two cases of neonatal infections associated with Cronobacter malonaticus and one case associated with Cronobacter sakazakii, two of them fatal, were reported in the same hospital. These are the first clinical isolates of Cronobacter spp. in Argentina. The objective of this work was to characterize and subtype clinical isolates of Cronobacter spp. in neonate patients, as well as to establish the genetic relationship between these isolates and the foodborne isolates previously identified in the country. Pulsed-field gel electrophoresis analysis showed a genetic relationship between the C. malonaticus isolates from two patients. Different results were found when the pulsed-field gel electrophoresis patterns of clinical isolates were compared with those deposited in the National Database of Cronobacter spp. PMID:24165138

  13. Happy bicentennial, electrophoresis!

    PubMed

    Righetti, Pier Giorgio

    2009-12-01

    A short survey of electrophoresis and a celebration of its bicentennial, with some remarkable mementos and a list of books that shaped the field. Where one also learns of a secret production plant with a huge-scale electrophoretic apparatus for skimming of latex from Hevea brasiliensis and keeping the wheels of the Ally Army running during World War II. And of cyber (mammoth) 2D gels of 1.5 x 1 m in size accommodating >12,000 spots. PMID:19938305

  14. Molecular Typing of Selected Enterococcus faecalis Isolates: Pilot Study Using Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis

    Microsoft Academic Search

    Sreedhar R. Nallapareddy; Ruay-Wang Duh; Kavindra V. Singh; Barbara E. Murray

    2002-01-01

    The present study compared the recently developed multilocus sequence typing (MLST) approach with a well-established molecular typing technique, pulsed-field gel electrophoresis (PFGE), for subspecies differen- tiation of Enterococcus faecalis isolates. We sequenced intragenic regions of three E. faecalis antigen-encoding genes (ace, encoding a collagen and laminin adhesin; efaA, encoding an endocarditis antigen; and salA, encoding a cell wall associated antigen)

  15. Repetitive Sequence-Based PCR versus Pulsed-Field Gel Electrophoresis for Typing of Enterococcus faecalis at the Subspecies Level

    Microsoft Academic Search

    KUMTHORN MALATHUM; KAVINDRA V. SINGH; GEORGE M. WEINSTOCK; BARBARA E. MURRAY

    1998-01-01

    Repetitive sequence-based PCR was compared to pulsed-field gel electrophoresis (PFGE) for the ability to discriminate Enterococcus faecalis isolates at the subspecies level. The BOXA2R primer, derived from repetitive sequences in Streptococcus pneumoniae, was applied to 41 isolates of E. faecalis collected from various sources. The REP1R-Dt and REP2-Dt primers, derived from the gram-negative repetitive extragenic palindromic element, were also applied

  16. Case study of the use of pulsed field gel electrophoresis in the detection of a food-borne outbreak.

    PubMed

    De Lappe, Niall; Cormican, Martin

    2015-01-01

    In early July 2008, a cluster of six Salmonella Agona was identified in the Republic of Ireland. A dispersed, common source outbreak was suspected. Later in July a further case was identified and the Health Protection Agency in the UK indicated that they had 32 cases of S. Agona since Feb 2008. This chapter discusses how pulsed field gel electrophoresis was used to help confirm an outbreak and to trace the source of the outbreak. PMID:25862046

  17. Detection of DNA damage in human lymphocytes by alkaline single cell gel electrophoresis after exposure to benzene or benzene metabolites

    Microsoft Academic Search

    Cristina Andreoli; Paola Leopardi; Riccardo Crebelli

    1997-01-01

    The alkaline single cell gel electrophoresis (Comet) assay was applied to study the occurrence of DNA damage in peripheral lymphocytes of human subjects with occupational exposure to low levels of benzene (twelve gasoline station attendants, with average benzene exposure of 0.3 mg\\/m3, 8 h TWA). The results obtained show a significant excess of DNA damage in lymphocytes of exposed workers,

  18. Characterization of Listeria monocytogenes from an ice cream plant by serotyping and pulsed-field gel electrophoresis

    Microsoft Academic Search

    Maria K Miettinen; K. Johanna Bjrkroth; Hannu J Korkeala

    1999-01-01

    One dominating strain of serotype 1\\/2b was found when serotyping and pulsed-field gel electrophoresis (PFGE) patterns were used for the characterization of 41 Listeria monocytogenes isolates originating from an ice cream plant. Samples were taken from the production environment, equipment and ice cream during the years 19901997. Serotyping divided the isolates into two serovars, 1\\/2b and 4b. Three rare-cutting enzymes

  19. Detection and Identification of Gastrointestinal Lactobacillus Species by Using Denaturing Gradient Gel Electrophoresis and Species-Specific PCR Primers

    Microsoft Academic Search

    J. Walter; G. W. Tannock; A. Tilsala-Timisjarvi; S. Rodtong; D. M. Loach; K. Munro; T. Alatossava

    2000-01-01

    Denaturing gradient gel electrophoresis (DGGE) of DNA fragments obtained by PCR amplification of the V2-V3 region of the 16S rRNA gene was used to detect the presence of Lactobacillus species in the stomach contents of mice. Lactobacillus isolates cultured from human and porcine gastrointestinal samples were identified to the species level by using a combination of DGGE and species-specific PCR

  20. PulseNet standardized protocol for subtyping Listeria monocytogenes by macrorestriction and pulsed-field gel electrophoresis

    Microsoft Academic Search

    Lewis M. Graves; Bala Swaminathan

    2001-01-01

    PulseNet is a national network of pubic health and food regulatory laboratories established in the US to detect clusters of foodborne disease and respond quickly to foodborne outbreak investigations. PulseNet laboratories currently subtype Escherichia coli O157:H7, non-typhoidal Salmonella, and Shigella isolates by a highly standardized 1-day pulsed-field gel electrophoresis (PFGE), and exchange normalized DNA fingerprint patterns via the Internet. We

  1. A public domain image-analysis program for the single-cell gel-electrophoresis (comet) assay

    Microsoft Academic Search

    Christoph Helma; Maria Uhl

    2000-01-01

    The single-cell gel electrophoresis (or comet) assay has gained widespread acceptance as a cheap and simple genotoxicity test, but it requires a computer-assisted image-analysis system. As commercial programs are expensive and inflexible, we decided to develop an image-analysis system based on public domain programs and make it publicly available for the scientific community. Our system is based on the scientific

  2. High-resolution capillary gel electrophoresis of reducing oligosaccharides labeled with 1-aminopyrene-3,6,8-trisulfonate.

    PubMed

    Guttman, A; Chen, F T; Evangelista, R A; Cooke, N

    1996-01-15

    High-resolution capillary gel electrophoresis was used for the separation of oligosaccharides labeled with a novel fluorophore 1-aminopyrene-3,6,8-trisulfonate (APTS) at the reducing termini by reductive amination. The APTS-saccharide adducts were detected by laser-induced fluorescence with excitation by the 488-nm Ar-ion laser and a 520-nm emission filter. The stoichiometry of labeling is such that only one molecule of fluorophore is attached to each molecule of oligosaccharide. Derivatization parameters, such as labeling reagent concentration, labeling temperature, and time as well as the influence of reaction solvent are thoroughly discussed. Desialylation of several sialylated oligosaccharides with different structures and linkages due to the effects of labeling temperature and time are also addressed. Employing the optimized conditions suggested in this paper, fluorophore labeling efficiency greater than 97% was achieved with no significant loss of sialic acid residues. The fluorescently labeled oligosaccharides are then separated and quantified by capillary gel electrophoresis. Practical examples of low-level derivatization and high-resolution capillary gel electrophoresis separation of N-linked glycans of ribonuclease-B and fetuin are also shown. PMID:8789724

  3. A comparison of three serological assays, protein gel electrophoresis and the polymerase chain reaction for the detection of Chlomydia psittici infections in pet birds

    E-print Network

    Hofle, Michael David

    1999-01-01

    : complement fixation (CF), elementary body agglutination (EBA), immunofluorescent antibodies (IFA), polymerase chain reaction (PCR), protein gel electrophoresis. Comparison of the results obtained showed a strong statistical association between EBA to CF, PCR...

  4. Real-time imaging of the reorientation mechanisms of YOYO-labelled DNA molecules during 90 and 120 pulsed field gel electrophoresis

    Microsoft Academic Search

    Sergio Gurrieri; Steven B. Smith; K. Sam Wells; Iain D. Johnson; Carlos Bustamante

    1996-01-01

    Pulsed field gel electrophoresis (PFGE) techniques have been developed to overcome the limitations of conventional electrophoresis and to increase the separ- ation to DNA chromosomes of few megabase pairs in size. Despite of the large success of these techniques, the various separation protocols employed for PFGE experiments have been determined empirically. How- ever, a deep understanding of the molecular mechan-

  5. Detection of antimicrobial (poly)peptides with Acid urea polyacrylamide gel electrophoresis followed by Western immunoblot.

    PubMed

    Porter, Edith; Valore, Erika V; Anouseyan, Rabin; Salzman, Nita H

    2015-01-01

    Antimicrobial (poly)peptides (AMPs) are ancient key effector molecules of innate host defense and have been identified in mammals, insects, plants, and even fungi (Nakatsuji and Gallo, J Invest Dermatol, 132: 887-895, 2012). They exhibit a cationic net charge at physiological pH and are rich in hydrophobic amino acids (Dufourc et al., Curr Protein Pept Sci, 13: 620-631, 2012). Their mode of action has been best investigated in bacteria. When assuming secondary structure the cationic and hydrophobic amino acids are sequestered creating a bipartitioned molecule in which the cationic amino acids mediate initial electrostatic interaction with the negatively charged bacterial surface and the hydrophobic amino acids mediate embedding into the bacterial membranes followed by a multitude of effects interfering with bacterial viability (Nicolas, FEBS J, 276: 6483-6496, 2009; Padovan et al., Curr Protein Pept Sci, 11: 210-219, 2010). However, immunomodulatory, antitumor, and other effects have been added to the ever increasing list of AMP functions (Pushpanathan et al., Int J Pept, 2013: 675391, 2013). Several classes of AMPs have been distinguished based on structure, namely anti-parallel beta-sheet, alpha-helical, circular, as well as disulfide bridge connectivity (Bond and Khalid, Protein Pept Lett, 17: 1313-1327, 2010). Many of the AMPs undergo posttranslational modification including further proteolysis. Biochemical analysis at the protein level is of great interest for a wide range of scientists and important when studying host-pathogen interaction, for example Salmonella invasion of the small intestine. Acid-urea polyacrylamide gel electrophoresis (AU-PAGE) followed by Western immunoblotting is an important tool for the identification and quantification of cationic AMPs. The protocol for these procedures outlined here describes, in detail, the necessary steps; including pouring the AU-gels, preparing the test samples, performing the electrophoretic separation and protein transfer to the membrane, and conducting the immunodetection using an alkaline phosphatase/NBT/BCIP system. A standard SDS-PAGE in comparison with AU-PAGE and the corresponding Western immunoblot are depicted in Fig. 1. PMID:25253251

  6. Detection and quantification of Vibrio populations using denaturant gradient gel electrophoresis.

    PubMed

    Eiler, Alexander; Bertilsson, Stefan

    2006-11-01

    Bacteria affiliated with the genus Vibrio are endemic in marine and estuarine ecosystems and are also found in many freshwater environments. Vibrios can enter viable but non-culturable states and since many species are pathogenic, there is a great need for culture-independent methods that identify and quantify multiple Vibrio populations. We adopted Vibrio-specific 16S rRNA-directed primers and a competitive PCR protocol (QC-PCR; [Thompson, J.R., Randa, M.A., Marcelino, L.A., Tomita-Mitchell, A., Lim, E., Polz, M.F., 2004b. Diversity and dynamics of a North Atlantic coastal Vibrio community. Appl. Environ. Microbiol. 70, 4103-4110]) for separation and quantification of Vibrio populations using denaturant gradient gel electrophoresis (DGGE). Sixteen Vibrio isolates and eight environmental samples were used to assess the precision and resolution of the method. A 45-70% gradient of Urea and formamide enabled separation of Vibrio populations with single nucleotide differences in the amplified fragment. A titration curve for the QC-PCR-DGGE, verified by amending surface water bacterioplankton samples with up to 3 x 10(5)Vibrio cholerae cells, could be approximated by a linear regression of log-transformed values (R(2)=0.96). The limit of detection for single populations was 180 cells per extracted sample or about 4 cells per PCR reaction. Environmental samples from the southern Stockholm archipelago in the Baltic Sea and the more saline coastal waters of Skagerrak each carried between 2 and 6 Vibrio populations, and there were major differences between the locations. Notably, multiple Vibrio populations could be detected and quantified against a background of native bacterioplankton exceeding Vibrio population abundance by more than 6 orders of magnitude. Putative identification based on migration in the DGGE gel was verified by parallel cloning and sequencing of PCR products, and representative clones were also characterized by DGGE. This general approach could also be useful for targeting other phylogenetically constrained bacterial groups and assess their abundance and distribution in complex environmental settings. PMID:16730823

  7. Detection of FLT3 Oncogene Mutations in Acute Myeloid Leukemia Using Conformation Sensitive Gel Electrophoresis

    PubMed Central

    Gari, Mamdooh; Abuzenadah, Adel; Chaudhary, Adeel; Al-Qahtani, Mohammed; Banni, Huda; Ahmad, Waseem; Al-Sayes, Fatin; Lary, Sahira; Damanhouri, Ghazi

    2008-01-01

    FLT3 (fms-related tyrosine kinase 3) is a receptor tyrosine kinase class III that is expressed on by early hematopoietic progenitor cells and plays an important role in hematopoietic stem cell proliferation, differentiation and survival. FLT3 is also expressed on leukemia blasts in most cases of acute myeloid leukemia (AML). In order to determine the frequency of FLT3 oncogene mutations, we analyzed genomic DNA of adult de novo acute myeloid leukemia (AML). Polymerase chain reaction (PCR) and conformation-sensitive gel electrophoresis (CSGE) were used for FLT3 exons 11, 14, and 15, followed by direct DNA sequencing. Two different types of functionally important FLT 3 mutations have been identified. Those mutations were unique to patients with inv(16), t(15:17) or t(8;21) and comprised fifteen cases with internal tandem duplication (ITD) mutation in the juxtamembrane domain and eleven cases with point mutation (exon 20, Asp835Tyr). The high frequency of the flt3 proto-oncogene mutations in acute myeloid leukemia AML suggests a key role for the receptor function. The association of FLT3 mutations with chromosomal abnormalities invites speculation as to the link between these two changes in the pathogenesis of acute myeloid leukemiaAML. Furthermore, CSGE method has shown to be a rapid and sensitive screening method for detection of nucleotide alteration in FLT3 gene. Finally, this study reports, for the first time in Saudi Arabia, mutations in the human FLT3 gene in acute myeloid leukemia AML patients. PMID:19330068

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

    PubMed Central

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

    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 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

  9. Pulsed-Field Gel Electrophoresis Diversity of Human and Bovine Clinical Salmonella Isolates

    PubMed Central

    Soyer, Ye?im; Alcaine, Samuel D.; Schoonmaker-Bopp, Dainna J.; Root, Timothy P.; Warnick, Lorin D.; McDonough, Patrick L.; Dumas, Nellie B.; Grhn, Yrjo T.

    2010-01-01

    Abstract Pulsed-field gel electrophoresis (PFGE) characterization of 335 temporally and spatially matched clinical, bovine, and human Salmonella enterica subsp. enterica isolates revealed 167 XbaI PFGE patterns. These isolates were previously classified into 51 serotypes and 73 sequence types, as determined by multilocus sequence typing. Discriminatory power of PFGE (Simpson's index, D?=?0.991) was considerably higher than that of multilocus sequence typing (D?=?0.920) or serotyping (D?=?0.913). Although 128 PFGE types each only represented a single isolate, 8 PFGE types represented >4 isolates, including (i) three serotype Enteritidis and Heidelberg patterns that were only identified among human isolates, (ii) two PFGE patterns (each representing serotypes Bardo and Newport) that were significantly more common among bovine isolates as compared with human isolates; (iii) two PFGE types that each includes two serotypes (4,5,12:i:- and Typhimurium; Thompson and 1,7:-:1,5); and (iv) one PFGE type that includes eight Typhimurium isolates from humans and cattle. Characterization of isolates collected over multiple farm visits indicated that given specific PFGE types persisted over time on 11 farms. On an additional seven farms, isolates with a given sequence type represented multiple PFGE type, which typically only differed by <3 bands, suggesting PFGE type diversification during strain persistence. Sixteen PFGE types were isolated from 2 or more farms, including two widely distributed serotype Newport-associated PFGE types each found on 10 farms. In six instances two or three human isolates collected in the same county in the same or consecutive months represented the same subtypes, suggesting small human case clusters. PFGE-based characterization and surveillance of human and animal isolates can provide improved understanding of Salmonella diversity and epidemiology, including identification of possible host-associated and common, widely distributed PFGE types. PMID:20180633

  10. Two-dimensional gel electrophoresis and immunoblotting of Campylobacter pylori proteins.

    PubMed

    Dunn, B E; Perez-Perez, G I; Blaser, M J

    1989-06-01

    Whole-cell, outer-membrane protein, flagellum-associated antigens and partially purified urease of Campylobacter pylori were analyzed by two-dimensional gel electrophoresis. C. pylori strains were readily distinguished from strains of Campylobacter jejuni, C. coli, and C. fetus by absence of major outer membrane proteins with Mrs of 41,000 to 45,000. C. pylori strains also lacked the acidic surface-array proteins at Mr 100,000 to 149,000 identified previously in serum-resistant strains of C. fetus. Surface labeling of intact C. pylori cells with 125I revealed two common major proteins, which we have designated protein 2 (pI 5.6 to 5.8, Mr 66,000) and protein 3 (pI 5.2 to 5.5, Mr 63,000). Proteins 2 and 3 were also the major components (subunits) observed in partially purified urease. Partially purified preparations of flagella consistently contained proteins 2 and 3. Thus, urease appears to be associated with both outer membranes and flagella of C. pylori. C. pylori strains also possessed an antigen at Mr 59,000 which was cross-reactive with antiserum against flagella of C. jejuni. However, the antigen did not appear to be associated with flagella per se in C. pylori. Protein 2 was unique to C. pylori among the Campylobacter species studied. It was not recognized by antibody against whole cells of C. jejuni or C. fetus or flagella of C. jejuni. Protein 3 was cross-reactive with antiserum against whole cells of C. jejuni and C. fetus, as were several other major protein antigens. Because protein 2 is a major outer membrane protein that is apparently unique to C. pylori, development of monospecific antibodies against this antigen may be useful for the identification of C. pylori in tissues, and purified antigen may be useful for serologic tests for specific diagnosis of C. pylori infections. PMID:2722241

  11. COMPARISON OF PROTEIN IDENTIFICATION BY MALDI-TOF/TOF AND LC ELECTROSPRAY IONIZATION TANDEM MS FOR 2D GEL SEPARATED CAULIFLOWER SAMPLES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In 2D-gel mapping, it is well-known that essentially identical proteins migrate to different positions in the gel (due to PTMs etc.) while some, seemingly, well resolved spots consist of multiple proteins. Clearly, this observation can have dire consequences for the validity of gel-based comparativ...

  12. A new method for assigning common spot boundaries for multiple gels in two-dimensional gel electrophoresis.

    PubMed

    Rye, Morten Beck; Faergestad, Ellen M; Alsberg, Bjrn K

    2008-03-01

    The benefits of defining common spot boundaries when several gels from 2-DE are compared and analyzed have lately been stressed by both commercial software producers and users of this software. Though the importance of common spot boundaries is clearly stated, few reports exist that target this issue explicitly. In this study a method for defining common spots boundaries is developed, called the spot density method. The method consists of the following steps: segmentation and spot identification on each individual gel, transferring the spot-center coordinates for all gels onto a single new gel, collecting spot centers clustered together in the new gel and finally assigning pixels and new spot boundaries based on the spots in each cluster. The method is compared to a synthetic gel approach, and validated by visual inspection of three representative areas in the gels. The gel images need to be aligned prior to segmentation and spot identification, but the method can be used regardless of the choice of segmentation procedure. This makes the method an easy extension to existing methods for spot identification and matching. Conclusions based on the visual inspection are that the spot density method identifies partly overlapping spots and low-intensity spots better than the synthetic gel approach. PMID:18348212

  13. Comparison of liver mitochondrial proteins derived from newborn cloned calves and from cloned adult cattle by two-dimensional differential gel electrophoresis.

    PubMed

    Takeda, Kumiko; Tasai, Mariko; Akagi, Satoshi; Watanabe, Shinya; Oe, Mika; Chikuni, Koichi; Ohnishi-Kameyama, Mayumi; Hanada, Hirofumi; Nakamura, Yoshiaki; Tagami, Takahiro; Nirasawa, Keijiro

    2011-04-01

    Aberrant reprogramming of donor somatic cell nuclei may result in many severe problems in animal cloning. The inability to establish functional interactions between donor nucleus and recipient mitochondria is also likely responsible for such a developmental deficiency. However, detailed knowledge of protein expression during somatic cell nuclear transfer (SCNT) in cattle is lacking. In the present study, variations in mitochondrial protein levels between SCNT-derived and control cattle, and from calves derived by artificial insemination were investigated. Mitochondrial fractions were prepared from frozen liver samples and subjected to two-dimensional (2-D) fluorescence differential gel electrophoresis (DIGE) using CyDye dyes. Protein expression changes were confirmed with a volume ratio greater than 2.0 (P?2D-DIGE analysis revealed differential expression of three proteins for SCNT cattle (n?=?4) and seven proteins for SCNT calves (n?=?6) compared to controls (P?

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

    PubMed Central

    2010-01-01

    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. Conclusions This work reports the first population dynamics study of complex smear ecosystems exhibiting in situ antilisterial activity. TTGE revealed the presence of marine lactic acid bacteria that are likely related to the strong Listeria inhibition, as their early development in the smear occurred simultaneously with a decrease in Listeria cell count. PMID:20222967

  15. Use of Pulsed-Field Gel Electrophoresis of Conserved XbaI Fragments for Identification of Swine Salmonella Serotypes?

    PubMed Central

    Gaul, Stephen B.; Wedel, Stephanie; Erdman, Matthew M.; Harris, D. L.; Harris, Isabel Turney; Ferris, Kathleen E.; Hoffman, Lorraine

    2007-01-01

    Swine Salmonella isolates (n = 674) from various locations throughout the United States and Canada were analyzed via pulsed-field gel electrophoresis (PFGE) with XbaI. PFGE subtypes were analyzed by cluster analysis and compared to conventional serotyping results. The analysis showed a correlation of serotype to PFGE subtype. In addition, conserved fragments were identified within the restriction patterns that were unique to each serotype. PFGE using XbaI restriction provided a possible alternative method for screening and identifying swine Salmonella serotypes. PMID:17166969

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

    PubMed Central

    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 ever appeared alone, but both high- and low-molecular-weight bands always appeared. It was concluded that the apparently high-molecular-weight bands represented aggregates of the two small polypeptides whose monomers formed the major bands. The preparation thus contained only two polypeptides. Images PMID:7411691

  17. Differentiation between fresh and frozen-thawed sea bass (Dicentrarchus labrax) fillets using two-dimensional gel electrophoresis.

    PubMed

    Ethuin, Pierrette; Marlard, Sylvain; Delosire, Mylne; Carapito, Christine; Delalande, Franois; Van Dorsselaer, Alain; Dehaut, Alexandre; Lencel, Valrie; Duflos, Guillaume; Grard, Thierry

    2015-06-01

    This study aimed to identify a protein marker that can differentiate between fresh skinless and frozen-thawed sea bass (Dicentrarchus labrax) fillets using the two-dimensional polyacrylamide gel electrophoresis (2-DE) technique. Distinct gel patterns, due to proteins with low molecular weight and low isoelectric points, distinguished fresh fillets from frozen-thawed ones. Frozen-thawed fillets showed two specific protein spots as early as the first day of the study. However, these spots were not observed in fresh fillets until at least 13days of storage between 0 and 4C, fillets were judged, beyond this period, fish were unfit for human consumption as revealed by complementary studies on fish spoilage indicators namely total volatile basic nitrogen and biogenic amines. Mass spectrometry identified the specific proteins as parvalbumin isoforms. Parvalbumins may thus be useful markers of differentiation between fresh and frozen-thawed sea bass fillets. PMID:25624236

  18. Separation of long linear polymers in gel electrophoresis with alternating electric fields: a theoretical study using the necklace model.

    PubMed

    Terranova, G R; Mrtin, H O; Aldao, C M

    2012-06-01

    The necklace model, which mimics the reptation of a chain of N beads in a square lattice, is used to study the drift velocity of charged linear polymers in gels under an applied electric field that periodically changes its direction. The characteristics of the model allow us to determine the effects of the alternating electric field on the chains' dynamics. We explain why chains of different N can be made to move in opposite directions with a nonuniform electric field with certain values of intensity and frequency. The key point is that, when alternating electric fields are applied, longer chains spend more time out of the steady-state regime than lower chains. Numerical results are obtained by means of Monte Carlo simulations and they are qualitatively in agreement with experiments of DNA migration in gel electrophoresis. PMID:23005118

  19. Separation of long linear polymers in gel electrophoresis with alternating electric fields: A theoretical study using the necklace model

    NASA Astrophysics Data System (ADS)

    Terranova, G. R.; Mrtin, H. O.; Aldao, C. M.

    2012-06-01

    The necklace model, which mimics the reptation of a chain of N beads in a square lattice, is used to study the drift velocity of charged linear polymers in gels under an applied electric field that periodically changes its direction. The characteristics of the model allow us to determine the effects of the alternating electric field on the chains dynamics. We explain why chains of different N can be made to move in opposite directions with a nonuniform electric field with certain values of intensity and frequency. The key point is that, when alternating electric fields are applied, longer chains spend more time out of the steady-state regime than lower chains. Numerical results are obtained by means of Monte Carlo simulations and they are qualitatively in agreement with experiments of DNA migration in gel electrophoresis.

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

    NASA Technical Reports Server (NTRS)

    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.

  1. Mass spectrometric protein structure characterization reveals cause of migration differences of haptoglobin alpha chains in two-dimensional gel electrophoresis.

    PubMed

    Mikkat, Stefan; Koy, Cornelia; Ulbrich, Markus; Ringel, Bruno; Glocker, Michael O

    2004-12-01

    Haptoglobin belongs to the major constituents of plasma and acts as hemoglobin-binding and acute-phase protein. Due to the occurrence of three major allelic variants and further structural modifications, the alpha chains of haptoglobin form varying spot patterns in two-dimensional gel electrophoresis (2-DE) gels, which is generally observed in differential proteome analyses using plasma or related body fluids of humans. In the present study plasma samples from 10 donors of initially unknown haptoglobin phenotype were separated by 2-DE and tryptic digests of excised haptoglobin alpha chain spots were analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and MALDI-quadrupole ion trap TOF-MS. Haptoglobin alpha1S, alpha1F, as well as alpha2 chains were found to occur each with at least three structurally differing protein species: (i) the unmodified form, which corresponds to the sequence database entries; (ii) derivatives, in which asparagine at position five is deamidated to aspartic acid; and (iii) derivatives with an additional C-terminal arginine residue. These structural variants account for the most commonly observed spot patterns of haptoglobin alpha chains in Coomassie-stained gels. Additionally, a minor derivative of the haptoglobin alpha2 chain carrying both modifications, deamidation at position five and the C-terminal arginine residue, was identified. Theoretical pI values of the characterized structural variants are, consistent with their observed migration in the 2-DE gels. PMID:15378693

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

    PubMed

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

    2012-01-01

    Currently, the effect of crude oil on ammonia-oxidizing bacterium communities from mangrove sediments is little understood. We studied the diversity of ammonia-oxidizing bacteria in mangrove microcosm experiments using mangrove sediments contaminated with 0.1, 0.5, 1, 2, and 5% crude oil as well as non-contaminated control and landfarm soil from near an oil refinery in Camamu Bay in Bahia, Brazil. The evolution of CO(2) production in all crude oil-contaminated microcosms showed potential for mineralization. Cluster analysis of denaturing gradient gel electrophoresis-derived samples generated with primers for gene amoA, which encodes the functional enzyme ammonia monooxygenase, showed differences in the sample contaminated with 5% compared to the other samples. Principal component analysis showed divergence of the non-contaminated samples from the 5% crude oil-contaminated sediment. A Venn diagram generated from the banding pattern of PCR-denaturing gradient gel electrophoresis was used to look for operational taxonomic units (OTUs) in common. Eight OTUs were found in non-contaminated sediments and in samples contaminated with 0.5, 1, or 2% crude oil. A Jaccard similarity index of 50% was found for samples contaminated with 0.1, 0.5, 1, and 2% crude oil. This is the first study that focuses on the impact of crude oil on the ammonia-oxidizing bacterium community in mangrove sediments from Camamu Bay. PMID:22370886

  3. A Chip-Capillary Hybrid Device for Automated Transfer of Sample Pre-Separated by Capillary Isoelectric Focusing to Parallel Capillary Gel Electrophoresis for Two-Dimensional Protein Separation

    PubMed Central

    Lu, Joann J.; Wang, Shili; Li, Guanbin; Wang, Wei; Pu, Qiaosheng; Liu, Shaorong

    2012-01-01

    In this report, we introduce a chip-capillary hybrid device to integrate capillary isoelectric focusing (CIEF) with parallel capillary sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or capillary gel electrophoresis (CGE) toward automating two-dimensional (2D) protein separations. The hybrid device consists of three chips that are butted together. The middle chip can be moved between two positions to re-route the fluidic paths, which enables the performance of CIEF and injection of proteins partially resolved by CIEF to CGE capillaries for parallel CGE separations in a continuous and automated fashion. Capillaries are attached to the other two chips to facilitate CIEF and CGE separations and to extend the effective lengths of CGE columns. Specifically, we illustrate the working principle of the hybrid device, develop protocols for producing and preparing the hybrid device, and demonstrate the feasibility of using this hybrid device for automated injection of CIEF-separated sample to parallel CGE for 2D protein separations. Potentials and problems associated with the hybrid device are also discussed. PMID:22830584

  4. Assessment of the 2-d gel-based proteomics application of clinically archived formalin-fixed paraffin embedded tissues.

    PubMed

    Davalieva, Katarina; Kiprijanovska, Sanja; Polenakovic, Momir

    2014-04-01

    Hospital tissue repositories possess a vast and valuable supply of disease samples with matched retrospective clinical information. Detection and characterization of disease biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues will greatly aid the understanding of the diseases mechanisms and help in the development of diagnostic and prognostic markers. In this study, the possibility of using full-length proteins extracted from clinically archived FFPE tissues in two-dimensional (2-D) gel-based proteomics was evaluated. The evaluation was done based on two types of tumor tissues (breast and prostate) and two extraction protocols. The comparison of the 2-D patterns of FFPE extracts obtained by two extraction protocols with the matching frozen tissue extracts showed that only 7-10% of proteins from frozen tissues can be matched to proteins from FFPE tissues. Most of the spots in the 2-D FFPE's maps had pl 4-6, while the percentages of proteins with pl above 6 were 3-5 times lower in comparison to the fresh/frozen tissue. Despite the three-fold lower number of the detected spots in FFPE maps compared to matched fresh/frozen maps, 67-78% of protein spots in FFPE could not be matched to the corresponding spots in the fresh/frozen tissue maps indicating irreversible protein modifications. In conclusion, the inability to completely reverse the cross-linked complexes and overcome protein fragmentation with the present day FFPE extraction methods stands in the way of effective use of these samples in 2-D gel based proteomics studies. PMID:24500075

  5. Lithium Dodecyl Sulfate\\/Polyacrylamide Gel Electrophoresis of Thylakoid Membranes at 4 degrees C: Characterizations of Two Additional Chlorophyll A-Protein Complexes

    Microsoft Academic Search

    Philippe Delepelaire; Nam-Hai Chua

    1979-01-01

    Lithium dodecyl sulfate\\/polyacrylamide gel electrophoresis of Chlamydomonas reinhardtii thylakoid membranes at room temperature gave two chlorophyll-protein complexes, CP I and CP II, as had been reported previously. However, when the electrophoresis was performed at 4 degrees C, there was an increase in the amount of chlorophyll associated with CP I and CP II, and in addition, three other chlorophyll-protein complexes

  6. Optimization of Terminal-Restriction Fragment Length Polymorphism Analysis for Complex Marine Bacterioplankton Communities and Comparison with Denaturing Gradient Gel Electrophoresis

    Microsoft Academic Search

    MARKUS M. MOESENEDER; JESUS M. ARRIETA; GERARD MUYZER; CHRISTIAN WINTER; GERHARD J. HERNDL

    1999-01-01

    The potential of terminal-restriction fragment length polymorphism (T-RFLP) and the detection of opera- tional taxonomic units (OTUs) by capillary electrophoresis (CE) to characterize marine bacterioplankton communities was compared with that of denaturing gradient gel electrophoresis (DGGE). A protocol has been developed to optimize the separation and detection of OTUs between 20 and 1,632 bp by using CE and laser-induced fluorescence

  7. Two-dimensional conformation-dependent electrophoresis (2D-CDE) to separate DNA fragments containing unmatched bulge from complex DNA samples

    PubMed Central

    Gunnarsson, Gudmundur H.; Thormar, Hans G.; Gudmundsson, Bjarki; Akesson, Lina; Jonsson, Jon J.

    2004-01-01

    DNA fragments containing mispaired and modified bases, bulges, lesions and specific sequences have altered conformation. Methods for separating complex samples of DNA fragments based on conformation but independent of length have many applications, including (i) separation of mismatched or unmatched DNA fragments from those perfectly matched; (ii) simultaneous, diagnostic, mismatch scanning of multiple fragments; (iii) isolation of damaged DNA fragments from undamaged fragments; and (iv) estimation of reannealing efficiency of complex DNA samples. We developed a two-dimensional conformation-dependent electrophoresis (2D-CDE) method for separating DNA fragments based on length and conformation in the first dimension and only on length in the second dimension. Differences in migration velocity due to conformation were minimized during second dimension electrophoresis by introducing an intercalator. To test the method, we constructed 298 bp DNA fragments containing cytosine bulges ranging from 1 to 5 nt. Bulge-containing DNA fragments had reduced migration velocity in the first dimension due to altered conformation. After 2D-CDE, bulge-containing DNA fragments had migrated in front of an arc comprising heterogeneous fragments with regular conformation. This simple and robust method could be used in both analytical and preparative applications involving complex DNA samples. PMID:14762200

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  9. High Resolution Capillary Electrophoresis of Forensic DNA Using a Non-Gel Sieving Buffer

    Microsoft Academic Search

    Bruce R. McCord; Janet M. Jung; Elizabeth A. Holleran

    1993-01-01

    For the routine application of capillary electrophoresis to forensic DNA analysis, a number of requirements must be met. In the analysis of DNA amplified by the polymerase chain reaction these requirements include high resolution and consistent and reproducible runs. Genetic markers of interest in this study reproducible runs more base pairs long with repeat unnits as small as 2 base

  10. Use of DNA Ladders for Reproducible Protein Fractionation by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

    E-print Network

    Chait, Brian T.

    . Keywords: DNA quantitation fractionation SILAC mass spectrometry proteomics Introduction In shotgun proteomics, protein digests are usually analyzed by liquid chromatography-tandem mass spectrometry with mass spectrometry. To evaluate the reproducibility of DNA-ladder-assisted gel cutting for quantitative

  11. Physical interpretation of the L(r) parameter in the theory for the gel electrophoresis of partially denatured DNA.

    PubMed

    Sean, David; Slater, Gary W

    2010-10-01

    Partial strand melting of dsDNA during gel electrophoresis typically results in an abrupt reduction of mobility. Several DNA analysis technologies are based on this phenomenon. Inspired by the de Gennes' theory for the reptation of branched polymers in gels, Lerman et al. (Ann. Rev. Biophys. Bioeng. 1984, 13, 399-423) proposed a mathematical expression to predict this reduced mobility. The latter contains only two parameters: the average number of denatured bases p (which can be obtained using a theory for DNA melting) and a constant L(r). However, there is confusion in the literature regarding the physical interpretation of L(r) and little is known about its dependence upon experimental parameters. The purpose of this short communication is to derive an explicit equation for the parameter L(r) from the de Gennes theory of reptation. Our derivation shines light on the meaning of L(r), clarifies the scope of the underlying approximations, and makes predictions about the dependence of L(r) upon the gel pore size and the persistence length of ssDNA. PMID:20922761

  12. Quantitative determination of human cytomegalovirus target sequences in peripheral blood leukocytes by nested polymerase chain reaction and temperature gradient gel electrophoresis

    Microsoft Academic Search

    P. Schafer; Riidiger W. Braun; K. Mohring; Karsten Henco; Jie Kang; Thomas Wendland; J. E. Kuhn

    1993-01-01

    A competitive nested PCR-temperature gradient gel electrophoresis protocol (nPCR\\/TGGE) has been es- tablished for the quantification of human cytomegalo- virus (HCMV) target sequences. The measurement was achieved by co-amplification of a defined copy number of an internal standard (st) and separation of st and wild- type (wt) amplimers by temperature gradient gel electro- phoresis (TGGE). The number of HCMV target

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

    PubMed Central

    2010-01-01

    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 understanding host proteins-virus interactions of these two PRRSV strains. PMID:20504321

  14. University of California, Irvine Environmental Health and Safety www.ehs.uci.edu Questions Call: (949) 824-6200 Version 2.1 Ethidium bromide is a commonly used stain for identifying nucleic acids in electrophoresis gels. It is

    E-print Network

    George, Steven C.

    in electrophoresis gels. It is known to be toxic and mutagenic and may be fatal if swallowed and harmful if inhaled Procedures Gels, filters, and other solids containing ethidium bromide must be managed as a hazardous for removing ethidium bromide from electrophoresis buffers through a bed of activated charcoal. Prior to drain

  15. Identification of plant mitochondrial proteins: A procedure linking two-dimensional gel electrophoresis to protein sequencing from PVDF membranes using a FastBlot cycle

    Microsoft Academic Search

    Bryan Dunbar; Thomas E. Elthon; John C. Osterman; Beth A. Whitaker; S. Brian Wilson

    1997-01-01

    Identification of the 329 spots visible in 2D gels of plant mitochondrial proteins is a challenge. This paper describes a\\u000a 2D mini-gel protocol involving free-radical scavengers and purified reagents to make it compatible with protein sequencing,\\u000a and evaluates its performance. The paper also describes a FastBlot sequencing cycle with the cycle time for protein sequencing\\u000a from PVDF membranes reduced to

  16. A Proposal for Source Tracking of Fecal Pollution in Recreational Waters by Pulsed-Field Gel Electrophoresis

    PubMed Central

    Furukawa, Takashi; Suzuki, Yoshihiro

    2013-01-01

    This study aimed to identify specific river sources of fecal contamination by applying pulsed-field gel electrophoresis (PFGE) to environmental water samples from a recreational beach in Japan. The genotypes of all Enterococcus faecium and Enterococcus faecalis strains used as indicators of fecal pollution on the recreational beach and rivers were analyzed by PFGE, and the PFGE profiles of the strains were classified at a 0.9 similarity level using dendrogram analysis. PFGE types of E. faecium isolated from Sakai River or urban drainage were classified in the same cluster. Therefore, the probable sources of fecal pollution on the recreational beach were Sakai River and urban drainage. The approaches for microbial source tracking employed in this study used PFGE with Enterococcus species as an indicator can be a potential tool to specify the source(s) of fecal pollution and contribute to improved public health in coastal environments. PMID:24256972

  17. Biofilm formation of salivary microbiota on dental restorative materials analyzed by denaturing gradient gel electrophoresis and sequencing.

    PubMed

    Wang, Shuai; Guo, Lihong; Seneviratne, Chaminda Jayampath; Huang, Bo; Han, Jianmin; Peng, Lei; Liu, Xiaodi; Zhang, Chengfei

    2014-01-01

    The microbial diversity of biofilms formed on the surfaces of amalgam, glass-ionomer cement, and resin composite was analyzed by denaturing gradient gel electrophoresis (DGGE). The V2-V3 region of salivary microbial 16S rDNA gene sequences of planktonic and biofilm bacteria, after 1 day and 1 week of incubation, was amplified by polymerase chain reaction (PCR) and analyzed by DGGE. The amounts of strongly adherent phylotypes after 1 day and 1 week on the three dental restorative materials were more than those on hydroxyapatite. Streptococcus salivarius was detected in both loosely adherent and strong adherent groups of all 1-day samples. At 1 week, the amounts of loosely adherent and strongly adherent phylotypes present on the three restorative materials ranked in this ascending order: glass-ionomer cement < resin composite < amalgam. Results of DGGE analysis suggested that glass-ionomer cement was the best material of choice in terms of suppressing bacterial phylotypes in biofilms. PMID:24598237

  18. A novel approach to pseudopodia proteomics: excimer laser etching, two-dimensional difference gel electrophoresis, and confocal imaging

    PubMed Central

    Mimae, Takahiro; Ito, Akihiko; Hagiyama, Man; Nakanishi, Jun; Hosokawa, Yoichiroh; Okada, Morihito; Murakami, Yoshinori; Kondo, Tadashi

    2014-01-01

    Pseudopodia are actin-rich ventral cellular protrusions shown to facilitate the migration and metastasis of tumor cells. Here, we present a novel approach to perform pseudopodia proteomics. Tumor cells growing on porous membranes extend pseudopodia into the membrane pores. In our method, cell bodies are removed by horizontal ablation at the basal cell surface with the excimer laser while pseudopodia are left in the membrane pores. For protein expression profiling, whole cell and pseudopodia proteins are extracted with a lysis buffer, labeled with highly sensitive fluorescent dyes, and separated by two-dimensional gel electrophoresis. Proteins with unique expression patterns in pseudopodia are identified by mass spectrometry. The effects of the identified proteins on pseudopodia formation are evaluated by measuring the pseudopodia length in cancer cells with genetically modified expression of target proteins using confocal imaging. This protocol allows global identification of pseudopodia proteins and evaluation of their functional significance in pseudopodia formation within one month. PMID:25309719

  19. Competitive adsorption of bovine serum albumin and lysozyme on characterized calcium phosphates by polyacrylamide gel electrophoresis method.

    PubMed

    Zhu, X D; Fan, H S; Zhao, C Y; Lu, J; Ikoma, T; Tanaka, J; Zhang, X D

    2007-11-01

    Characterizations of hydroxyapatite (HA), biphasic calcium phosphate (BCP) and beta tricalcium phosphate (beta-TCP) ceramic particles were carried out using X-ray diffusion (XRD), Scanning electron micrograph (SEM), Particle Sizer and Zeta potential analyzer. Competitive adsorption of bovine serum albumin (BSA) and lysozyme (LSZ) on the three calcium phosphates were investigated by polyacrylamide gel electrophoresis (PAGE) method. The results showed that HA, BCP and beta-TCP ceramic particles with irregular shapes and similar size distributions all had negative surface net charges in pH7.4 phosphate buffered saline (PBS) solution and exhibited alike behaviors of BSA and LSZ adsorption. LSZ had higher affinity for calcium phosphate ceramics than BSA and its adsorption on them didn't be almost influenced by the increasing of BSA concentration in the solution. Electrostatic interaction played an important role on the competitive adsorption of BSA and LSZ on the surface of calcium phosphate ceramic particles. PMID:17619993

  20. Proteins synthesized in African swine fever virus-infected cells analyzed by two-dimensional gel electrophoresis.

    PubMed

    Urzainqui, A; Tabars, E; Carrasco, L

    1987-09-01

    At least 74 acidic and 37 basic proteins are synthesized in African swine fever virus (ASFV)-infected monkey cells not detected in uninfected cells analyzed by two-dimensional gel electrophoresis. Essentially all the proteins synthesized early during infection are also observed at late times. The use of inhibitors such as cycloheximide and phosphonoacetate has led to the identification of 34 immediate early and 13 delayed early polypeptides. Therefore 64 proteins were classified as late polypeptides. Several ASFV-induced proteins are phosphorylated as proteins a1, a4, a20, a41, a48, a49, a51, a52, a55, a58, a67, b2, b12, b28, and b32. PMID:3629977

  1. Comparison of restriction enzyme analysis and pulsed-field gradient gel electrophoresis as typing systems for Candida albicans.

    PubMed Central

    Vazquez, J A; Beckley, A; Sobel, J D; Zervos, M J

    1991-01-01

    Candida species are an important cause of infection in immunocompromised hosts and the leading cause of nosocomial fungal infections. Study of the epidemiology of Candida infection has been difficult because of lack of a reliable typing system. We describe a typing system utilizing contour-clamped homogeneous electric fields (CHEF), which is a modified version of pulsed-field gradient gel electrophoresis, and compared it with restriction enzyme analysis (REA) of genomic DNA. The study was done with 35 Candida albicans clinical isolates from separate patients. CHEF and REA were performed on each isolate, and the patterns were compared. The REA procedure revealed 17 strain types while the CHEF procedure was able to distinguish 23 strain types of C. albicans. The CHEF technique yields unique patterns of chromosomal bands that can be used to distinguish clinical isolates and demonstrates greater sensitivity than REA. Images PMID:1647409

  2. Analysis of haemorrhagic septicaemia-causing isolates of Pasteurella multocida by ribotyping and field alternation gel electrophoresis (FAGE).

    PubMed

    Townsend, K M; Dawkins, H J; Papadimitriou, J M

    1997-10-16

    Ribotyping and field alternation gel electrophoresis (FAGE) were used to examine 19 Pasteurella multocida isolates, and to assess the ability of these techniques to differentiate P. multocida strains that cause haemorrhagic septicaemia (HS). Reproducible patterns were obtained from both methods, with FAGE demonstrating greater discriminatory power than ribotyping. FAGE analysis was particularly useful in distinguishing North American cultures originating from the 1922 Yellowstone National Park Buffalo 'B' strain, demonstrating the ability to detect genetic alterations induced by repeated subculture. A remarkable homogeneity was observed among Asian HS strains following ribotyping and FAGE analysis, with a clear distinction observed between virulent and avirulent HS isolates. This study has illustrated the value of genomic fingerprinting methods in distinguishing strains of similar serotype, and the capability of these methods to produce detailed characterisation of P. multocida isolates. PMID:9444075

  3. Investigating Freshwater Periphyton Community Response to Uranium with Phospholipid Fatty Acid and Denaturing Gradient Gel Electrophoresis Analyses

    SciTech Connect

    Small, Jack A.; Bunn, Amoret L.; McKinstry, Craig A.; Peacock, A. D.; Miracle, Ann L.

    2008-04-01

    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 exposure on periphyton. Laboratory exposures of periphyton in river water amended with uranium were performed for 5 days, followed by 2 days of uranium depuration in unamended river water. Productivity as measured by biomass was not affected by concentrations up to 100 g L-1 uranium. Phospholipid fatty acid (PLFA) profiles and denaturing gradient gel electrophoresis (DGGE) banding patterns found no changes in community or genetic structure related to uranium exposure. We suggest that the periphyton community as a whole is not impacted by exposures of uranium up to a dose of 100 g L-1. These findings have significance for the assessment and prediction of uranium impacts on aquatic ecosystems.

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

    PubMed

    Gornatti Churria, Carlos D; Vigo, Germn B; Origlia, Javier; Campos, Josefina; Caffer, Mara; Pscopo, 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

  5. Identification of velvet antler by random amplified polymorphism DNA combined with non-gel sieving capillary electrophoresis.

    PubMed

    Yuan, Guangxin; Sun, Jiyan; Li, Hongyu; Fu, Guilian; Xu, Guangyu; Li, Mingcheng; Zhang, Lihua; Fan, Xintian

    2014-08-01

    Abstract Mitochondrial DNA of velvet antler was amplified with random amplified polymorphic DNA (RAPD) technique and the PCR products were detected with non-gel sieving capillary electrophoresis to establish a RAPD-HPCE method used for identifying the authenticity of velvet antler or it counterfeits. Factors that could affect the PCR amplification and capillary electrophoresis were optimized. Under the optimized conditions, namely, 20?mmol?L(-1) NaH2PO4-Na2HPO4-2?mmolL(-1) EDTA buffer solution [0.8% (W/V) HPMC, 15?mmol?L(-1) TBAP and pH 7.3], -10?kV injection voltage and -8?kV separation voltage, Cervus nippon Temminck antler, Cervus elaphus Linnaeus antler, Rangifer tarandus antler, Cervus canadensis antler and Elaphurus davidianus antler were analyzed. The analysis on the similarity of obtained elctrophoretograms showed that there were significant differences in similarities of different velvet antlers, which could be used for the quick identification of the authenticity of velvet antler samples. It can be found that the technique of RAPD combined with HPCE is advantageous in rich polymorphism, high detection rate, simple and convenient performance, high efficiency, rapidness and sensitivity, indicating that it should be suitable for the quick identification of the authenticity of velvet antler samples. PMID:25103424

  6. Amino Acid Composition, Molecular Weight Distribution and Gel Electrophoresis of Walnut (Juglans regia L.) Proteins and Protein Fractionations

    PubMed Central

    Mao, Xiaoying; Hua, Yufei; Chen, Guogang

    2014-01-01

    As a by-product of oil production, walnut proteins are considered as an additional source of plant protein for human food. To make full use of the protein resource, a comprehensive understanding of composition and characteristics of walnut proteins are required. Walnut proteins have been fractionated and characterized in this study. Amino acid composition, molecular weight distribution and gel electrophoresis of walnut proteins and protein fractionations were analyzed. The proteins were sequentially separated into four fractions according to their solubility. Glutelin was the main component of the protein extract. The content of glutelin, albumin, globulin and prolamin was about 72.06%, 7.54%, 15.67% and 4.73% respectively. Glutelin, albumin and globulin have a balanced content of essential amino acids, except for methionine, with respect to the FAO pattern recommended for adults. SDS-PAGE patterns of albumin, globulin and glutelin showed several polypeptides with molecular weights 14.4 to 66.2 kDa. The pattern of walnut proteins in two-dimension electrophoresis (2-DE) showed that the isoelectric point was mainly in the range of 4.86.8. The results of size exclusion chromatogram indicated molecular weight of the major components of walnut proteins were between 3.54 and 81.76 kDa. PMID:24473146

  7. Assessing the stability of cystatin/cysteine proteinase complexes using mildly-denaturing gelatin-polyacrylamide gel electrophoresis.

    PubMed

    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

  8. Definition of Mycobacterium tuberculosis Culture Filtrate Proteins by Two-Dimensional Polyacrylamide Gel Electrophoresis, N-Terminal Amino Acid Sequencing, and Electrospray Mass Spectrometry

    Microsoft Academic Search

    MICHAEL G. SONNENBERG; JOHN T. BELISLE

    1997-01-01

    A number of the culture filtrate proteins secreted by Mycobacterium tuberculosis are known to contribute to the immunology of tuberculosis and to possess enzymatic activities associated with pathogenicity. However, a complete analysis of the protein composition of this fraction has been lacking. By using two-dimensional polyacrylamide gel electrophoresis, detailed maps of the culture filtrate proteins of M. tuberculosis H37Rv were

  9. Pseudomonas aeruginosa Endophthalmitis after Penetrating Keratoplasty Transmitted from the Same Donor to Two Recipients Confirmed by Pulsed-Field Gel Electrophoresis ?

    PubMed Central

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

    2011-01-01

    Two devastating cases of multidrug-resistant Pseudomonas aeruginosa endophthalmitis after keratoplasty as the result of transmission from the same donor were confirmed by pulsed-field gel electrophoresis. Strategies for preventing donor-to-host transmission, such as the use of antimicrobial agents of greater efficacy and better methods for detecting microorganisms in preservation medium, could minimize this type of transmission. PMID:21775545

  10. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

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

    ERIC Educational Resources Information Center

    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

  12. Detection by Denaturing Gradient Gel Electrophoresis of pncA Mutations Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates from the United States-Mexico Border Region

    Microsoft Academic Search

    Mark T. McCammon; John S. Gillette; Derek P. Thomas; Srinivas V. Ramaswamy; Ishmael I. Rosas; Edward A. Graviss; Jan Vijg; Teresa N. Quitugua

    2005-01-01

    Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with pyrazin- amide (PZA) resistance in the pncA gene of Mycobacterium tuberculosis. DGGE scans for mutations across large regions of DNA and rivals sequencing in its ability to detect DNA alterations. Specific mutations can often be recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint.

  13. A comparison of non-typhoidal Salmonella from humans and food animals using pulsed-field gel electrophoresis and antimicrobial susceptibility patterns

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  14. 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

    ERIC Educational Resources Information Center

    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

  15. USE OF PULSED-FIELD GEL ELECTROPHORESIS TO CHARACTERIZE THE HETEROGENEITY AND CLONALITY OF SALMONELLA ISOLATES OBTAINED FROM THE CARCASS AND FECES OF SWINE AT SLAUGHTER

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In a previous study, Salmonella were recovered from swine at a collaborating processing plant over a two month period in the spring of 2000. In the present study, molecular subtyping by pulsed-field gel electrophoresis (PFGE) was performed on the 581 confirmed Salmonella isolates from the 84 Salmon...

  16. A comparison of BOX-PCR and pulsed-field gel electrophoresis to determine genetic relatedness of enterococci from different environments

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  17. Mapping of polar fox renal cortex proteins using two-dimensional gel electrophoresis and mass spectrometry--a preliminary study.

    PubMed

    Ciechanowicz, A K; Ozgo, M; Sta?ski, ? R; Herosimczyk, A; Piotrowska, A; Szymeczko, R; Laszczy?ska, M; Skrzypczak, W F

    2014-01-01

    The aim of the present study was to establish protein map of polar fox (Alopex lagopus) renal cortex. Kidney cortex proteins of isoelectric point ranging from 3 to 10 were analysed using two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Sixteen protein spots corresponding to thirteen different gene products were identified. These proteins were divided into following groups: lipid and fatty acid metabolism, amino acid metabolism, energetic pathways, regulatory proteins, transport proteins and structural proteins. This is the first attempt to create reproducible 2-D map, of renal cortex proteins characteristic for polar foxes, used as animal model for carnivores. It is worth emphasizing that the results of this study may broaden currently available protein databases. PMID:24988848

  18. Identification of protein binding sites in genomic DNA by two-dimensional gel electrophoresis.

    PubMed Central

    Boffini, A; Prentki, P

    1991-01-01

    We describe a simple two-dimensional electrophoresis procedure to identify the recognition sites of DNA-binding proteins within large DNA molecules. Using this approach, we have mapped E. coli IHF (Integration Host Factor) binding sites within phage Lambda (48 kb) and phage Mu (39 kb) DNA. We are also able to visualize IHF binding sites in E. coli chromosomal DNA (4,700 kb). We present an extension of this technique using direct amplification by PCR of the isolated restriction fragments, which should permit the cloning of a collection of recognition sequences for DNA binding proteins in complex genomes. Images PMID:1827523

  19. Impact of DNA extraction method on bacterial community composition measured by denaturing gradient gel electrophoresis

    Microsoft Academic Search

    Julia R. de Lipthay; Christiane Enzinger; Kaare Johnsen; Jens Aamand; Sren J. Srensen

    2004-01-01

    The impact of DNA extraction protocol on soil DNA yield and bacterial community composition was evaluated. Three different procedures to physically disrupt cells were compared: sonication, grindingfreezingthawing, and bead beating. The three protocols were applied to three different topsoils. For all soils, we found that each DNA extraction method resulted in unique community patterns as measured by denaturing gradient gel

  20. Identification of cultivars of Stylosanthes capitata Vog. by polyacrylamide gel electrophoresis of seed proteins

    Microsoft Academic Search

    A. Hussain; H. Ramirez; W. Bushuk; W. M. Roca

    1988-01-01

    A method was developed for identification of cultivars of the pasture legume, Stylosanthes capitata Vog., using electrophoretic patterns of seed proteins in polyacrylamide gels as the genotypic markers. The method can be used for accurate identification of cultivars in germplasm banks, in selecting parents for development of new varieties, and in registering new cultivars for proprietary purposes.

  1. Detection of specific sequences among DNA fragments separated by gel electrophoresis

    Microsoft Academic Search

    E. M. Southern

    1975-01-01

    This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters. The fragments can then be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography. The method is illustrated by analyses of restriction fragments complementary to ribosomal RNAs from Escherichia coli and Xenopus laevis, and from several mammals.

  2. An Economical Electrophoresis Apparatus

    ERIC Educational Resources Information Center

    Andrews, I. M.

    1975-01-01

    Describes the production of an electrophoresis apparatus from commonly discarded articles. Outlines paper and gel electrophoresis and its application to the separation of amino acids and intestinal enzymes. (GS)

  3. Proteomic analysis of peach fruit mesocarp softening and chilling injury using difference gel electrophoresis (DIGE)

    Microsoft Academic Search

    Ricardo Nilo; Carlos Saffie; Kathryn Lilley; Ricardo Baeza-Yates; Vernica Cambiazo; Reinaldo Campos-Vargas; Mauricio Gonzlez; Lee A Meisel; Julio Retamales; Herman Silva; Ariel Orellana

    2010-01-01

    BACKGROUND: Peach fruit undergoes a rapid softening process that involves a number of metabolic changes. Storing fruit at low temperatures has been widely used to extend its postharvest life. However, this leads to undesired changes, such as mealiness and browning, which affect the quality of the fruit. In this study, a 2-D DIGE approach was designed to screen for differentially

  4. Comparing SILAC and two-dimensional gel electrophoresis image analysis for profiling urokinase plasminogen activator signaling in ovarian cancer cells.

    PubMed

    Uitto, Pauliina M; Lance, Braddon K; Wood, Graham R; Sherman, James; Baker, Mark S; Molloy, Mark P

    2007-06-01

    Two-dimensional gel electrophoresis (2-DE) image analysis is conventionally used for comparative proteomics. However, there are a number of technical difficulties associated with 2-DE protein separation that limit the depth of proteome coverage, and the image analysis steps are typically labor-intensive and low-throughput. Recently, mass spectrometry-based quantitation strategies have been described as alternative differential proteome analysis techniques. In this study, we investigated changes in protein expression using an ovarian cancer cell line, OVMZ6, 24 h post-stimulation with the relatively weak agonist, urokinase-type plasminogen activator (uPA). Quantitative protein profiles were obtained by MALDI-TOF/TOF from stable isotope-labeled cells in culture (SILAC), and these results were compared to the quantitative ratios obtained using 2-DE gel image analysis. MALDI-TOF/TOF mass spectrometry showed that differential quantitation using SILAC was highly reproducible (approximately 8% coefficient of variation (CV)), and this variance was considerably lower than that achieved using automated 2-DE image analysis strategies (CV approximately 25%). Both techniques revealed subtle alterations in cellular protein expression following uPA stimulation. However, due to the lower variances associated with the SILAC technique, smaller changes in expression of uPA-inducible proteins could be found with greater certainty. PMID:17472359

  5. Comparison of bacterial community changes in fermenting kimchi at two different temperatures using a denaturing gradient gel electrophoresis analysis.

    PubMed

    Hong, Yeun; Yang, Hee-Seok; Chang, Hae-Choon; Kim, Hae-Yeong

    2013-01-01

    A polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique followed by sequencing of the 16S rDNA fragments eluted from the bands of interest on denaturing gradient gels was used to monitor changes in the bacterial microflora of two commercial kimchi, salted cabbage, and ingredient mix samples during 30 days of fermentation at 4C and 10C. Leuconostoc (Lc.) was the dominant lactic acid bacteria (LAB) over Lactobacillus (Lb.) species at 4C. Weissella confusa was detected in the ingredient mix and also in kimchi samples throughout fermentation in both samples at 4C and 10C. Lc. gelidum was detected as the dominant LAB at 4C in both samples. The temperature affected the LAB profile of kimchi by varing the pH, which was primarily caused by the temperature-dependent competition among different LAB species in kimchi. At 4C, the sample variations in pH and titratable acidity were more conspicuous owing to the delayed growth of LAB. Temperature affected only initial decreases in pH and initial increases in viable cell counts, but affected both the initial increases and final values of titratable acidity. The initial microflora in the kimchi sample was probably determined by the microflora of the ingredient mix, not by that of the salted cabbage. The microbial distributions in the samples used in this study resembled across the different kimchi samples and the different fermentation temperatures as the numbers of LAB increased and titratable acidity decreased. PMID:23314371

  6. Analysis of Mutant SOD1 Electrophoretic Mobility by Blue Native Gel Electrophoresis; Evidence for Soluble Multimeric Assemblies

    PubMed Central

    Brown, Hilda H.; Borchelt, David R.

    2014-01-01

    Mutations in superoxide dismutase 1 (SOD1) cause familial forms of amyotrophic lateral sclerosis (fALS). Disease causing mutations have diverse consequences on the activity and half-life of the protein, ranging from complete inactivity and short half-life to full activity and long-half-life. Uniformly, disease causing mutations induce the protein to misfold and aggregate and such aggregation tendencies are readily visualized by over-expression of the proteins in cultured cells. In the present study we have investigated the potential of using immunoblotting of proteins separated by Blue-Native gel electrophoresis (BNGE) as a means to identify soluble multimeric forms of mutant protein. We find that over-expressed wild-type human SOD1 (hSOD1) is generally not prone to form soluble high molecular weight entities that can be separated by BNGE. For ALS mutant SOD1, we observe that for all mutants examined (A4V, G37R, G85R, G93A, and L126Z), immunoblots of BN-gels separating protein solubilized by digitonin demonstrated varied amounts of high molecular weight immunoreactive entities. These entities lacked reactivity to ubiquitin and were partially dissociated by reducing agents. With the exception of the G93A mutant, these entities were not reactive to the C4F6 conformational antibody. Collectively, these data demonstrate that BNGE can be used to assess the formation of soluble multimeric assemblies of mutant SOD1. PMID:25121776

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

    PubMed Central

    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

  8. Protein Extraction for Two-Dimensional Gel Electrophoresis of Proteomic Profiling in Turfgrass

    Microsoft Academic Search

    Chenping Xu; Yan Xu; Bingru Huang

    2008-01-01

    Protein extraction for two-dimensional gel elec- trophoresis (2-DE) from plant samples is chal- lenging due to low protein content and high level of contaminants. Proteomic research in turfgrass is limited by the lack of effi cient protein extrac- tion methods. To establish an effective protocol of protein extraction suitable for 2-DE analysis in turfgrasses, four protein extraction meth- ods (chloroform\\/acetone,

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

    NASA Astrophysics Data System (ADS)

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

    1997-12-01

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

  10. Effects of Clostridium difficile Toxin A on the proteome of colonocytes studied by differential 2D electrophoresis.

    PubMed

    Zeiser, Johannes J; Klodmann, Jennifer; Braun, Hans-Peter; Gerhard, Ralf; Just, Ingo; Pich, Andreas

    2011-12-21

    Clostridium difficile is a spore-forming anaerobic pathogen, commonly associated with severe diarrhea or life-threatening pseudomembraneous colitis. Its main virulence factors are the single-chain, multi-domain toxin A (TcdA) and B (TcdB). Their glucosyltransferase domain selectively inactivates Rho proteins leading to a reorganization of the cytoskeleton. To study exclusively glucosyltransferase-dependent molecular effects of TcdA, human colonic cells (Caco-2) were treated with recombinant wild type TcdA and the glucosyltransferase deficient variant of the toxin, TcdA(gd) for 24h. Changes in the protein pattern of the colonic cells were investigated by 2-D DIGE and LCMS/MS methodology combined with detailed proteome mapping. gdTcdA did not induce any detectable significant changes in the protein pattern. Comparing TcdA-treated cells with a control group revealed seven spots of higher and two of lower intensity (p<0.05). Three proteins are involved in the assembly of the cytoskeleton (?-actin, ezrin, and DPYL2) and four are involved in metabolism and/or oxidative stress response (ubiquitin, DHE3, MCCB, FABPL) and two in regulatory processes (FUBP1, AL1A1). These findings correlate well to known effects of TcdA like the reorganization of the cytoskeleton and stress the importance of Rho protein glucosylation for the pathogenic effects of TcdA. PMID:21890007

  11. The Arabidopsis thaliana 2-D gel mitochondrial proteome: Refining the value of reference maps for assessing protein abundance, contaminants and post-translational modifications.

    PubMed

    Taylor, Nicolas L; Heazlewood, Joshua L; Millar, A Harvey

    2011-05-01

    Mitochondria undertake the process of oxidative phosphorylation yielding ATP for plant cell maintenance and growth. The principles of isolation and fractionation of plant mitochondrial proteins have been improved over decades, and surveys of the mitochondrial proteome in a number of plants species have been performed. Over time, many quantitative analyses of changes in the plant mitochondrial proteome have been performed by 2-D gel analyses revealing the induction, degradation and modification of mitochondrial proteins in responses to mutation, stress and development. Here, we present a saturating MS analysis of 2-D gel separable protein spots from a typical purification of Arabidopsis mitochondria identifying 264 proteins, alongside an LC-MS/MS survey by non-gel methods identifying 220 proteins. This allowed us to characterise the major mitochondrial proteins that are not observed on 2-D gels, the common contaminants and the abundance of the protein machinery of key mitochondrial biochemical pathways, and consider the impact of N-terminal pre-sequence cleavage and phosphorylation as explanations of multiple protein spots and the co-ordinates of proteins on 2-D gels. PMID:21472856

  12. Supplementary Materials for 2D-PCR: A Method of Mapping DNA in Tissue Sections

    E-print Network

    Shapiro, Benjamin

    and thermocycling. #12;1.2 Electrophoresis Gels for Validation of 2D-PCR Method Our tissue DNA extraction protocolSupplementary Materials for 2D-PCR: A Method of Mapping DNA in Tissue Sections Michael Armaniabeg-derived DNA extract. The PCR DNA amplification protocol in the agarose-filled aluminum miniature wells

  13. SEPARATION AND IDENTIFICATION OF SOYBEAN LEAF PROTEINS BY 2D-PAGE AND MASS SPECTROMETRY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To establish a proteomic reference map for soybean leaves, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). We detected approximately 700 protein spots by 2D-PAGE using a pH 3-10 immobilized pH gradient (IPG) st...

  14. Using single-strand conformational polymorphism gel electrophoresis to analyze mutually exclusive alternative splicing.

    PubMed

    Celotto, Alicia M; Graveley, Brenton R

    2004-01-01

    Single-strand conformational polymorphism analysis has been used successfully to identify single nucleotide changes within sequences based on the fact that multidetection enhancement gels will separate molecules based on their conformation rather than their size. We have expanded the utility of this technique to analyze easily the alternative splicing of pre-mRNAs containing multiple mutually exclusive exons of the same size. We have used this technique to study the Caenorhabditis elegans let-2 gene containing two alternative exons and the Drosophilia melanogaster Dscam gene, which contains 12 mutually exclusive exons. The ease and the quantitative nature of this technique should be very useful. PMID:14769996

  15. Changes on the Caco-2 Secretome through Differentiation Analyzed by 2-D Differential In-Gel Electrophoresis (DIGE)

    PubMed Central

    Garca-Lorenzo, Andrs; Rodrguez-Pieiro, Ana M.; Rodrguez-Berrocal, Francisco J.; de la Cadena, Mara Pez; Martnez-Zorzano, Vicenta S.

    2012-01-01

    Colorectal cancer is still a major health burden worldwide, and its diagnosis has not improved in recent years due to a lack of appropriate diagnostic serum markers. Aiming to find new diagnostic proteins, we applied the proteomic DIGE technology to analyze changes in the secretome before/after differentiation of the colon adenocarcinoma Caco-2 cell line, an accepted in vitro model to study colorectal tumorigenesis. When the secretomes from undifferentiated (tumor-like) and differentiated cells (resembling healthy enterocytes) were compared, we found 96 spots differentially expressed. After MS/MS analysis, 22 spots corresponding to 15 different proteins were identified. Principal component analysis demonstrated these 22 spots could serve as a discriminatory panel between the tumor-like and normal-like cells. Among the identified proteins, the translationally-controlled tumor protein (TCTP), the transforming growth factor-beta-induced protein ig-h3 (TGF?Ip), and the Niemann-Pick disease type C2 protein (NPC2) are interesting candidates for future studies focused on their utility as serum biomarkers of colorectal cancer. PMID:23203071

  16. Brownian Dynamics Studies on DNA Gel Electrophoresis. I. Numerical Method and Quasi-Periodic Behavior of Elongation-Contraction Motions

    E-print Network

    Ryuzo Azuma; Hajime Takayama

    2001-11-07

    Dynamics of individual DNA undergoing constant field gel electrophoresis (CFGE) is studied by a Brownian dynamics (BD) simulation method which we have developed. The method simulates electrophoresis of DNA in a 3 dimensional (3D) space by a chain of electrolyte beads of hard spheres. Under the constraint that the separation of each pair of bonded beads is restricted to be less than a certain fixed value, as well as with the excluded volume effect, the Langevin equation of motion for the beads is solved by means of the Lagrangian multiplier method. The resultant mobilities, $\\mu$, as a function of the electric field coincide satisfactorily with the corresponding experimental results, once the time, the length and the field of the simulation are properly scaled. In relatively strong fields quasi-periodic behavior is found in the chain dynamics, and is examined through the time evolution of the radius of the longer principal axis, $R_l(t)$. It is found that the mean width of a peak in $R_l(t)$, or a period of one elongation-contraction process of the chain, is proportional to the number of beads in the chain, $M$, while the mean period between two such adjacent peaks is proportional to $M^0$ for large $M$. These results, combined with the observation that the chain moves to the field direction by the distance proportional to $M$ in each elongation-contraction motion, yield $\\mu \\propto M^0$. This explains why CFGE cannot separate DNA according to their size $L (\\propto M)$ for large $L$.

  17. Electrophoresis. [in microgravity environment

    NASA Technical Reports Server (NTRS)

    Bier, M.

    1977-01-01

    Ground-based techniques for electrophoresis take account of the need either to circumvent the effects of gravity to prevent convection, or to use gravity for fluid stabilization through artificial density gradients. The microgravity environments of orbiting spacecraft provides a new alternative for electrophoresis by avoiding the need for either of these two approaches. The paper presents some theoretical considerations concerning electrophoresis, examines certain experimental techniques (zone and high density gel electrophoresis, isoelectric focusing and isotachophoresis), and examines the electrophoresis of living cells.

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

    SciTech Connect

    Carrasco, L.; Bravo, R.

    1986-05-01

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

  19. Double-stranded cucumovirus associated RNA 5: which sequence variations may be detected by optical melting and temperature-gradient gel electrophoresis?

    PubMed Central

    Steger, G; Po, T; Kaper, J; Riesner, D

    1987-01-01

    Sequence variants of the double-stranded form of satellite RNAs of cucumber mosaic virus (dsCARNA 5) were analyzed for the possibility to experimentally detect minor nucleotide sequence changes. Denaturation maps (helix-probability versus position of the nucleotide in the sequence versus temperature) were calculated applying the Poland algorithm. Optical denaturation curves and temperature-gradient gel mobility curves were simulated using the denaturation maps and were compared with experimental results from optical melting and temperature-gradient gel electrophoresis (Tien Po et al., accompanying paper). Melting of the dsRNAs starts from both ends of the molecule in two transitions of low co-operativity, continues in the right part in a highly co-operative transition, and is finished in another highly co-operative transition including strand-separation. Whereas all parts of the molecule contribute uniformly to the optical melting curve, opening of the ends predominates in the retardation transition in gel electrophoresis. Detailed discussion of the influence of base pair changes in the sequence shows that a single base pair change may be detected by temperature-gradient gel electrophoresis, if it is located in certain favorable locations, whereas its detection in optical melting curves is possible only in very special cases. The systematic differences found in the accompanying paper between necrogenic and non-necrogenic dsCARNA 5 could be interpreted on the basis of such nucleotide sequence differences. Images PMID:3601668

  20. Analysis of proteins expressed in rat plasma exposed to dioxin using 2-dimensional gel electrophoresis.

    PubMed

    Son, Won-Kyu; Lee, Do-Youn; Lee, Sung-Han; Joo, Won-A; Kim, Chan-Wha

    2003-12-01

    Dioxins are a class of polyhalogenated aromatic hydrocarbons that induces a wide spectrum of toxic responses in animals. In this study, two groups of Sprague-Dawley rats were exposed to 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD); one group received short-term exposure at a single dose of 1, 10, 20 or 50 microg/kg body weight and the other received long-term exposure to a daily low dose of 0.01, 0.1, 1 or 2.5 microg/kg body weight for one month. Two-dimensional electrophoresis was utilized to resolve the protein profile of rat plasma exposed to TCDD at different doses. One novel and three volume-increased spots were identified and characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and electrospray-ionization on quadropole-TOF2 mass spectrometry. The novel protein was identified as plasma glutathione peroxidase precursor and the volume-increased proteins were cytokeratin 8 polypeptide, Ig lambda-1 chain C region and Ig lambda-2 chain C region. These proteins may be used as biomarkers to diagnose dioxin exposure and may help in understanding the toxic effects of dioxins. PMID:14673789

  1. Assessment of the Red Cell Proteome of Young Patients with Unexplained Hemolytic Anemia by Two-Dimensional Differential In-Gel Electrophoresis (DIGE)

    PubMed Central

    von Lhneysen, Katharina; Scott, Thomas M.; Soldau, Katrin; Xu, Xiuling; Friedman, Jeffrey S.

    2012-01-01

    Erythrocyte cytosolic protein expression profiles of children with unexplained hemolytic anemia were compared with profiles of close relatives and controls by two-dimensional differential in-gel electrophoresis (2D-DIGE). The severity of anemia in the patients varied from compensated (i.e., no medical intervention required) to chronic transfusion dependence. Common characteristics of all patients included chronic elevation of reticulocyte count and a negative workup for anemia focusing on hemoglobinopathies, morphologic abnormalities that would suggest a membrane defect, immune-mediated red cell destruction, and evaluation of the most common red cell enzyme defects, glucose-6-phosphate dehydrogenase and pyruvate kinase deficiency. Based upon this initial workup and presentation during infancy or early childhood, four patients classified as hereditary nonspherocytic hemolytic anemia (HNSHA) of unknown etiology were selected for proteomic analysis. DIGE analysis of red cell cytosolic proteins clearly discriminated each anemic patient from both familial and unrelated controls, revealing both patient-specific and shared patterns of differential protein expression. Changes in expression pattern shared among the four patients were identified in several protein classes including chaperons, cytoskeletal and proteasome proteins. Elevated expression in patient samples of some proteins correlated with high reticulocyte count, likely identifying a subset of proteins that are normally lost during erythroid maturation, including proteins involved in mitochondrial metabolism and protein synthesis. Proteins identified with patient-specific decreased expression included components of the glutathione synthetic pathway, antioxidant pathways, and proteins involved in signal transduction and nucleotide metabolism. Among the more than 200 proteins identified in this study are 21 proteins not previously described as part of the erythrocyte proteome. These results demonstrate the feasibility of applying a global proteomic approach to aid characterization of red cells from patients with hereditary anemia of unknown cause, including the identification of differentially expressed proteins as potential candidates with a role in disease pathogenesis. PMID:22509282

  2. Western Blotting For Protein Analysis Part 1: Laemmli Gel Electrophoresis Using Mini-PROTEAN II

    E-print Network

    Shoubridge, Eric

    persulfate 50 µL TEMED 10 µL Total 10 mL Resolving Gel Preparation - 0.375 M Tris, pH 8.8 5 % 7.5% 1 2 % 1 5L 4.0 mL 5.0 mL 6.67 mL 10% Ammonium persulfate 50 µL 50 µL 50 µL 50 µL 50 µL TEMED 5 µL 5 µL 5 µL 5 µL centrifuge tubes combine all the solutions except the ammonium persulfate (APS) and the TEMED. #12;2. Place

  3. Nanorods of Various Oxides and Hierarchically Structured Mesoporous Silica by Sol-Gel Electrophoresis

    SciTech Connect

    Limmer, Steven J.; Hubler, Timothy L.; Cao, Guozhong

    2003-01-02

    In this paper, we report the template-based growth of nanorods of oxides and hierarchically structured mesoporous silica, formed by means of a combination of sol-gel processing and elecrophoretic deposition. Both single metal oxides (TiO2) and complex oxides (Pb(Zr0.52Ti0.48)O3) have been grown by this method. This method has also been applied to the growth of nanorods of mesoporous silica having an ordered pore structure, where the pores are aligned parallel to the long axis of the nanorod. Uniformly sized nanorods of about 125-200 nm in diameter and 10 um in length were grown over large areas with near unidirectional alignment. Appropriate sol preparation yielded the desired stoichiometric chemical composition and crystal structure of the oxide nanorods, with a heat treatment (500-700 C for 15-30 min) for crystallization, densification and any necessary pyrolysis.

  4. Utilization of denaturing gradient gel electrophoresis for diagnosis of {beta}-thalassemia and ascertainment of new mutations

    SciTech Connect

    Ngo, K.Y.; Liu, D.; Lee, J. [Univ. of California, San Diego (United States)] [and others

    1994-09-01

    During the past two years we have tested 2,300 Southeast Asians for alpha- and beta-thaleassemia mutations. We found the incidence of hemoglobin E ({beta}{sup 26}) to be 47% among Laotians and 38% among Cambodians. The incidence of beta thalassemia trait is 9% for Laotians and 6% for Cambodians. Thus, the risk for hemoglobin E/{beta}{sup 26} thalassemia, a transfusion-dependent disorder, is increased in these two population groups. Denaturing gradient gel electrophoresis (DGGE) has proven to be useful in testing for beta-thalassemia carriers and identifying new mutations in the beta globin gene. DNA was extracted from venous blood obtained from patients with elevated Hgb A2 (>4%). Five DNA fragments, encompassing the beta globin gene cluster, were amplified by PCR and analyzed, along with known beta gene mutations as controls, by DGGE using different denaturing gradient concentrations. Different mutations at the same nucleotide position can be distinguished by migration pattern on the DGGE (e.g., in IVS-I-1, G{r_arrow}A and T). Compound heterozygotes for {beta}-thalassemia can be detected on the same gel (e.g., HbE/mutation codon 17). New mutations are identified by their migration pattern compared with controls and determined by subsequent sequencing. We have identified three new mutations: codon 82 CAA{r_arrow}AAA in one Cambodian patient; IVS-II-667, T{r_arrow}C and IVS-II-672, A{r_arrow}C in two Laotian patients. When the parent`s genotypes are known, prenatal diagnosis can be obtained within 24 hours. Thus, PCR/DGGE combination is a rapid and reliable diagnostic approach to clinically significant {beta}-thalassemia. The most important steps are carefully designed primers and predetermined gradient concentrations for DGGE.

  5. Proteomic analysis of peach fruit mesocarp softening and chilling injury using difference gel electrophoresis (DIGE)

    PubMed Central

    2010-01-01

    Background Peach fruit undergoes a rapid softening process that involves a number of metabolic changes. Storing fruit at low temperatures has been widely used to extend its postharvest life. However, this leads to undesired changes, such as mealiness and browning, which affect the quality of the fruit. In this study, a 2-D DIGE approach was designed to screen for differentially accumulated proteins in peach fruit during normal softening as well as under conditions that led to fruit chilling injury. Results The analysis allowed us to identify 43 spots -representing about 18% of the total number analyzed- that show statistically significant changes. Thirty-nine of the proteins could be identified by mass spectrometry. Some of the proteins that changed during postharvest had been related to peach fruit ripening and cold stress in the past. However, we identified other proteins that had not been linked to these processes. A graphical display of the relationship between the differentially accumulated proteins was obtained using pairwise average-linkage cluster analysis and principal component analysis. Proteins such as endopolygalacturonase, catalase, NADP-dependent isocitrate dehydrogenase, pectin methylesterase and dehydrins were found to be very important for distinguishing between healthy and chill injured fruit. A categorization of the differentially accumulated proteins was performed using Gene Ontology annotation. The results showed that the 'response to stress', 'cellular homeostasis', 'metabolism of carbohydrates' and 'amino acid metabolism' biological processes were affected the most during the postharvest. Conclusions Using a comparative proteomic approach with 2-D DIGE allowed us to identify proteins that showed stage-specific changes in their accumulation pattern. Several proteins that are related to response to stress, cellular homeostasis, cellular component organization and carbohydrate metabolism were detected as being differentially accumulated. Finally, a significant proportion of the proteins identified had not been associated with softening, cold storage or chilling injury-altered fruit before; thus, comparative proteomics has proven to be a valuable tool for understanding fruit softening and postharvest. PMID:20082721

  6. Detection of seminal fluid proteins in the bed bug, Cimex lectularius, using two-dimensional gel electrophoresis and mass spectrometry

    PubMed Central

    REINHARDT, K.; WONG, C. H.; GEORGIOU, A. S.

    2008-01-01

    SUMMARY The global increase of the human parasite, the common bed bug Cimex lectularius, calls for specific pest control target sites. The bed bug is also a model species for sexual conflict theory which suggests seminal fluids may be highly diverse. The species has a highly unusual sperm biology and seminal proteins may have unique functions. 1-D PAGE gels showed 40 to 50% band sharing between C. lectularius and another cimicid species, Afrocimex constrictus. However, adult, sexually rested C. lectularius males were found to store 5 to 7?g of seminal protein and with only 60?g of protein we obtained informative 2-D PAGE gels. These showed 79% shared protein spots between two laboratory populations, and more than half of the shared protein spots were detected in the mated female. Further analysis using liquid chromatography electrospray ionisation tandem mass spectrometry revealed that 26.5% of the proteins had matches among arthropods in data bases and 14.5% matched Drosophila proteins. These included ubiquitous proteins but also those more closely associated with reproduction such as moj 29, ubiquitin, the stress-related elongation factor EF-1alpha, a protein disulfide isomerase and an antioxidant, Peroxiredoxin 6. PMID:19091156

  7. Detection of seminal fluid proteins in the bed bug, Cimex lectularius, using two-dimensional gel electrophoresis and mass spectrometry.

    PubMed

    Reinhardt, K; Wong, C H; Georgiou, A S

    2009-03-01

    The global increase of the human parasite, the common bed bug Cimex lectularius, calls for specific pest control target sites. The bed bug is also a model species for sexual conflict theory which suggests that seminal fluids may be highly diverse. The species has a highly unusual sperm biology and seminal proteins may have unique functions. One-dimensional PAGE gels showed 40-50% band sharing between C. lectularius and another cimicid species, Afrocimex constrictus. However, adult, sexually rested C. lectularius males were found to store 5-7 microg of seminal protein and with only 60 microg of protein we obtained informative 2-D PAGE gels. These showed 79% shared protein spots between 2 laboratory populations, and more than half of the shared protein spots were detected in the mated female. Further analysis using liquid chromatography electrospray ionization tandem mass spectrometry revealed that 26.5% of the proteins had matches among arthropods in databases and 14.5% matched Drosophila proteins. These included ubiquitous proteins but also those more closely associated with reproduction such as moj 29, ubiquitin, the stress-related elongation factor EF-1 alpha, a protein disulfide isomerase and an antioxidant, Peroxiredoxin 6. PMID:19091156

  8. Detritus-dependent development of the microbial community in an experimental system: qualitative analysis by denaturing gradient gel electrophoresis.

    PubMed

    van Hannen, E J; Mooij, W; van Agterveld, M P; Gons, H J; Laanbroek, H J

    1999-06-01

    Correlations between the biomass of phytoplankton and the biomass of bacteria and between the biomass of bacteria and the biomass of protozoans suggest that there is coupling between these compartments of the "microbial loop." To investigate this coupling on the species level, bacteria and protozoans from untreated lake water inocula were allowed to grow on detritus of the green alga Ankistrodesmus falcatus or the cyanobacterium Oscillatoria limnetica in continuous-flow systems for 1 month. Denaturing gradient gel electrophoresis (DGGE) of the 16S and 18S rRNA genes was used to monitor the development of the bacterial community structure and the eukaryotic community structure, respectively. Nonmetric multidimensional scaling of the DGGE profiles revealed the changes in the microbial community structure. This analysis showed that significantly different bacterial communities developed on the green algal detritus and on the cyanobacterial detritus. Although similar results were obtained for the eukaryotic communities, the differences were not significant. Hence, our findings indicate that the origin of detritus can affect the structure of at least the bacterial community. A phylogenetic analysis of 20 18S ribosomal DNA clones that were isolated from the continuous cultures revealed that many sequences were related to the sequences of bacterivorous protozoans (members of the Ciliophora, Rhizopoda, Amoeba, and Kinetoplastida). One clone grouped in a recently established clade whose previously described members are all parasites. The affiliations of about 20% of the clones could not be determined. PMID:10347030

  9. 16S rRNA PCR-Denaturing Gradient Gel Electrophoresis of Oral Lactobacillus casei Group and Their Phenotypic Appearances

    PubMed Central

    Piwat, S.; Teanpaisan, R.

    2013-01-01

    This study aimed to develop a 16S rRNA PCR-denaturing gradient gel electrophoresis (DGGE) to identify the species level of Lactobacillus casei group and to investigate their characteristics of acid production and inhibitory effect. PCR-DGGE has been developed based on the 16S rRNA gene, and a set of HDA-1-GC and HDA-2, designed at V2-V3 region, and another set of CARP-1-GC and CARP-2, designed at V1 region, have been used. The bacterial strains included L. casei ATCC 393, L. paracasei CCUG 32212, L. rhamnosus ATCC 7469, L. zeae CCUG 35515, and 46 clinical strains of L. casei/paracasei/rhamnosus. Inhibitory effect against Streptococcus mutans and acid production were examined. Results revealed that each type species strain and identified clinical isolate showed its own unique DGGE pattern using CARP1-GC and CARP2 primers. HDA1-GC and HDA2 primers could distinguish the strains of L. paracasei from L. casei. It was found that inhibitory effect of L. paracasei was stronger than L. casei and L. rhamnosus. The acid production of L. paracasei was lower than L. casei and L. rhamnosus. In conclusion, the technique has been proven to be able to differentiate between closely related species in L. casei group and thus provide reliable information of their phenotypic appearances. PMID:24191230

  10. Two-Dimensional Gel Electrophoresis Analysis of the Response of Pseudomonas putida KT2442 to 2-Chlorophenol

    PubMed Central

    Lupi, C. G.; Colangelo, T.; Mason, C. A.

    1995-01-01

    The effects of exposure of Pseudomonas putida KT2442 to 2-chlorophenol as a model for the chemical stress response were examined by two-dimensional polyacrylamide gel electrophoresis. Individual protein concentrations were determined at 45, 65, and 95 min following the addition of 2-chlorophenol at a concentration of 1.63 mM to exponentially growing cultures of P. putida KT2442 by silver staining the separated proteins. The changes in the protein concentrations could be classified into four categories, namely those which increased continuously during exposure, those which decreased in concentration, those which showed a concentration peak at some point following exposure, and those which were essentially unaffected. Thirty proteins with isoelectric points between pH 4 and 6 increased in concentration, 27 decreased, and 90 had a concentration maximum or minimum between 45 and 95 min. Of those proteins with isoelectric points between 5.5 and 10, 68 increased in concentration, 39 decreased in concentration, and 47 showed a concentration peak in the middle of the sampling period. Thus, in the evaluation of the stress response, a functional description requires an understanding both of proteins which are required at higher concentrations and of those whose presence appears to be no longer essential. PMID:16535093

  11. Temporal and spatial distribution of Cronobacter isolates in a milk powder processing plant determined by pulsed-field gel electrophoresis.

    PubMed

    Hein, Ingeborg; Gadzov, Boris; Schoder, Dagmar; Foissy, Helmut; Malorny, Burkhard; Wagner, Martin

    2009-03-01

    A milk powder processing line was sampled for the presence of Enterobacteriaceae and the opportunistic neonatal pathogen Cronobacter at six different sampling sites during an 11-month period. The highest number of Enterobacteriaceae-positive samples was recovered from the raw milk concentrate before pasteurization (78.2%) and from nonproduct samples of the processing line (86.5%), which included swabs from the drying tower and screw conveyers, swabs from the explosion chamber, waste water after the automated cleaning-in-place procedure, air filter cut-outs, and floor samples underneath the outlet of the packaging machine. The prepackaged and packaged final product was contaminated at a rate of 6.6% and 7.1%, respectively. The prevalence of Cronobacter in the prefinal product and the prepackaged and packaged final product was 14.3%, 3.8%, and 2.1%, respectively. Pulsed-field gel electrophoresis (PFGE) analysis of 133 Cronobacter isolates yielded 40 different PFGE profiles. Long-term persistence in the processing line of some of these PFGE profiles was observed. Comparison of the PFGE profiles recovered at different sampling sites revealed the supply air as a potential source for extrinsic Cronobacter contamination. In addition, recovery of the same PFGE profiles before and after CIP events followed by heat treatment indicated the inefficiency of these hygiene measures to completely eliminate Cronobacter from all areas of the processing line. This information provides an essential basis for developing control and prevention strategies concerning this opportunistic pathogen. PMID:19245339

  12. Multilocus Sequence Typing Lacks the Discriminatory Ability of Pulsed-Field Gel Electrophoresis for Typing Salmonella enterica Serovar Typhimurium

    PubMed Central

    Fakhr, Mohamed K.; Nolan, Lisa K.; Logue, Catherine M.

    2005-01-01

    Nontyphoidal salmonellae are among the leading causes of food-borne disease in the United States. Because of the importance of Salmonella enterica in food-borne disease, numerous typing methodologies have been developed. Among the several molecular typing methods, pulsed-field gel electrophoresis (PFGE) is currently considered the gold standard technique in typing Salmonella. The aim of this study was to compare the discriminatory power of PFGE to multilocus sequence typing (MLST) in typing Salmonella enterica serovar Typhimurium clinical isolates. A total of 85 Salmonella Typhimurium clinical isolates from cattle were used in this study. PFGE using XbaI was performed on the 85 isolates by the Centers for Disease Control and Prevention method, and data were analyzed using the BioNumerics software package. Fifty PFGE profiles were observed among the isolates, and these grouped into three major clusters. For the MLST analysis, the manB, pduF, glnA, and spaM genes were amplified by PCR from the same 85 isolates. DNA sequencing of these four genes, manB, pduF, glnA, and spaM, showed no genetic diversity among the isolates tested, with a 100% identity in nucleotide sequence. Moreover, the DNA sequences of the aforementioned genes showed 100% identity to the sequence reported in GenBank for the S. enterica serovar Typhimurium LT2 strain. Therefore, MLST, using these genes, lacks the discriminatory power of PFGE for typing Salmonella enterica serovar Typhimurium. PMID:15872244

  13. Multilocus sequence typing lacks the discriminatory ability of pulsed-field gel electrophoresis for typing Salmonella enterica serovar Typhimurium.

    PubMed

    Fakhr, Mohamed K; Nolan, Lisa K; Logue, Catherine M

    2005-05-01

    Nontyphoidal salmonellae are among the leading causes of food-borne disease in the United States. Because of the importance of Salmonella enterica in food-borne disease, numerous typing methodologies have been developed. Among the several molecular typing methods, pulsed-field gel electrophoresis (PFGE) is currently considered the "gold standard" technique in typing Salmonella. The aim of this study was to compare the discriminatory power of PFGE to multilocus sequence typing (MLST) in typing Salmonella enterica serovar Typhimurium clinical isolates. A total of 85 Salmonella Typhimurium clinical isolates from cattle were used in this study. PFGE using XbaI was performed on the 85 isolates by the Centers for Disease Control and Prevention method, and data were analyzed using the BioNumerics software package. Fifty PFGE profiles were observed among the isolates, and these grouped into three major clusters. For the MLST analysis, the manB, pduF, glnA, and spaM genes were amplified by PCR from the same 85 isolates. DNA sequencing of these four genes, manB, pduF, glnA, and spaM, showed no genetic diversity among the isolates tested, with a 100% identity in nucleotide sequence. Moreover, the DNA sequences of the aforementioned genes showed 100% identity to the sequence reported in GenBank for the S. enterica serovar Typhimurium LT2 strain. Therefore, MLST, using these genes, lacks the discriminatory power of PFGE for typing Salmonella enterica serovar Typhimurium. PMID:15872244

  14. Denaturing Gradient Gel Electrophoresis and Barcoded Pyrosequencing Reveal Unprecedented Archaeal Diversity in Mangrove Sediment and Rhizosphere Samples

    PubMed Central

    Pires, Ana C. C.; Cleary, Daniel F. R.; Almeida, Adelaide; Cunha, ngela; Dealtry, Simone; Mendona-Hagler, Leda C. S.; Smalla, Kornelia

    2012-01-01

    Mangroves are complex ecosystems that regulate nutrient and sediment fluxes to the open sea. The importance of bacteria and fungi in regulating nutrient cycles has led to an interest in their diversity and composition in mangroves. However, very few studies have assessed Archaea in mangroves, and virtually nothing is known about whether mangrove rhizospheres affect archaeal diversity and composition. Here, we studied the diversity and composition of Archaea in mangrove bulk sediment and the rhizospheres of two mangrove trees, Rhizophora mangle and Laguncularia racemosa, using denaturing gradient gel electrophoresis (DGGE) and pyrosequencing of archaeal 16S rRNA genes with a nested-amplification approach. DGGE profiles revealed significant structural differences between bulk sediment and rhizosphere samples, suggesting that roots of both mangrove species influence the sediment archaeal community. Nearly all of the detected sequences obtained with pyrosequencing were identified as Archaea, but most were unclassified at the level of phylum or below. Archaeal richness was, furthermore, the highest in the L. racemosa rhizosphere, intermediate in bulk sediment, and the lowest in the R. mangle rhizosphere. This study shows that rhizosphere microhabitats of R. mangle and L. racemosa, common plants in subtropical mangroves located in Rio de Janeiro, Brazil, hosted distinct archaeal assemblages. PMID:22660713

  15. Detection and identification of transgenic elements by fluorescent-PCR-based capillary gel electrophoresis in genetically modified cotton and soybean.

    PubMed

    Basak, Sanjay; Ehtesham, Nasreen Z; Sesikeran, Boindala; Ghosh, Sudip

    2014-01-01

    A detection method for genetically modified foods is an essential regulatory requirement for many countries. The present study is aimed at developing a qualitative method for detection of genetically modified organisms by combining PCR methodology with capillary gel electrophoresis (PCR-CGE) in a sequencing platform to detect Bacillus thuringiensis (Bt)-cotton (MON 531) and Roundup Ready (RR) soybean (GTS 40-3-2). A sensitive duplex PCR-CGE method was developed in which target DNA sequences (35S and Nos) were separated both by size and color to detect 0.01% Cry1Ac DNA (w/w) in Bt-cotton. A multiplex PCR-CGE method was developed to simultaneously detect four targets such as Sad1, Cry1Ac, 35S, and Nos in Bt-cotton. Four novel PCR primers were designed to customize amplicon size for multiplexing for better visualization of multiple peaks. The LOD for CrylAc DNA specific PCR was 0.01% for Bt-cotton. The LOD for multiplex PCR assay was 0.05% for Bt-cotton. A singleplex PCR-CGE method was developed to detect Lec, 35S and Nos in a trace sample of RR soybean grain powder (0.1%, w/w). This study demonstrates a PCR-CGE-based method for the qualitative detection of 35S, Nos and Cry1Ac targets associated with genetically modified products. PMID:24672872

  16. Monitoring of the microbial communities involved in the soy sauce manufacturing process by PCR-denaturing gradient gel electrophoresis.

    PubMed

    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

  17. Evaluation of sample preparation methods from rice seeds and seedlings suitable for two-dimensional gel electrophoresis.

    PubMed

    Wongpia, Aphinya; Mahatheeranont, Sugunya; Lomthaisong, Khemika; Niamsup, Hataichanoke

    2015-01-01

    In a proteomic study, sample preparation is very important because it affects the quality of protein profiles on two-dimensional gel electrophoresis (2-DE). This study investigated the suitability of four protein extraction methods-direct lysis buffer extraction, trichloroacetic acid (TCA)/acetone precipitation, phenol extraction, and polyethylene glycol (PEG) fractionation-from rice seeds and seedlings (Oryza sativa L. ssp. indica cv. Khao Dawk Mali 105). The effectiveness of these methods was evaluated by the protein quantity and the 2-DE profiling quality. This included the number of protein spots, the consistency and uniqueness of protein spots, and their distribution in different ranges of pI and molecular weight (M r ). For protein quantity, the phenol and direct lysis extraction methods gave the highest protein yield in rice seeds and rice seedlings, respectively. However, in terms of the quality of 2-DE profiles, samples prepared by the TCA/acetone and phenol methods exhibited higher protein resolution and more spots than the protein profile derived from direct lysis extract. In addition, TCA/acetone method had the efficiency for high M r protein detection. PEG fractionation provided the best 2-DE pattern in terms of resolution, number of spots, minimal streaking, and reproducibility. Moreover, PEG fractionation was better for determining low M r basic proteins. PMID:25355004

  18. 16S rRNA PCR-Denaturing Gradient Gel Electrophoresis of Oral Lactobacillus casei Group and Their Phenotypic Appearances.

    PubMed

    Piwat, S; Teanpaisan, R

    2013-01-01

    This study aimed to develop a 16S rRNA PCR-denaturing gradient gel electrophoresis (DGGE) to identify the species level of Lactobacillus casei group and to investigate their characteristics of acid production and inhibitory effect. PCR-DGGE has been developed based on the 16S rRNA gene, and a set of HDA-1-GC and HDA-2, designed at V2-V3 region, and another set of CARP-1-GC and CARP-2, designed at V1 region, have been used. The bacterial strains included L. casei ATCC 393, L. paracasei CCUG 32212, L. rhamnosus ATCC 7469, L. zeae CCUG 35515, and 46 clinical strains of L. casei/paracasei/rhamnosus. Inhibitory effect against Streptococcus mutans and acid production were examined. Results revealed that each type species strain and identified clinical isolate showed its own unique DGGE pattern using CARP1-GC and CARP2 primers. HDA1-GC and HDA2 primers could distinguish the strains of L. paracasei from L. casei. It was found that inhibitory effect of L. paracasei was stronger than L. casei and L. rhamnosus. The acid production of L. paracasei was lower than L. casei and L. rhamnosus. In conclusion, the technique has been proven to be able to differentiate between closely related species in L. casei group and thus provide reliable information of their phenotypic appearances. PMID:24191230

  19. Tetrabutylammonium phosphate-assisted separation of multiplex polymerase chain reaction products in non-gel sieving capillary electrophoresis.

    PubMed

    Zhang, Sheng; Jiang, Cheng; Jia, Li

    2011-01-15

    A method based on non-gel sieving capillary electrophoresis (NGS-CE) with ultraviolet (UV) detection has been developed for the separation of multiplex polymerase chain reaction (PCR) products of three pathogenic bacteria in which hydroxypropylmethylcellulose was used as the sieving medium and dynamic capillary coating. In the method, an ion pair reagent, tetrabutylammonium phosphate (TBAP), was first used in NGS-CE to improve the detection sensitivities and resolutions of DNA fragments. The interaction of TBAP and DNA was proved using the UV spectra of DNA with and without TBAP. Field-enhanced sample injection was used as an on-line preconcentration method to improve the detection sensitivity. The separation of DNA fragments ranging from 100 to 1000 bp was accomplished in 30 min. Three pairs of primers and three PCR products of bacteria were successfully separated in 25 min using the developed method. The intraday relative standard deviations (RSDs) for the migration time and peak area for each PCR product were less than 2.4% (n=5), and the interday RSDs were less than 6.1% (n=15). PMID:20868644

  20. Yeast involved in fermentation of Coffea arabica in East Africa determined by genotyping and by direct denaturating gradient gel electrophoresis.

    PubMed

    Masoud, Wafa; Cesar, Lene Bjrg; Jespersen, Lene; Jakobsen, Mogens

    2004-05-01

    Samples of Coffea arabica were collected during the different stages of the fermentation from two production sites in Tanzania. The yeasts community was identified by genotyping using ITS-PCR and sequence analysis of the D1/D2 domain of the 26S rRNA gene. For confirmation, denaturating gradient gel electrophoresis (DGGE) of PCR-amplified 26S rRNA gene was performed to detect yeast directly from coffee samples without cultivation. Yeast counts were in the range 4.0 x 10(4) - 5.0 x 10(7) CFU/g with an increase during fermentation. Three yeasts species were dominant. The predominant yeast found during fermentation and drying was Pichia kluyveri. Pichia anomala was found in high numbers during drying of coffee beans. Hanseniaspora uvarum was the predominant yeast during fermentation but decreased during drying. Kluyveromyces marxianus, Candida pseudointermedia, Issatchenkia orientalis, Pichia ohmeri and Torulaspora delbrueckii occurred in concentrations of 10(3) CFU/g or below in coffee samples. Saccharomyces cerevisiae and Candida xestobii were not isolated by cultivation, but by the DGGE technique. A good agreement was found between the sequence analysis of the D1/D2 domain of the 26S rRNA gene and sequencing of the DGGE bands. PMID:15164358

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

    PubMed Central

    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 the prevailing ecological conditions in the different sourdough fermentations, only a few Lactobacillus species were found to be competitive and became dominant. In sourdough A (traditional process with rye flour), Lactobacillus sanfranciscensis and a new species, L. mindensis, were detected. In rye flour sourdoughs B and C, which differed in the process temperature, exclusively L. crispatus and L. pontis became the predominant species in sourdough B and L. crispatus, L. panis, and L. frumenti became the predominant species in sourdough C. On the other hand, in sourdough D (corresponding to sourdough C but produced with rye bran), L. johnsonii and L. reuteri were found. The results of PCR-DGGE were consistent with those obtained by culturing, except for sourdough B, in which L. fermentum was also detected. Isolates of the species L. sanfranciscensis and L. fermentum were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively. PMID:12514030

  2. Monitoring the bacterial population dynamics in sourdough fermentation processes by using PCR-denaturing gradient gel electrophoresis.

    PubMed

    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 the prevailing ecological conditions in the different sourdough fermentations, only a few Lactobacillus species were found to be competitive and became dominant. In sourdough A (traditional process with rye flour), Lactobacillus sanfranciscensis and a new species, L. mindensis, were detected. In rye flour sourdoughs B and C, which differed in the process temperature, exclusively L. crispatus and L. pontis became the predominant species in sourdough B and L. crispatus, L. panis, and L. frumenti became the predominant species in sourdough C. On the other hand, in sourdough D (corresponding to sourdough C but produced with rye bran), L. johnsonii and L. reuteri were found. The results of PCR-DGGE were consistent with those obtained by culturing, except for sourdough B, in which L. fermentum was also detected. Isolates of the species L. sanfranciscensis and L. fermentum were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively. PMID:12514030

  3. Fate of a metal-resistant inoculum in contaminated and pristine soils assessed by denaturing gradient gel electrophoresis

    SciTech Connect

    Stephen, J.R.; Chang, Y.J.; MacNaughton, S.J.; Leung, K.T.; Flemming, C.A. (Univ. of Tennessee, Knoxville, TN (United States). Center for Environmental Biotechnology); Whitaker, S.L.; Hicks, C.L. (Microbial Insights, Rockford, TN (United States)); White, D.C. (Univ. of Tennessee, Knoxville, TN (United States). Center for Environmental Biotechnology Oak Ridge National Lab., TN (United States). Environmental Sciences Div.)

    1999-06-01

    Cesium, cadmium, cobalt, and strontium are four contaminants frequently found in soils at biotoxic levels. Introduction of certain nongenetically modified bacteria has been frequently suggested as a method for the immobilization of heavy metal contaminants in soil, thereby reducing runoff and bioavailability. In this study, the authors have used the polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) to track the survival of the five bacterial species added to soil microcosms with and without the addition of a mixture of these metals. The PCR primers targeted conserved regions of the 165 rDNA molecular present in all bacteria. The reaction products were shown to reflect the relative abundance of the bacteria both in mixtures of pure cultures and against a background of all the eubacterial species present in the soil following inoculation. Three of the species (Pseudomonas aeruginosa FRD-1, Shewanella putrifaciens 200, and Desulfovibrio vulgaris Hildenborough) decreased rapidly following inoculation into both soils. The proportion of Alcaligenes eutrophus CH34 remained at a constant level throughout the 8-week experiment in both soil treatments. Sphingomonas aromaticivorans B0695 showed toxic metal-dependent survival in that its relative abundance dropped rapidly in pristine soil but remained at approximately inoculation levels throughout the experiment in contaminated microcosms.

  4. Diversity of pulsed-field gel electrophoresis patterns of cereulide-producing isolates of Bacillus cereus and Bacillus weihenstephanensis.

    PubMed

    Castiaux, Virginie; N'guessan, Elise; Swiecicka, Izabela; Delbrassinne, Laurence; Dierick, Katelijne; Mahillon, Jacques

    2014-04-01

    Bacillus cereus is an important foodborne pathogen causing diarrhoea, emesis and in, rare cases, lethal poisonings. The emetic syndrome is caused by cereulide, a heat-stable toxin. Originally considered as a rather homogenous group, the emetic strains have since been shown to display some diversity, including the existence of two clusters of mesophilic B. cereus and psychrotolerant B. weihenstephanensis. Using pulsed-field gel electrophoresis (PFGE) analysis, this research aimed to better understand the diversity and spatio-temporal occurrence of emetic strains originating from environmental or food niches vs. those isolated from foodborne cases. The diversity was evaluated using a set of 52 B. cereus and B. weihenstephanensis strains isolated between 2000 and 2011 in ten countries. PFGE analysis could discriminate 17 distinct profiles (pulsotypes). The most striking observations were as follows: (1) more than one emetic pulsotype can be observed in a single outbreak; (2) the number of distinct isolates involved in emetic intoxications is limited, and these potentially clonal strains frequently occurred in successive and independent food poisoning cases; (3) isolates from different countries displayed identical profiles; and (4) the cereulide-producing psychrotolerant B. weihenstephanensis were, so far, only isolated from environmental niches. PMID:24627989

  5. Variations among Japanese of the factor IX gene (F9) detected by PCR-denaturing gradient gel electrophoresis

    SciTech Connect

    Satoh, Chiyoko; Takahashi, Norio; Asakawa, Junichi; Hiyama, Keiko; Kodaira, Meiko (Radiation Effects Research Foundation, Hiroshima (Japan))

    1993-01-01

    In the course of feasibility studies to examine the efficiencies and practicalities of various techniques for screening for genetic variations, the human coagulation factor IX (F9) genes of 63 Japanese families were examined by PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Four target sequences with lengths of 983-2,891 bp from the F9 genes of 126 unrelated individuals from Hiroshima and their 100 children were amplified by PCR, digested with restriction enzymes to approximately 500-bp fragments, and examined by DGGE - a total of 6,724 bp being examined per individual. GC-rich sequences (GC-clamps) of 40 bp were attached to both ends of the target sequences, as far as was feasible. Eleven types of new nucleotide substitutions were detected in the population, none of which produced RFLPs or caused hemophilia B. By examining two target sequences in a single lane, approximately 8,000 bp in a diploid individual could be examined. This approach is very effective for the detection of variations in DNA and is applicable to large-scale population studies. 46 refs., 3 figs., 1 tab.

  6. Effect of Natural and Semisynthetic Pseudoguianolides on the Stability of NF-?B:DNA Complex Studied by Agarose Gel Electrophoresis

    PubMed Central

    Villagomez, Rodrigo; Hatti-Kaul, Rajni; Sterner, Olov; Almanza, Giovanna; Linares-Pastn, Javier A.

    2015-01-01

    The nuclear factor ?B (NF-?B) is a promising target for drug discovery. NF-?B is a heterodimeric complex of RelA and p50 subunits that interact with the DNA, regulating the expression of several genes; its dysregulation can trigger diverse diseases including inflammation, immunodeficiency, and cancer. There is some experimental evidence, based on whole cells studies, that natural sesquiterpene lactones (Sls) can inhibit the interaction of NF-?B with DNA, by alkylating the RelA subunit via a Michael addition. In the present work, 28 natural and semisynthetic pseudoguianolides were screened as potential inhibitors of NF-?B in a biochemical assay that was designed using pure NF-?B heterodimer, pseudoguianolides and a ~1000 bp palindromic DNA fragment harboring two NF-?B recognition sequences. By comparing the relative amount of free DNA fragment to the NF-?B DNA complex, in a routine agarose gel electrophoresis, the destabilizing effect of a compound on the complex is estimated. The results of the assay and the following structure-activity relationship study, allowed the identification of several relevant structural features in the pseudoguaianolide skeleton, which are necessary to enhance the dissociating capacity of NF-?BDNA complex. The most active compounds are substituted at C-3 (?-carbonyl), in addition to having the ?-methylene-?-lactone moiety which is essential for the alkylation of RelA. PMID:25615602

  7. Succession of bacterial community structure along the Changjiang River determined by denaturing gradient gel electrophoresis and clone library analysis.

    PubMed

    Sekiguchi, Hiroyuki; Watanabe, Masataka; Nakahara, Tadaatsu; Xu, Baohua; Uchiyama, Hiroo

    2002-10-01

    Bacterial community structure along the Changjiang River (which is more than 2,500 km long) was studied by using denaturing gradient gel electrophoresis (DGGE) and clone library analysis of PCR-amplified 16S ribosomal DNA (rDNA) with universal bacterial primer sets. DGGE profiles and principal-component analysis (PCA) demonstrated that the bacterial community gradually changed from upstream to downstream in both 1998 and 1999. Bacterial diversity, as determined by the Shannon index (H'), gradually decreased from upstream to downstream. The PCA plots revealed that the differences in the bacterial communities among riverine stations were not appreciable compared with the differences in two adjacent lakes, Lake Dongting and Lake Poyang. The relative stability of the bacterial communities at the riverine stations was probably due to the buffering action of the large amount of water flowing down the river. Clone library analysis of 16S rDNA revealed that the dominant bacterial groups changed from beta-proteobacteria and the Cytophaga-Flexibacter-Bacteroides group upstream to high-G+C-content gram-positive bacteria downstream and also that the bacterial community structure differed among the stations in the river and the lakes. The results obtained in this study should provide a reference for future changes caused by construction of the Three Gorges Dam. PMID:12324365

  8. Characterization of proteins in latex of the opium poppy (Papaver somniferum) using two-dimensional gel electrophoresis and microsequencing.

    PubMed

    Decker, G; Wanner, G; Zenk, M H; Lottspeich, F

    2000-10-01

    The opium poppy (Papaver somniferum) belongs to the group of latex-containing plants. Latex is the milky-like fluid within laticifer cells. In this study, poppy latex was analyzed with respect to ultrastructure, alkaloid, and protein content. The main goal of this project was the examination of the proteins by two-dimensional gel electrophoresis. In a proteomics approach, we investigated two main fractions of the latex, namely the cytosolic serum and the sedimented fraction containing the alkaloid-accumulating vesicles. Of the serum, representing the protein-rich part of the latex, 75 spots were analyzed by internal peptide microsequencing, followed by a database searching. For 69 proteins a function could be assigned due to homology to known proteins, whereas six spots could not be identified. Furthermore, codeinone reductase, a representative of the specific enzyme system in morphine biosynthesis, could be detected within the cytosolic serum fraction. In the vesicle-containing pellet, 23 protein spots were analyzed. An attempt was also made to separate the vesicle pellet by density centrifugation, followed by investigation of the alkaloid content, ultrastructure, and protein pattern. This study describes the first database of soluble proteins present in the latex of P. somniferum PMID:11079569

  9. Detritus-Dependent Development of the Microbial Community in an Experimental System: Qualitative Analysis by Denaturing Gradient Gel Electrophoresis

    PubMed Central

    van Hannen, Erik J.; Mooij, Wolf; van Agterveld, Miranda P.; Gons, Herman J.; Laanbroek, Hendrikus J.

    1999-01-01

    Correlations between the biomass of phytoplankton and the biomass of bacteria and between the biomass of bacteria and the biomass of protozoans suggest that there is coupling between these compartments of the microbial loop. To investigate this coupling on the species level, bacteria and protozoans from untreated lake water inocula were allowed to grow on detritus of the green alga Ankistrodesmus falcatus or the cyanobacterium Oscillatoria limnetica in continuous-flow systems for 1 month. Denaturing gradient gel electrophoresis (DGGE) of the 16S and 18S rRNA genes was used to monitor the development of the bacterial community structure and the eukaryotic community structure, respectively. Nonmetric multidimensional scaling of the DGGE profiles revealed the changes in the microbial community structure. This analysis showed that significantly different bacterial communities developed on the green algal detritus and on the cyanobacterial detritus. Although similar results were obtained for the eukaryotic communities, the differences were not significant. Hence, our findings indicate that the origin of detritus can affect the structure of at least the bacterial community. A phylogenetic analysis of 20 18S ribosomal DNA clones that were isolated from the continuous cultures revealed that many sequences were related to the sequences of bacterivorous protozoans (members of the Ciliophora, Rhizopoda, Amoeba, and Kinetoplastida). One clone grouped in a recently established clade whose previously described members are all parasites. The affiliations of about 20% of the clones could not be determined. PMID:10347030

  10. Comparison of pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus PCR and biochemical tests to characterize Lactococcus garvieae.

    PubMed

    Ture, M; Altinok, I; Capkin, E

    2015-01-01

    Biochemical test, pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) were used to compare 42 strains of Lactococcus garvieae isolated from different regions of Turkey, Italy, France and Spain. Twenty biotypes of L. garvieae were formed based on 54 biochemical tests. ERIC-PCR of genomic DNA from different L. garvieae strains resulted in amplification of multiple fragments of DNA in sizes ranging between 200 and 5000 bp with various band intensities. After cutting DNA with ApaI restriction enzyme and running on the PFGE, 1122 resolvable bands ranging from 2 to 194 kb were observed. Turkish isolates were grouped into two clusters, and only A58 (Italy) strain was connected with Turkish isolates. Similarities between Turkish, Spanish, Italian and French isolates were <50% except 216-6 Rize strain. In Turkey, first lactococcosis occurred in Mugla, and then, it has been spread all over the country. Based on ERIC-PCR, Spanish and Italian strains of L. garvieae were related to Mugla strains. Therefore, after comparing PFGE profiles, ERIC-PCR profiles and phenotypic characteristics of 42 strains of L. garvieae, there were no relationships found between these three typing methods. PFGE method was more discriminative than the other methods. PMID:25664362

  11. Brownian dynamic simulations of electrophoresis and electro-stretching of DNA molecules in polymer gels.

    NASA Astrophysics Data System (ADS)

    Larson, Ronald; Graham, Richard

    2006-03-01

    We derive a model for the motion of long DNA chains entangled in a concentrated gel matrix in the presence of a strong electric field. The model is adapted from a tube-based slip-link approach, which was originally intended to model the rheology of entangled polymer fluids, and is suitable for solution by Brownian dynamic simulation. We account for the constraining effect of the surrounding matrix, motion due to the electric field and finite extensibility of the DNA chain. We are able investigate the effect of molecular weight and field strength on the DNA drift velocity in a constant electric field, along with molecular stretching in an oscillating field. Both examples have applications in DNA separation and sequencing. Our approach includes a detailed treatment of the chain end motion through the matrix, which our simulations demonstrate has a significant role in the DNA dynamics, particularly in oscillating fields. The model provides a convenient formalism for further refinements. For example, large fields may tend to cause hernia-like chain loops to protrude from the main tube. Furthermore, to model matrices comprised of linear polymers we can include the effect of constraint release, in which the confinement experienced by the DNA is diminished by the motion of the matrix chains.

  12. Coupling of Chlorophyll Metabolism with Submembrane Chloroplast Particles, Isolated with Digitonin and Gel Electrophoresis

    PubMed Central

    Fradkin, Leonid I.; Chkanikova, Rinata A.; Shlyk, Alexander A.

    1981-01-01

    An unusual set of submembrane particles is obtained from digitonintreated barley chloroplasts as five gel-electrophoretic zones. Four of them are photochemically active, whereas the most mobile fifth zone has essential traits of the light-harvesting complexes. All of the particles contain the well-known chlorophyll-protein complexes and represent an intermediate level of membrane organization. When isolated from plants fed ?-aminolevulinate in the dark, the fifth zone is characterized by a high level of protochlorophyllide, which is also present to a lesser extent in all the other zones. When [14C]aminolevulinate was fed in the dark, followed by exposing the plants to light, the same pattern of the distribution was observed for [14C]chlorophyll a. Thus, particles of all the types are involved in chlorophyll formation and the fifth zone is the most distinct in this respect. Its material seems to originate from the most intensely developing areas of the metabolically heterogeneous chloroplast membrane system. PMID:16661713

  13. Agarose gel electrophoresis of joint fluid using Hyrys-Hydrasys SEBIA system as a new prognostic tool for periprosthetic osteolysisin revision arthroplasty

    PubMed Central

    Chiva, A

    2013-01-01

    Rationale. Prevention of wear-mediated osteolysis, the most common complication in total joint arthroplasty, is a great challenge for orthopedic surgery. Despite the diversity of current biomarkers of periprosthetic osteolysis (products of wear, bone turnover and inflammatory biomarkers), the major interferences and the great amount of sample necessary for analysis limit their use in clinical practice. Objective. The aim of this paper is to present three new electrophoretic methods using Hyrys-Hydrasys SEBIA system that have been used for the first time in Electrophoresis Laboratory of our hospital in the analysis of joint fluid for the prevention of periprosthetic osteolysis in revision arthroplasty. Methods and results. Analytical aspects of agarose gel electrophoresis of joint fluid proteins and lipoproteins as well as SDS-agarose gel electrophoresis of joint fluid proteins, their performances and clinical value are presented. The decreased level of albumin and increased level of alpha1 and alpha2 globulins were frequent changes detected on SEBIA electropherograms and good indicator for the presence of an inflammatory reaction generated by particle debris. In addition, a slightly increase of LDL mobility could provide good information about a high oxidative stress. Moreover, the Ig G assessed by using SDS-agarose gel electrophoresis could be a potential biomarker for an immunological reaction towards orthopedic implants. Discussion. Electrophoresis of joint fluid using Hyrys-Hydrasys SEBIA France system is a new analytical technique able to remove the most of current biomarkers disadvantages due to the determination of particular proteins (acute phase proteins, albumin, lipoproteins, and immunoglobulins) by using minimal amounts of joint fluid with minor interferences, minimal cost and rapid results. Abbreviations CTX, crosslinked C-telopeptide; IL- interleukins; Ig G, immunoglobulin G; LDL, low density lipoprotein; NTX, crosslinked N-telopeptide; PICP, procollagen I C terminal extension peptide; SDS, sodium dodecyl sulphate PMID:24146682

  14. Simulating Electrophoresis.

    ERIC Educational Resources Information Center

    Moertel, Cheryl; Frutiger, Bruce

    1996-01-01

    Describes a DNA fingerprinting simulation that uses vegetable food coloring and plastic food containers instead of DNA and expensive gel electrophoresis chambers. Allows students to decipher unknown combinations of dyes in a method similar to that used to decipher samples of DNA in DNA fingerprint techniques. (JRH)

  15. Comparison of Pulsed-Field Gel Electrophoresis and Coagulase Gene Restriction Profile Analysis Techniques in the Molecular Typing of Staphylococcus aureus

    Microsoft Academic Search

    CHIEN-SHUN CHIOU; HSIAO-LUN WEI; LI-CHU YANG

    2000-01-01

    Pulsed-field gel electrophoresis (PFGE) and coagulase gene restriction profile (CRP) analysis techniques were used to analyze 71 Staphylococcus aureus isolates recovered from nine food-borne disease outbreaks. Twenty-two PFGE profiles and 11 CRPs were identified, with discrimination indices of 0.86 and 0.72, respec- tively. In addition, the variable regions of the coagulase genes of 39 isolates were sequenced and showed extensive

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

    Microsoft Academic Search

    Wei Wang; Jibin Sun; Manfred Nimtz; Wolf-Dieter Deckwer; An-Ping Zeng

    2003-01-01

    Separation of proteins by two-dimensional gel electrophoresis (2-DE) coupled with identification of proteins through peptide mass fingerprinting (PMF) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is the widely used technique for proteomic analysis. This approach relies, however, on the presence of the proteins studied in public-accessible protein databases or the availability of annotated genome sequences of an

  17. Population dynamics of free-floating and attached bacteria in a styrene-degrading biotrickling filter analyzed by denaturing gradient gel electrophoresis

    Microsoft Academic Search

    O. Tresse; M.-J. Lorrain; D. Rho

    2002-01-01

    . Population dynamics was studied in a 52-l biotrickling filter (BTF) operated for 182days and used to clean air contaminated\\u000a with styrene vapors. In the BTF, biomass grew either as free-floating (planktonic) or attached (sessile) microorganisms. PCR-amplified\\u000a 16S rDNA fragments from planktonic and sessile cells within the bioreactor were analyzed using denaturing gradient gel electrophoresis\\u000a (DGGE). The results indicated that

  18. Evaluation of denaturing gradient gel electrophoresis in the detection of 16S rDNA sequence variation in rhizobia and methanotrophs

    Microsoft Academic Search

    Tatiana Vallaeys; Edward Topp; Gerard Muyzer; Valrie Macheret; Gisle Laguerre; Annabel Rigaud; Guy Soulas

    1997-01-01

    The ability of denaturing gradient gel electrophoresis (DGGE) technique to resolve 16S rDNA products generated from two different collections of bacteria using universal 16S primers was investigated. Alignments of 16S rDNA sequences of known species of rhizobia and methanotrophs were performed in order to determine the genetic variations within a 200 bp product obtained with PCR primers which amplify the

  19. Diversity of Listeria monocytogenes isolates of human and food origin studied by serotyping, automated ribotyping and pulsed-field gel electrophoresis

    Microsoft Academic Search

    S. Lukinmaa; K. Aarnisalo; M.-L. Suihko; A. Siitonen

    2004-01-01

    Automated ribotyping, pulsed-field gel electrophoresis (PFGE) and serotyping were evaluated for the epidemiological study of isolates of Listeria monocytogenes collected in Finland in 1997-1999 from human blood (n 116) and the food industry (n 72). The isolates divided into six serotypes, 23 EcoRI ribotypes, 54 AscI PFGE types, and 57 final subtypes if all results were combined. The

  20. Unveiling contamination sources and dissemination routes of Salmonella sp. in pigs at a Portuguese slaughterhouse through macrorestriction profiling by pulsed-field gel electrophoresis

    Microsoft Academic Search

    Madalena Vieira-Pinto; Rogrio Tenreiro; Conceio Martins

    2006-01-01

    Sixty-nine isolates of Salmonella sp. isolated from the ileum, tonsils, carcass and mandibular and ileocolic lymph nodes of individual pigs slaughtered for consumption in one abattoir were analyzed using serotyping and macrorestriction profiling by Pulsed-Field Gel Electrophoresis (RFLP-PFGE), in order to identify clonal relationships. XbaI macrorestriction was able to distinguish 18 genotypes among the eight identified serotypes: Salmonella Typhimurium (4

  1. Easy DNA extraction method and optimisation of PCR-Temporal Temperature Gel Electrophoresis to identify the predominant high and low GC-content bacteria from dairy products

    Microsoft Academic Search

    Sandrine Parayre; Hlne Falentin; Marie-Nolle Madec; Katia Sivieri; Anne-Sophie Le Dizes; Danile Sohier; Sylvie Lortal

    2007-01-01

    Molecular fingerprinting of bacterial ecosystems has recently increased in food microbiology. The aim of this work was to develop a rapid and easy method to extract DNA from various cheeses, and to optimize the separation of low and high GC-content bacteria by PCR-Temporal Temperature Gel Electrophoresis (PCR-TTGE).Seventy six strains belonging to 50 of the most frequently encountered bacterial species in

  2. DNA heterogeneity of Staphylococcus aureus strains evaluated by Sma I and Sgr AI pulsed-field gel electrophoresis in patients with impetigo

    Microsoft Academic Search

    Ettore Capoluongo; Amalia Giglio; Francesco Leonetti; Mauro Belardi; Alberto Giannetti; Federico Caprilli; Franco Ameglio

    2000-01-01

    To our knowledge, no studies have previously been carried out on the heterogeneity and intrafamily colonization of impetigo Staphylococcus aureus strains obtained by powerful discriminating methods such as pulsed-field gel electrophoresis (PFGE). To explore this topic, macrorestriction patterns of S. aureus strains were analyzed after SmaI and SgrAI digestion. The two enzymes provided superimposable results. A total of ninety-seven S.

  3. Acid and Base-Induced Proteins during Aerobic and Anaerobic Growth of Escherichia coli Revealed by Two-Dimensional Gel Electrophoresis

    Microsoft Academic Search

    DARCY BLANKENHORN; JUDITH PHILLIPS; JOAN L. SLONCZEWSKI

    1999-01-01

    Proteins induced by acid or base, during long-term aerobic or anaerobic growth in complex medium, were identified in Escherichia coli. Two-dimensional gel electrophoresis revealed pH-dependent induction of 18 pro- teins, nine of which were identified by N-terminal sequencing. At pH 9, tryptophan deaminase (TnaA) was induced to a high level, becoming one of the most abundant proteins observed. TnaA may

  4. Microchip gel electrophoresis with programmed field strength gradients for ultra-fast detection of canine T-cell lymphoma in dogs

    Microsoft Academic Search

    Kumar K. Suresh; Mi-Jin Lee; Jinho Park; Seong Ho Kang

    2008-01-01

    This paper describes the applicability of microchip gel electrophoresis using a programmed field strength gradients (MGE-PFSG) method coupled with a polymerase chain reaction (PCR) for the ultra-fast diagnosis of canine T-cell lymphoma. The variable region in the T-cell receptor ? (TCR?) gene from a T-cell lymphoma was used in PCR amplification. The contributions of the various parameters, including the effects

  5. A Single-Sample Method for Determination of Carbohydrate and Protein Contents Glycoprotein Bands Separated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

    Microsoft Academic Search

    Ewa Zdebska; Jerzy Ko?cielak

    1999-01-01

    A method is described for determination of carbohydrate and protein contents of glycoproteins separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and then electroblotted onto polyvinylidene difluoride (PVDF) membranes. Blots were stained, and appropriate pieces of PVDF membranes were excised, destained, and subjected to sequential hydrolysis with 0.2 M trifluoroacetic acid (TFA) for 1 h at 80C, then with 2 M

  6. Two-dimensional gel electrophoresis analysis of the proteomes expressed in the human hepatoma cell line BEL7404 and normal liver cell line L-02

    Microsoft Academic Search

    Lirong Yu; Nan Wang; Gaode Wu; Yonghua Xu; Qichang Xia

    2000-01-01

    Proteome analysis technology has been used extensively in conducting discovery research of biology and has become one of the\\u000a most essential technologies in functional genomics. The proteomes of the human hepatoma cell line BEL-7404 and the normal\\u000a human liver cell line L-02 have been separated by high resolution two-dimensional gel electrophoresis (2-DE) with immobilized\\u000a pH gradient isoelectric focusing (IPG-IEF) in

  7. Dominant bacterioplankton populations in the meromictic Lake Suigetsu as determined by denaturing gradient gel electrophoresis of 16S rRNA gene fragments

    Microsoft Academic Search

    Ryuji Kondo; Akie Nakagawa; Lisa Mochizuki; Kyoko Osawa; Yukiyasu Fujioka; Junki Butani

    2009-01-01

    Lake Suigetsu is a typical meromictic lake in Japan characterized by a permanent chemocline at a depth of between 3 and 8m\\u000a separating the oxic freshwater mixolimnion from anoxic saline sulfidogenic monimolimnion. Dominant bacterioplankton populations\\u000a in Lake Suigetsu were investigated using PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. The\\u000a bacterial population was vertically stratified, and temporal shifts

  8. Specific detection of different phylogenetic groups of chemocline bacteria based on PCR and denaturing gradient gel electrophoresis of 16S rRNA gene fragments

    Microsoft Academic Search

    Jrg Overmann; Marco J. L. Coolen; Christian Tuschak

    1999-01-01

    Specific amplification of 16S rRNA gene fragments in combination with denaturing gradient gel electrophoresis (DGGE) was\\u000a used to generate fingerprints of Chromatiaceae, green sulfur bacteria, Desulfovibrionaceae, and ?-Proteobacteria. Sequencing\\u000a of the gene fragments confirmed that each primer pair was highly specific for the respective phylogenetic group. Applying\\u000a the new primer sets, the bacterial diversity in the chemoclines of a eutrophic

  9. Application of polymerase chain reaction-denaturing gradient gel electrophoresis for comparison of direct and indirect extraction methods of soil DNA used for microbial community fingerprinting

    Microsoft Academic Search

    J. Kozdrj; J. D. van Elsas

    2000-01-01

    We used polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to compare bacterial community patterns\\u000a obtained with target DNA extracted from a soil by direct and indirect methods. For this purpose, two direct extraction methods,\\u000a i.e. cell lysis by bead beating and cell disruption by grinding in liquid N, and two indirect methods, i.e. cell extraction\\u000a followed by DNA extraction, and

  10. Proteomic Analysis of Seed Filling in Brassica napus. Developmental Characterization of Metabolic Isozymes Using High-Resolution Two-Dimensional Gel Electrophoresis

    Microsoft Academic Search

    Martin Hajduch; Jill E. Casteel; Katherine E. Hurrelmeyer; Zhao Song; Ganesh Kumar Agrawal; Jay J. Thelen

    2006-01-01

    Brassica napus (cultivar Reston) seed proteins were analyzed at 2, 3, 4, 5, and 6 weeks after flowering in biological quadruplicate using two-dimensional gel electrophoresis. Developmental expression profiles for 794 protein spot groups were established and hierarchical cluster analysis revealed 12 different expression trends. Tryptic peptides from each spot group were analyzed in duplicate using matrix-assisted laser desorption ionization time-of-flight

  11. Seasonal Distributions of Dominant 16S rRNA-Defined Populations in a Hot Spring Microbial Mat Examined by Denaturing Gradient Gel Electrophoresis

    Microsoft Academic Search

    M. J. FERRIS; ANDD. M. WARD

    1997-01-01

    Denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA gene segments was used to examine the distributions of bacterial populations within a hot spring microbial mat (Octopus Spring, Yellowstone National Park). Populations at sites along the thermal gradient of the spring's effluent channel weresurveyedatseasonalintervals.Noshiftinthethermalgradientwasdetected,andpopulationsatspatially or temperature-defined sites exhibited only slight changes over the annual sampling period. A new cyanobac- terial

  12. Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella Species in Human Feces by Using Group-Specific PCR Primers and Denaturing Gradient Gel Electrophoresis

    Microsoft Academic Search

    JENS WALTER; CHRISTIAN HERTEL; GERALD W. TANNOCK; CLAUDIA M. LIS; KAREN MUNRO; WALTER P. HAMMES

    2001-01-01

    Denaturing gradient gel electrophoresis (DGGE) of DNA fragments generated by PCR with 16S ribosomal DNA-targeted group-specific primers was used to detect lactic acid bacteria (LAB) of the genera Lactobacillus, Pediococcus, Leuconostoc, and Weissella in human feces. Analysis of fecal samples of four subjects revealed individual profiles of DNA fragments originating not only from species that have been described as intestinal

  13. Identification of and Spatio-Temporal Differences between Microbial Assemblages from Two Neighboring Sulfurous Lakes: Comparison by Microscopy and Denaturing Gradient Gel Electrophoresis

    Microsoft Academic Search

    EMILIO O. CASAMAYOR; HENDRIK SCHAFER; LLUIS BANERAS; CARLOS PEDROS-ALIO; G. Muyzer

    2000-01-01

    The microbial assemblages of Lake Cisoand Lake Vilar (Banyoles, northeast Spain) were analyzed in space and time by microscopy and by performing PCR-denaturing gradient gel electrophoresis (DGGE) and se- quence analysis of 16S rRNA gene fragments. Samples obtained from different water depths and at two different times of the year (in the winter during holomixis and in the early spring

  14. Strain identification of probiotic Lactobacillus casei-related isolates with randomly amplified polymorphic DNA and pulsed-field gel electrophoresis methods

    Microsoft Academic Search

    Denis Roy; Pierre Ward; Daniel Vincent

    1999-01-01

    Typing of reference strains and isolates identified as Lactobacillus casei, Lactobacillus paracasei or Lactobacillus rhamnosus was carried out using randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) analyses. Strains of L.paracasei were mainly grouped in the same cluster as those of L.casei. The RAPD fingerprints of strains ATCC 393 and ATCC 15820 differ from those of the L.rhamnosus

  15. Phylogenetic relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments

    Microsoft Academic Search

    Gerard Muyzer; Andreas Teske; Carl O. Wirsen; Holger W. Jannasch

    1995-01-01

    Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments was used to explore the genetic diversity\\u000a of hydrothermal vent microbial communities, specifically to determine the importance of sulfur-oxidizing bacteria therein.\\u000a DGGE analysis of two different hydrothermal vent samples revealed one PCR band for one sample and three PCR bands for the\\u000a other sample, which probably correspond to the dominant

  16. Development and Validation of PCR Primers To Assess the Diversity of Clostridium spp. in Cheese by Temporal Temperature Gradient Gel Electrophoresis

    Microsoft Academic Search

    Anne-Gaelle Le Bourhis; Katiana Saunier; Joel Dore; Jean-Philippe Carlier; Jean-Francois Chamba; Michel-Robert Popoff; Jean-Luc Tholozan

    2005-01-01

    A nested-PCR temporal temperature gradient gel electrophoresis (TTGE) approach was developed for the detection of bacteria belonging to phylogenetic cluster I of the genus Clostridium (the largest clostridial group, which represents 25% of the currently cultured clostridial species) in cheese suspected of late blowing. Primers were designed based on the 16S rRNA gene sequence, and the specificity was confirmed in

  17. Pulsed-Field Gel Electrophoresis Typing of Oxacillin-Resistant Staphylococcus aureus Isolates from the United States: Establishing a National Database

    Microsoft Academic Search

    Linda K. McDougal; Christine D. Steward; George E. Killgore; Jasmine M. Chaitram; Sigrid K. McAllister; Fred C. Tenover

    2003-01-01

    Received 2 June 2003\\/Returned for modification 10 July 2003\\/Accepted 22 August 2003 Oxacillin-resistant Staphylococcus aureus (ORSA) is a virulent pathogen responsible for both health care- associated and community onset disease. We used SmaI-digested genomic DNA separated by pulsed-field gel electrophoresis (PFGE) to characterize 957 S. aureus isolates and establish a database of PFGE patterns. In addition to PFGE patterns of

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

    Microsoft Academic Search

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

    2005-01-01

    We have developed a novel method for the ultra-fast analysis of genetically modified organisms (GMOs) in soybeans by microchip capillary gel electrophoresis (MCGE) using programmed field strength gradients (PFSG) in a conventional glass double-T microchip. Under the programmed electric field strength and 0.3% poly(ethylene oxide) sieving matrix, the GMO in soybeans was analyzed within only 11s of the microchip. The

  19. Temperature gradient gel electrophoresis analysis of 16S rRNA from human fecal samples reveals stable and host-specific communities of active bacteria

    Microsoft Academic Search

    ERWIN G. ZOETENDAL; ANTOON D. L. AKKERMANS; Vos de W. M

    1998-01-01

    The diversity of the predominant bacteria in the human gastrointestinal tract was studied by using 16S rRNA-based approaches. PCR amplicons of the V6 to V8 regions of fecal 16S rRNA and ribosomal DNA (rDNA) were analyzed by temperature gradient gel electrophoresis (TGGE). TGGE of fecal 16S rDNA ampli- cons from 16 individuals showed different profiles, with some bands in common.

  20. Evaluation of the taxonomic utility of six-enzyme pulsed-field gel electrophoresis in reconstructing Salmonella subspecies phylogeny.

    PubMed

    Trujillo, Socrates; Keys, Christine E; Brown, Eric W

    2011-01-01

    Pulsed-field gel electrophoresis (PFGE) remains an important tool in the molecular epidemiological evaluation of strains emerging from disease outbreak clusters. Recent studies of Escherichia coli O157:H7 and Salmonella Enteritidis have noted marked improvements in the discriminatory power of PFGE when combining band profiles from up to six restriction enzyme datasets into a single concatenated analysis. This approach has provided more accurate assignments of genetic relationships among closely related strains and allowed effective phylogenetic inference of host and geographical reservoirs. Although this approach enhances epidemiological congruence among closely related strains, it remains unclear to what extent six-enzyme PFGE pattern similarity reiterates evolutionary relatedness among more distantly related Salmonella strains (i.e., serovar or subspecies levels). Here, taxonomic accuracy of six-enzyme PFGE is tested phylogenetically across two distinct Salmonella enterica populations-Salmonella reference collection B (SARB), representing the breadth of taxonomic diversity of S. enterica subspecies I only, and Salmonella reference collection C (SARC), comprising the seven disparate subspecies of S. enterica plus S. bongori. Cladistic analysis of SAR strains revealed substantial polyphyly between the two strain collections such that numerous SARB strains clustered more closely with diverged SARC subspecies rather than with other members of subspecies I. Also, in several cases, SARC sibling strains from the same subspecies were evolutionary obscured-broken into distant locales on the most parsimonious six-enzyme trees. Genetic diversity among SARB and SARC strains was comparable at 45% and 47%, respectively, while congruence testing revealed discordance among individual enzyme datasets. While six-enzyme PFGE is effective in ascertaining accurate genetic relationships for more closely related strains (e.g., strains within the same serovar), reconstitution of evolutionarily meaningful strain groupings may be elusive for Salmonella at the serovar level and above. Thus, caution is warranted when applying PFGE with a limited number of enzymes as the primary phylogenetic marker in these instances. PMID:20959148

  1. High-Resolution Differentiation of Cyanobacteria by Using rRNA-Internal Transcribed Spacer Denaturing Gradient Gel Electrophoresis

    PubMed Central

    Janse, Ingmar; Meima, Marion; Kardinaal, W. Edwin A.; Zwart, Gabriel

    2003-01-01

    For many ecological studies of cyanobacteria, it is essential that closely related species or strains can be discriminated. Since this is often not possible by using morphological features, cyanobacteria are frequently studied by using DNA-based methods. A powerful method for analysis of the diversity and dynamics of microbial populations and for checking the purity and affiliation of cultivated strains is denaturing gradient gel electrophoresis (DGGE). We realized high-resolution discrimination of a variety of cyanobacteria by means of DGGE analysis of sections of the internal transcribed spacer between the 16S and 23S rRNA genes (rRNA-ITS). A forward primer specific for cyanobacteria, targeted at the 3? end of the 16S rRNA gene, was designed. The combination of this primer and three different reverse primers targeted to the rRNA-ITS or to the 23S rRNA gene yielded PCR products of different sizes from cultures of all 16 cyanobacterial genera that were tested but not from other bacteria. DGGE profiles produced from the shortest section of rRNA-ITS consisted of one band for all but one cyanobacterial genera, and those generated from longer stretches of rRNA-ITS yielded DGGE profiles containing one to four bands. The suitability of DGGE for detecting intrageneric and intraspecific variation was tested by using strains of the genus Microcystis. Many strains could be discriminated by means of rRNA-ITS DGGE, and the resolution of this method was strikingly higher than that obtained with previously described methods. The applicability of the developed DGGE assays for analysis of cyanobacteria in field samples was demonstrated by using samples from freshwater lakes. The advantages and disadvantages associated with the use of each developed primer set are discussed. PMID:14602623

  2. Comparison of multilocus sequence typing and pulsed-field gel electrophoresis for Salmonella spp. identification in surface water

    NASA Astrophysics Data System (ADS)

    Kuo, Chun Wei; Hao Huang, Kuan; Hsu, Bing Mu; Tsai, Hsien Lung; Tseng, Shao Feng; Kao, Po Min; Shen, Shu Min; Chou Chiu, Yi; Chen, Jung Sheng

    2013-04-01

    Salmonella is one of the most important pathogens of waterborne diseases with outbreaks from contaminated water reported worldwide. In addition, Salmonella spp. can survive for long periods in aquatic environments. To realize genotypes and serovars of Salmonella in aquatic environments, we isolated the Salmonella strains by selective culture plates to identify the serovars of Salmonella by serological assay, and identify the genotypes by Multilocus sequence typing (MLST) based on the sequence data from University College Cork (UCC), respectively. The results show that 36 stream water samples (30.1%) and 18 drinking water samples (23.3%) were confirmed the existence of Salmonella using culture method combined PCR specific invA gene amplification. In this study, 24 cultured isolates of Salmonella from water samples were classified to fifteen Salmonella enterica serovars. In addition, we construct phylogenetic analysis using phylogenetic tree and Minimum spanning tree (MST) method to analyze the relationship of clinical, environmental, and geographical data. Phylogenetic tree showed that four main clusters and our strains can be distributed in all. The genotypes of isolates from stream water are more biodiversity while comparing the Salmonella strains genotypes from drinking water sources. According to MST data, we can found the positive correlation between serovars and genotypes of Salmonella. Previous studies revealed that the result of Pulsed field gel electrophoresis (PFGE) method can predict the serovars of Salmonella strain. Hence, we used the MLST data combined phylogenetic analysis to identify the serovars of Salmonella strain and achieved effectiveness. While using the geographical data combined phylogenetic analysis, the result showed that the dominant strains were existed in whole stream area in rainy season. Keywords: Salmonella spp., MLST, phylogenetic analysis, PFGE

  3. Protein composition of wheat gluten polymer fractions determined by quantitative two-dimensional gel electrophoresis and tandem mass spectrometry

    PubMed Central

    2014-01-01

    Background Certain wheat gluten proteins form large protein polymers that are extractable in 0.5% SDS only after sonication. Although there is a strong relationship between the amounts of these polymers in the flour and bread-making quality, the protein components of these polymers have not been thoroughly investigated. Results Flour proteins from the US bread wheat Butte 86 were extracted in 0.5% SDS using a two-step procedure with and without sonication. Proteins were further separated by size exclusion chromatography (SEC) into monomeric and polymeric fractions and analyzed by quantitative two-dimensional gel electrophoresis (2-DE). When proteins in select 2-DE spots were identified by tandem mass spectrometry (MS/MS), overlapping spots from the different protein fractions often yielded different identifications. Most high-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) partitioned into the polymer fractions, while most gliadins were found in the monomer fractions. The exceptions were alpha, gamma and omega gliadins containing odd numbers of cysteine residues. These proteins were detected in all fractions, but comprised the largest proportion of the SDS-extractable polymer fraction. Several types of non-gluten proteins also were found in the polymer fractions, including serpins, triticins and globulins. All three types were found in the largest proportions in the SDS-extractable polymer fraction. Conclusions This is the first study to report the accumulation of gliadins containing odd numbers of cysteine residues in the SDS-extractable glutenin polymer fraction, supporting the hypothesis that these gliadins serve as chain terminators of the polymer chains. These data make it possible to formulate hypotheses about how protein composition influences polymer size and structure and provide a foundation for future experiments aimed at determining how environment affects glutenin polymer distribution. In addition, the analysis revealed additional layers of complexity to the wheat flour proteome that should be considered when evaluating quantitative 2-DE data. PMID:24517725

  4. Bulk and Rhizosphere Soil Bacterial Communities Studied by Denaturing Gradient Gel Electrophoresis: Plant-Dependent Enrichment and Seasonal Shifts Revealed

    PubMed Central

    Smalla, K.; Wieland, G.; Buchner, A.; Zock, A.; Parzy, J.; Kaiser, S.; Roskot, N.; Heuer, H.; Berg, G.

    2001-01-01

    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

  5. Genomic Diversity within the Genus Pediococcus as Revealed by Randomly Amplified Polymorphic DNA PCR and Pulsed-Field Gel Electrophoresis

    PubMed Central

    Simpson, P. J.; Stanton, C.; Fitzgerald, G. F.; Ross, R. P.

    2002-01-01

    The genomic diversity of 33 previously assigned strains from six species within the genus Pediococcus was assessed by randomly amplified polymorphic DNA (RAPD) PCR and pulsed-field-gel electrophoresis (PFGE). The RAPD PCR patterns produced by two separate random primers, termed P1 (ACGCGCCCT) and P2 (ATGTAACGCC), were compared by the Pearson correlation coefficient and the unweighted pair group method with arithmetic averages clustering algorithm. Pattern variations between repeat samples set a strain discrimination threshold of less than 70% similarity. P1 and P2 primers alone and in combination produced 14, 21, and 28 distinct patterns, respectively. When each strain was assigned with a type strain with which it shared the highest level of similarity, both primers grouped 17 of the 27 strains to their proposed species. PFGE following genomic digestion with the restriction enzymes ApaI, NotI, and AscI produced 30, 32, and 28 distinct macrorestriction patterns, respectively. Specific DNA fragments within the NotI and AscI macrorestriction patterns for each strain were observed that allowed 27 of the 33 strains to be assigned to their proposed species. For example, following digestion with AscI, all Pediococcus parvulus strains were characterized by two DNA fragments, one of approximately 220 kb and another between 700 and 800 kb. The exceptions correlated with those observed with both RAPD PCR primers and included three P. damnosus and two P. pentosaceus strains that grew at temperatures regarded as nonpermissive for their proposed species but not for those with which they grouped. PMID:11823217

  6. Diversity and Seasonal Variability of ?-Proteobacteria in Biofilms of Polluted Rivers: Analysis by Temperature Gradient Gel Electrophoresis and Cloning

    PubMed Central

    Brmmer, I. H. M.; Felske, A.; Wagner-Dbler, I.

    2003-01-01

    The ?-subgroup of the Proteobacteria has been shown to be important in aquatic habitats and was investigated in depth here by molecular 16S rRNA techniques in biofilms of the Elbe River and its polluted tributary, the Spittelwasser River. The bacterial 16S rRNA genes were cloned from each site, screened for ?-proteobacterial clones and sequenced. River biofilm clones from both rivers grouped into 9 clusters (RBFs). RBFs 1, 2, and 3 fell into the recently described ?I cluster of cosmopolitan freshwater bacteria, where they represented new species related to Rhodoferax, Aquaspirillum, and Hydrogenophaga. RBFs 4 to 7 affiliated with Aquabacterium commune, Ideonella dechloratans, and Sphaerotilus natans, respectively. The two remaining RBFs were uncultivated clusters, one of them being distantly related to Gallionella ferruginea. Seasonal changes in the relative intensity of the ?-proteobacterial 16S rRNA genes of biofilms harvested monthly for 1 year were determined by specific amplification and separation by temperature gradient gel electrophoresis (TGGE). Bands were identified by comparison of clones to community fingerprints by TGGE. Eight of 13 identified bands were shared by both habitats but showed different relative abundance and seasonal variability in the two rivers, probably caused by differences in temperature and pollutants. The data indicate new not-yet-cultivated clusters of river biofilm organisms, some of them probably distributed globally. They confirm the importance of certain known freshwater genera in river biofilms. The high phylogenetic resolution obtained by clone library analysis combined with the high temporal resolution obtained by TGGE suggest that the observed microdiversity in the river biofilm clone libraries might be caused by phylogenetically closely related microbial populations which are adapted to ecological parameters. PMID:12902230

  7. Single-cell pulsed-field gel electrophoresis to detect the early stage of DNA fragmentation in human sperm nuclei.

    PubMed

    Kaneko, Satoru; Yoshida, Joji; Ishikawa, Hiromichi; Takamatsu, Kiyoshi

    2012-01-01

    Single-cell pulsed-field gel electrophoresis (SCPFGE) with dual electrode pairs was developed to detect the early stage of DNA fragmentation in human sperm. The motile sperm were purified by the commonly used density-gradient centrifugation technique and subsequent swim-up. The sperm were embedded in a thin film of agarose containing bovine trypsin (20 g/mL) and were then lysed. Prior to SCPFGE, proteolysis of DNA-binding components, such as protamine and the nuclear matrix was essential to separate the long chain fibers from the fibrous and granular fragments derived from a single nucleus. The overall electrophoretic profiles elucidated the course of DNA fragmentation. A few large fibrous fragments were observed at the beginning of the process, however, as the fragmentation advanced, the long chain fibers decreased and shortened, and, conversely, the granular fragments increased until finally almost all the DNA was shredded. Although the ejaculate contained sperm with heterogeneous stages, the purified motile sperm exhibited several dozens of uniformly elongated fibers arising from the tangled DNA at the origin, whereas a part of these fibers gave rise to fibrous fragments beyond the tip of the elongated fibers, and their numbers and sizes varied among the sperm. Conventional intra-cytoplasmic sperm injection (ICSI) usually depends on intra-operative light microscopic observation to select a sperm for injection. The present results revealed that sperm motility could not give full assurance of DNA integrity. SCPFGE is likely to serve an important role in the preoperative differential diagnosis to determine the competence of the sperm population provided for injection. PMID:22848752

  8. Directions for in-gel tryptic digestions of coomassie-stained 1D Bands and 2D Spots. NOTE: Although nearly any SDS-PAGE system can be utilized upstream of an LC-MS analysis, the DPCF

    E-print Network

    Richardson, David

    Directions for in-gel tryptic digestions of coomassie-stained 1D Bands and 2D Spots. NOTE: Although nearly any SDS-PAGE system can be utilized upstream of an LC-MS analysis, the DPCF recommends Invitrogen's NuPAGE Bis-Tris mini-gel system. A good general purpose gel covering a large MW range (6-200 k

  9. AGAROSE GEL PREPARATION AND DNA QUANTIFICATION

    E-print Network

    Gill, Kulvinder

    in the electrophoresis tank. Use 1X TBE as electrophoresis buffer, add just enough to cover the gel surface. 4. Load 5 µ are expected to come in lower yields (nano gram amounts). 6. Electrophoresis-: 20 minutes at 100 V for mini-gel migrated into the gel; long electrophoresis is not necessary. 7. After electrophoresis, bring gel to the UV

  10. Electrophoresis. Author manuscript A versatile electrophoresis system for the analysis of high-and

    E-print Network

    Paris-Sud XI, Université de

    sulfatepolyacrylamide gel electrophoresis of proteins in the relative molecular weight Mw range of 300,000-3000 Da processes. MESH Keywords Buffers ; Electrophoresis, Gel, Two-Dimensional ; methods ; Electrophoresis weight. To be analyzed with this electrophoresis system, high concentration gels are needed (example

  11. Recent advances in preparative electrophoresis

    NASA Technical Reports Server (NTRS)

    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.

  12. Two Electrophoresis Experiments for Freshmen in the Health Professions.

    ERIC Educational Resources Information Center

    Brabson, G. Dana; Waugh, David S.

    1986-01-01

    Describes procedures involved with paper electrophoresis separation of amino acids, gel electrophoresis separation of DNA, and design of an electrophoresis tank. Describes experiments using paper (amino acids) and gel (deoxyribonucleic acid fragments). Provides material lists, procedures, and discussion. (JM)

  13. Application of two-dimensional gel electrophoresis in the study of cytoskeletal protein regulation during growth activation and differentiation.

    PubMed

    Ben-Ze'ev, A

    1990-03-01

    Two-dimensional gel electrophoresis was used to study the regulation of cytoskeletal protein synthesis during growth activation and development of the differentiated phenotype. We demonstrated a correlation between the state of organization and the expression of the respective cytoskeletal protein by showing that depolymerization of microtubules leads to a rapid decrease in new tubulin synthesis. We found that the synthesis of vimentin in both fibroblasts and epithelial cells correlates with extensive cell spreading on the substrate, while cytokeratin synthesis is maximal when cell to cell contacts are abundant. The analysis of cytoskeletal elements, involved directly in the formation of cell contacts, revealed that the level of vinculin synthesis is dependent on the extent of adherent type of cell contacts formed. Moreover, we found that the transient disappearance of vinculin from adhesion plaques of quiescent fibroblasts in response to serum factors was followed by an induction of vinculin mRNA and protein synthesis. The morphological changes associated with establishment of the differentiated phenotype were also found to include changes in the expression of the cytoskeletal-extracellular matrix complex. This was demonstrated in several differentiating systems: in 3T3 preadipocytes which change their shape from a fibroblastic to a spherical shape when stimulated to differentiate with adipogenic medium, we observed a decrease in mRNA levels and in the synthesis of fibronectin, beta-integrin, and the microfilament proteins, vinculin, alpha-actinin, tropomyosin and actin. The culturing of these cells on a certain extracellular matrix prevented the morphological changes occurring in the presence of adipogenic medium and blocked the shifts in cytoskeletal- and differentiation-related gene expression. Similar changes in the organization and expression of cytoskeletal proteins were identified during maturation of primary ovarian granulosa cell cultures, stimulated with gonadotropic hormones to form highly steroidogenic cells. The cell rounding and aggregation occurring during this process were associated with a decreased synthesis of vinculin, alpha-actinin, actin and the nonmuscle tropomyosins. The physiological relevance of these changes was suggested by the observation that the level of tropomyosin mRNA was lower in follicles of animals at late stages of granulosa cell maturation when compared to earlier stages. The expression of tissue-specific and cytoskeletal proteins was also determined in primary cultures of liver hepatocytes, maintained under conditions either favorable for growth or for expression of liver-specific functions. When DNA synthesis was elevated, cytoskeletal protein synthesis was high and that of liver-specific proteins was low.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2188832

  14. Proteomic analysis of surface proteins of Trichinella spiralis muscle larvae by two-dimensional gel electrophoresis and mass spectrometry

    PubMed Central

    2013-01-01

    Background Trichinella spiralis is a zoonotic tissue-dwelling parasitic nematode that infects humans and other mammals. Its surface proteins are recognized as antigenic in many infected hosts, being directly exposed to the hosts immune system and are the main target antigens that induce the immune responses. The larval surface proteins may also interact with intestinal epithelial cells and may play an important role in the invasion and development process of T. spiralis. The purpose of this study was to analyze and characterize the surface proteins of T. spiralis muscle larvae by two-dimensional gel electrophoresis (2-DE) and mass spectrometry. Methods The surface proteins of T. spiralis muscle larvae were stripped from the cuticle of live larvae by the cetyltrimethylammonium bromide (CTAB) and sodium deoxycholate. The surface protein stripping was examined by an immunofluorescent test (IFT). The surface proteins were analyzed by SDS-PAGE and Western blotting, and then identified by 2-DE and MALDI-TOF/TOF mass spectrometry analysis. Results The IFT results showed that the surface proteins-stripped larvae were not recognized by sera of mice immunized with surface antigens. Western blotting showed 7 of 12 protein bands of the surface proteins were recognized by mouse infection sera at 18 dpi and at 42 dpi. The 2-DE results showed that a total of approximately 33 proteins spots were detected with molecular weights varying from 10 to 66kDa and isoelectric point (pI) from 4 to 7. Twenty-seven of 33 protein spots were identified and characterized to correlate with 15 different proteins. Out of the 14 proteins identified as T. spiralis proteins, 5 proteins (partial P49 antigen, deoxyribonuclease II family protein, two serine proteases, and serine proteinase) had catalytic and hydrolase activity. All of these 5 proteins were also associated with metabolic processes and 2 of the five proteins were associated with cellular processes. Conclusions In this study, T. spiralis muscle larval surface proteins have been identified, which will provide useful information to elucidate the host-parasite interaction, identify the invasion-related proteins, early diagnostic antigens and the targets for a vaccine. PMID:24330777

  15. Rapid Separation and Quantification of Major Caseins and Whey Proteins of Bovine Milk by Polyacrylamide Gel Electrophoresis

    Microsoft Academic Search

    K. F. Ng-Kwai-Hang; E. M. Kroeker

    1984-01-01

    A rapid polyacrylamide gel electro- phoretic method was developed for separating and quantifying major pro- teins in casein and whey protein fractions of bovine milk. For casein separation, best results were achieved by an 8% poly- acrylamide gel containing 4 M urea and a top layer of large pore sample gel; for whey protein the most_ satisfactory separation was with

  16. Assessment of resolution and intercenter reproducibility of results of genotyping Staphylococcus aureus by pulsed-field gel electrophoresis of SmaI macrorestriction fragments: a multicenter study

    Microsoft Academic Search

    Belkum van A. F; S. Salmenlinna; M. Kooistra; BARRY COOKSON; W. Witte; N. El Solh; F. Forey; J. Etienne; R. Goering; A. Morvan; M. Struelens; J. Vuopio-Varkila; F. C. Tenover; C. Steward; N. Legakis; A. Talens; F. O'Brien; P. Tassios; Ryck de R; W. Grubb; M. E. Kaufmann; H. A. Verbrugh; Leeuwen van W. B

    1998-01-01

    Twenty well-characterized isolates of methicillin-resistant Staphylococcus\\u000a aureus were used to study the optimal resolution and interlaboratory\\u000a reproducibility of pulsed-field gel electrophoresis (PFGE) of DNA\\u000a macrorestriction fragments. Five identical isolates (one PFGE type), 5\\u000a isolates that produced related PFGE subtypes, and 10 isolates with unique\\u000a PFGE patterns were analyzed blindly in 12 different laboratories by\\u000a in-house protocols. In several laboratories a

  17. Conservation of infectivity in purified fibrillary extracts of scrapie-infected hamster brain after sequential enzymatic digestion or polyacrylamide gel electrophoresis.

    PubMed Central

    Brown, P; Liberski, P P; Wolff, A; Gajdusek, D C

    1990-01-01

    Infectious extracts of scrapie-infected hamster brain enriched for scrapie-associated fibrils and scrapie amyloid protein (PrP) were partially denatured and subjected to either polyacrylamide gel electrophoresis with subsequent isolation of the PrP band or sequential enzymatic digestion with deglycosidase, phospholipase, proteinase, and several different nucleases. Infectivity measurements of these various specimens revealed a convincing association between infectivity and scrapie amyloid protein, with or without its sugar chains and disulfide bonds, and did not support the hypothesis that nucleic acid is involved in replication. Images PMID:2119503

  18. Proteins pattern alteration in AZT-treated K562 cells detected by two-dimensional gel electrophoresis and peptide mass fingerprinting

    PubMed Central

    D'Andrea, Gabriele; Lizzi, Anna R; Venditti, Sara; Di Francesco, Laura; Giorgi, Alessandra; Mignogna, Giuseppina; Oratore, Arduino; Bozzi, Argante

    2006-01-01

    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. PMID:16571109

  19. Copper(II)-Alizarin Red S complex as an efficient chemiluminescent probe for the detection of human serum proteins after polyacrylamide gel electrophoresis.

    PubMed

    Wang, Zhenzhen; Liu, Xia; Baeyens, Willy R G; Delanghe, Joris R; Ouyang, Jin

    2008-12-01

    A novel chemiluminescent probe, copper(II)-Alizarin Red S (ARS) complex, for the detection of human serum proteins after polyacrylamide gel electrophoresis (PAGE) is described. The detection is based on the binding of the copper(II)-ARS complex to proteins and the catalytic activity of copper(II) in the luminol-hydrogen peroxide chemiluminescence (CL) system. Various proteins are directly detected in polyacrylamide gels, avoiding tedious transferring procedures. In the present study, the possible reaction mechanism and sensitivity evaluation are analyzed. The experimental conditions such as solution concentration, complex ratio, and washing reagents are likewise optimized. The proposed method offers simple, fast, and sensitive detection of serum proteins. As a novel chemiluminescent detection method, it shows significant analytical potential in biochemistry. PMID:19367697

  20. Isolation and characterization by immunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western blot, restriction fragment length polymorphism-PCR, 16S rRNA gene sequencing, and pulsed-field gel electrophoresis of Rochalimaea quintana from a patient with bacillary angiomatosis.

    PubMed Central

    Maurin, M; Roux, V; Stein, A; Ferrier, F; Viraben, R; Raoult, D

    1994-01-01

    Rochalimaea quintana was isolated from the blood of a French human immunodeficiency virus-infected patient with bacillary angiomatosis. The isolate showed the typical growth characteristics of Rochalimaea species and was inert when typical biochemical testing was used. The purpose of the present work was to characterize and compare this new isolate with reference strains of R. quintana, Rochalimaea vinsonii, and Rochalimaea henselae by using immunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot (immunoblot), restriction fragment length polymorphism-PCR of the citrate synthase gene, 16S rRNA gene sequencing, and pulsed-field gel electrophoresis. SDS-PAGE, Western blot, restriction fragment length polymorphism-PCR with TaqI enzyme, and 16S rRNA gene sequencing could differentiate the three Rochalimaea species and allowed characterization of the French isolate as R. quintana. However, identification of the Rochalimaea isolate to the species level was more easily obtained by immunofluorescence with specific murine antisera. Pulsed-field gel electrophoresis allowed differentiation of the French R. quintana isolate from R. quintana Fuller and may serve as an epidemiological tool. Images PMID:7519628

  1. Differential Single Nucleotide Polymorphism-Based Analysis of an Outbreak Caused by Salmonella enterica Serovar Manhattan Reveals Epidemiological Details Missed by Standard Pulsed-Field Gel Electrophoresis.

    PubMed

    Scaltriti, Erika; Sassera, Davide; Comandatore, Francesco; Morganti, Marina; Mandalari, Carmen; Gaiarsa, Stefano; Bandi, Claudio; Zehender, Gianguglielmo; Bolzoni, Luca; Casadei, Gabriele; Pongolini, Stefano

    2015-04-01

    We retrospectively analyzed a rare Salmonella enterica serovar Manhattan outbreak that occurred in Italy in 2009 to evaluate the potential of new genomic tools based on differential single nucleotide polymorphism (SNP) analysis in comparison with the gold standard genotyping method, pulsed-field gel electrophoresis. A total of 39 isolates were analyzed from patients (n = 15) and food, feed, animal, and environmental sources (n = 24), resulting in five different pulsed-field gel electrophoresis (PFGE) profiles. Isolates epidemiologically related to the outbreak clustered within the same pulsotype, SXB_BS.0003, without any further differentiation. Thirty-three isolates were considered for genomic analysis based on different sets of SNPs, core, synonymous, nonsynonymous, as well as SNPs in different codon positions, by Bayesian and maximum likelihood algorithms. Trees generated from core and nonsynonymous SNPs, as well as SNPs at the second and first plus second codon positions detailed four distinct groups of isolates within the outbreak pulsotype, discriminating outbreak-related isolates of human and food origins. Conversely, the trees derived from synonymous and third-codon-position SNPs clustered food and human isolates together, indicating that all outbreak-related isolates constituted a single clone, which was in line with the epidemiological evidence. Further experiments are in place to extend this approach within our regional enteropathogen surveillance system. PMID:25653407

  2. Introducing basic molecular biology to Turkish rural and urban primary school children via hands-on PCR and gel electrophoresis activities.

    PubMed

    Selli, Cigdem; Y?ld?r?m, Gokce; Kaymak, Aysegul; Karacicek, Bilge; Ogut, Deniz; Gungor, Turkan; Erem, Erdem; Ege, Mehmet; Bmen, Nilay; Tosun, Metiner

    2014-01-01

    This study includes the results of a 2-day education project titled "Molecular Biology Laboratory Summer School, MoBiLYO." The project was held at a University Research Center by scientists from Department of Pharmacology and graduate students. The project was composed of introductory lectures, model construction, DNA isolation, polymerase chain reaction (PCR), and gel electrophoresis. The participants were 13-year-old eighth-graders attending primary schools affiliated with Ministry of National Education in urban and rural areas of Izmir, Turkey. The purpose of this study was to introduce basic molecular biology concepts through individually performed experiments such as PCR and gel electrophoresis integrated with creative drama. The students were assessed at the beginning and the end of each project day via mini-tests, experimental and presentation skills evaluation forms. Data showed that students' knowledge about DNA structure and basic molecular biology techniques significantly increased. On the basis of experimental and presentational skills, there was no significant difference between kids from urban and rural schools or between public and boarding public schools, whereas the average score of girls was significantly higher than that of boys. In conclusion, individually performed experiments integrated with creative drama significantly increased students' perception of complex experimental procedures on basic molecular biology concepts. Data suggests that integration of these concepts into the science and technology curriculum of Turkish primary education may support the recruitment of future scientists who can handle rapidly developing genomic techniques that will affect our everyday life. PMID:24474053

  3. Tracing outbreaks of Streptococcus equi infection (strangles) in horses using sequence variation in the seM gene and pulsed-field gel electrophoresis.

    PubMed

    Lindahl, Susanne; Sderlund, Robert; Frosth, Sara; Pringle, John; Bverud, Viveca; Aspn, Anna

    2011-11-21

    Strangles is a serious respiratory disease in horses caused by Streptococcus equi subspecies equi (S. equi). Transmission of the disease occurs by direct contact with an infected horse or contaminated equipment. Genetically, S. equi strains are highly homogenous and differentiation of strains has proven difficult. However, the S. equi M-protein SeM contains a variable N-terminal region and has been proposed as a target gene to distinguish between different strains of S. equi and determine the source of an outbreak. In this study, strains of S. equi (n=60) from 32 strangles outbreaks in Sweden during 1998-2003 and 2008-2009 were genetically characterized by sequencing the SeM protein gene (seM), and by pulsed-field gel electrophoresis (PFGE). Swedish strains belonged to 10 different seM types, of which five have not previously been described. Most were identical or highly similar to allele types from strangles outbreaks in the UK. Outbreaks in 2008/2009 sharing the same seM type were associated by geographic location and/or type of usage of the horses (racing stables). Sequencing of the seM gene generally agreed with pulsed-field gel electrophoresis profiles. Our data suggest that seM sequencing as a epidemiological tool is supported by the agreement between seM and PFGE and that sequencing of the SeM protein gene is more sensitive than PFGE in discriminating strains of S. equi. PMID:21511406

  4. A Bayesian filtering approach to incorporate 2D/3D time-lapse confocal images for tracking angiogenic sprouting cells interacting with the gel matrix.

    PubMed

    Ong, Lee-Ling S; Dauwels, Justin; Ang, Marcelo H; Asada, H Harry

    2014-01-01

    We present a new approach to incorporating information from heterogeneous images of migrating cells in 3D gel. We study 3D angiogenic sprouting, where cells burrow into the gel matrix, communicate with other cells and create vascular networks. We combine time-lapse fluorescent images of stained cell nuclei and transmitted light images of the background gel to track cell trajectories. The nuclei images are sampled less frequently due to photo toxicity. Hence, 3D cell tracking can be performed more reliably when 2D sprout profiles, extracted from gel matrix images, are effectively incorporated. We employ a Bayesian filtering approach to optimally combine the two heterogeneous images with different sampling rates. We construct stochastic models to predict cell locations and sprout profiles and condition the likelihood of nuclei location by the sprout profile. The conditional distribution is non-Gaussian and the cell dynamics is non-linear. To jointly update cell and sprout estimates, we use a Rao-Blackwell particle filter. Simulation and experimental results show accurate tracking of multiple cells along with sprout formation, demonstrating synergistic effects of incorporating the two types of images. PMID:24239653

  5. Spot quantification in two dimensional gel electrophoresis image analysis: comparison of different approaches and presentation of a novel compound fitting algorithm

    PubMed Central

    2014-01-01

    Background Various computer-based methods exist for the detection and quantification of protein spots in two dimensional gel electrophoresis images. Area-based methods are commonly used for spot quantification: an area is assigned to each spot and the sum of the pixel intensities in that area, the so-called volume, is used a measure for spot signal. Other methods use the optical density, i.e. the intensity of the most intense pixel of a spot, or calculate the volume from the parameters of a fitted function. Results In this study we compare the performance of different spot quantification methods using synthetic and real data. We propose a ready-to-use algorithm for spot detection and quantification that uses fitting of two dimensional Gaussian function curves for the extraction of data from two dimensional gel electrophoresis (2-DE) images. The algorithm implements fitting using logical compounds and is computationally efficient. The applicability of the compound fitting algorithm was evaluated for various simulated data and compared with other quantification approaches. We provide evidence that even if an incorrect bell-shaped function is used, the fitting method is superior to other approaches, especially when spots overlap. Finally, we validated the method with experimental data of urea-based 2-DE of A? peptides andre-analyzed published data sets. Our methods showed higher precision and accuracy than other approaches when applied to exposure time series and standard gels. Conclusion Compound fitting as a quantification method for 2-DE spots shows several advantages over other approaches and could be combined with various spot detection methods. The algorithm was scripted in MATLAB (Mathworks) and is available as a supplemental file. PMID:24915860

  6. A coupling system of capillary gel electrophoresis with inductively coupled plasma-mass spectrometry for the determination of double stranded DNA fragments.

    PubMed

    Fujii, Shin-ichiro; Inagaki, Kazumi; Miyashita, Shin-ichi; Nagasawa, Keisuke; Chiba, Koichi; Takatsu, Akiko

    2013-05-01

    The coupling system of capillary gel electrophoresis (CGE) and inductively coupled plasma-mass spectrometry (ICP-MS) was newly developed and successfully applied to the double-stranded (ds) DNA quantification. The developed system combines the separation technique for large biomolecules and element selective detection of ICP-MS. This coupling was achieved by using the modified high performance concentric nebulizer (HPCN) with the PTFE tube (HPCN-PT), which can produce the liquid jet by the flow focusing effect. The HPCN-PT effectively nebulizes the highly viscous solution containing gel buffer even at a low flow rate. At a liquid flow rate of 0.010 mL min(-1) and a nebulizer gas flow rate of 1 L min(-1), the Sauter mean diameter (D3,2) of primary aerosols generated by the HPCN-PT was 3.4 ?m, and over 90% (v/v) of the aerosol droplets were less than 10 ?m in diameter. The electrophoresis capillary filled with gel buffer was connected to the HPCN-PT via the interface. This interface has two connectors and an electrode that can connect CE and ICP-MS. After the electrophoretic separation at atmospheric pressure, the samples were transferred to the ICP-MS through the interface by applying additional pressure. Fragments of dsDNA, which were commercially available as a ladder marker solution, were successfully separated and analyzed by measuring (31)P(+) with CGE-ICP-MS, and a linear calibration curve of the phosphorus standard solution (R(2) = 0.999) was obtained from 2.7 to 27 mg kg(-1). The detection limit (LOD) and absolute detection limit of P were 3.7 ?g kg(-1) and 0.6 pg (equivalent to 6 pg of DNA), respectively. This absolute detection limit value was equal to the conventional fluorescence determination of DNA. PMID:23604270

  7. A novel (18)O inverse labeling-based workflow for accurate bottom-up mass spectrometry quantification of proteins separated by gel electrophoresis.

    PubMed

    Santos, Hugo M; Glez-Pea, Daniel; Reboiro-Jato, Miguel; Fdez-Riverola, Florentino; Diniz, Mrio S; Lodeiro, Carlos; Capelo-Martnez, Jos-Luis

    2010-10-01

    In the present work we report on a novel and fast protocol for accurate bottom-up protein quantification that overcomes the drawbacks of in-gel digestion and MALDI analysis, while maintaining their benefits. It relies on the following steps: (i) gel electrophoresis separation of proteins, (ii) fast in-gel protein digestion with trypsin, (iii) (18)O-labeling through the decoupled method, (iv) quantification through selected peptides previously chosen using the (18)O inverse labeling approach and that, finally, (v) it takes advantage of software specifically developed to select the peptides that will drive the quantification of the protein in an automated mode. We have accurately quantified the following six proteins: glycogen phosphorylase, BSA, ovalbumin, carbonic anhydrase, trypsin inhibitor, and ?-lactalbumin. As a case study we have quantified the protein vitellogenin in plasma of Cyprinus carpio exposed to high levels of estrogens. The proposed new protocol was validated against the traditional ELISA method; both were found to provide comparable results (non-parametric Mann-Whitney U-test). PMID:20882554

  8. A Prototype Two-Dimensional Capillary Electrophoresis System Fabricated in

    E-print Network

    Prentiss, Mara

    gel electrophoresis in a capillary format is presented. In this method, separation in the first electrophoresis.6,7 One-dimensional (1D) gel electrophoresis (in the form of SDS-PAGE) is still used for separations of proteins, but 1D gels are increasingly being replaced by capillary electrophoresis.8

  9. Dosimetric verification for intensity-modulated arc therapy plans by use of 2D diode array, radiochromic film and radiosensitive polymer gel

    PubMed Central

    Hayashi, Naoki; Malmin, Ryan L.; Watanabe, Yoichi

    2014-01-01

    Several tools are used for the dosimetric verification of intensity-modulated arc therapy (IMAT) treatment delivery. However, limited information is available for composite on-line evaluation of these tools. The purpose of this study was to evaluate the dosimetric verification of IMAT treatment plans using a 2D diode array detector (2D array), radiochromic film (RCF) and radiosensitive polymer gel dosimeter (RPGD). The specific verification plans were created for IMAT for two prostate cancer patients by use of the clinical treatment plans. Accordingly, the IMAT deliveries were performed with the 2D array on a gantry-mounting device, RCF in a cylindrical acrylic phantom, and the RPGD in two cylindrical phantoms. After the irradiation, the planar dose distributions from the 2D array and the RCFs, and the 3D dose distributions from the RPGD measurements were compared with the calculated dose distributions using the gamma analysis method (3% dose difference and 3-mm distance-to-agreement criterion), dose-dependent dose difference diagrams, dose difference histograms, and isodose distributions. The gamma passing rates of 2D array, RCFs and RPGD for one patient were 99.5%, 96.5% and 93.7%, respectively; the corresponding values for the second patient were 97.5%, 92.6% and 92.9%. Mean percentage differences between the RPGD measured and calculated doses in 3D volumes containing PTVs were 0.29 7.1% and 0.97 7.6% for the two patients, respectively. In conclusion, IMAT prostate plans can be delivered with high accuracy, although the 3D measurements indicated less satisfactory agreement with the treatment plans, mainly due to the dosimetric inaccuracy in low-dose regions of the RPGD measurements. PMID:24449714

  10. Microscale 2D separation systems for proteomic analysis

    PubMed Central

    Xu, Xin; Liu, Ke; Fan, Z. Hugh

    2012-01-01

    Microscale 2D separation systems have been implemented in capillaries and microfabricated channels. They offer advantages of faster analysis, higher separation efficiency and less sample consumption than the conventional methods, such as liquid chromatography (LC) in a column and slab gel electrophoresis. In this article, we review their recent advancement, focusing on three types of platforms, including 2D capillary electrophoresis (CE), CE coupling with capillary LC, and microfluidic devices. A variety of CE and LC modes have been employed to construct 2D separation systems via sophistically designed interfaces. Coupling of different separation modes has also been realized in a number of microfluidic devices. These separation systems have been applied for the proteomic analysis of various biological samples, ranging from a single cell to tumor tissues. PMID:22462786

  11. Use of pulsed-field gel electrophoresis for detecting differences in Staphylococcus aureus strain populations between dairy herds with different cattle importation practices.

    PubMed Central

    Middleton, J. R.; Fox, L. K.; Gay, J. M.; Tyler, J. W.; Besser, T. E.

    2002-01-01

    The hypothesis tested was that dairy herds which import cattle for replacement or expansion have a higher prevalence of Staphylococcus aureus mastitis and a greater number of new Staphylococcus aureus strains enter their herds than closed herds. Fifteen commercial dairy herds were divided into four groups based on cattle importation practices. Composite foremilk samples were collected at 4-monthly intervals for 1 year from all lactating cattle. Additionally, foremilk samples were collected from cattle at parturition and skin swabs were taken from the udder of primiparous heifers. All samples were cultured for Staphylococcus aureus and isolates were strain-typed using pulsed-field gel electrophoresis. Herds that purchased replacement heifers had a higher prevalence of Staphylococcus aureus mastitis than herds that purchased lactating cattle for expansion (P = 0.02). Herds that purchased replacement heifers had more total strains of Staphylococcus aureus (P = 0.01) and more new strains (P = 0.04) enter the herd than closed herds. PMID:12403115

  12. Application of denaturing gradient gel electrophoresis for detection of bacterial and yeast communities along a salinity gradient in the estuary of the Cachoeira River in Brazil.

    PubMed

    Rodrigues, C S P; Souza, S S; Rezende, R P; Silva, A; Andrioli, J L; Costa, H; Fontana, R; Dias, J C T

    2013-01-01

    An estuary is a transition zone between freshwater and marine ecosystems, resulting in dilution of seawater. Estuaries are also considered environments of intense biological activity related to the processes of nutrient cycling. The aim of this study was to evaluate the microbial community composition along a salinity gradient in the estuary of the Cachoeira River, located in southern Bahia, Brazil. The analysis of bacterial and yeast communities was performed by determining the denaturing gradient gel electrophoresis band richness. Formation of zones with similar profiles of bands was observed, and the increasing richness at the intermediate zone demonstrated a clear spatial distinction of communities depending on salinity. In addition, the dissolved oxygen content, temperature, pH, salinity, and dissolved inorganic nutrient contents (NH3(+), NO2(-), NO3(-), PO4(-)) were determined. Nutrients were distributed in similar patterns, with decreasing concentrations as the salinity increases. PMID:23765981

  13. Study of yeast mitochondrial tRNAs by two-dimensional polyacrylamide gel electrophoresis: characterization of isoaccepting species and search for imported cytoplasmic tRNAs.

    PubMed Central

    Martin, R P; Schneller, J M; Stahl, A J; Dirheimer, G

    1977-01-01

    By two-dimensional polyacrylamide gel electrophoresis, yeast mitochondrial tRNA is fractionated into 27 major species. All but 6 of them migrate distinctly from cytoplasmic tRNAs. Migration of mitochondrial DNA-coded mitochondrial tRNAs shows the occurence of only one cytoplasmic tRNA in mitochondria. Several mitochondrial tRNA spots are identified on the electrophoregrams, some of them show isoaccepting species (Val, Ser, Met, Leu). It is suggested that there are sufficient mitochondrial tRNA genes on yeast mitochondrial DNA to allow mitochondrial protein biosynthesis by the mitochondrial tRNAs alone. Guanosine + Cytidine content and rate base composition are reported for some individual species. Mitochondrial tRNAPhe lacks Ribothymidine. Images PMID:337238

  14. Multilocus sequence typing and pulsed-field gel electrophoresis analysis of Oenococcus oeni from different wine-producing regions of China.

    PubMed

    Wang, Tao; Li, Hua; Wang, Hua; Su, Jing

    2015-04-16

    The present study established a typing method with NotI-based pulsed-field gel electrophoresis (PFGE) and stress response gene schemed multilocus sequence typing (MLST) for 55 Oenococcus oeni strains isolated from six individual regions in China and two model strains PSU-1 (CP000411) and ATCC BAA-1163 (AAUV00000000). Seven stress response genes, cfa, clpL, clpP, ctsR, mleA, mleP and omrA, were selected for MLST testing, and positive selective pressure was detected for these genes. Furthermore, both methods separated the strains into two clusters. The PFGE clusters are correlated with the region, whereas the sequence types (STs) formed by the MLST confirm the two clusters identified by PFGE. In addition, the population structure was a mixture of evolutionary pathways, and the strains exhibited both clonal and panmictic characteristics. PMID:25625911

  15. The use of pulsed-field gel electrophoresis to examine the epidemiology of Bordetella bronchiseptica isolated from cats and other species.

    PubMed Central

    Binns, S. H.; Speakman, A. J.; Dawson, S.; Bennett, M.; Gaskell, R. M.; Hart, C. A.

    1998-01-01

    A collection (164) of isolates of Bordetella bronchiseptica made predominantly from cats (132) but also from dogs (15), pigs (12) and other species was examined by pulsed field gel electrophoresis following macrorestriction digestion with XbaI. Each isolate was analysed twice and the patterns were entirely reproducible. The isolates fell into 17 different strains (> 3 bands different) and within strains there were numerous subtypes. Feline isolates fell into 12 of the 17 strains. In general, cats housed together had similar or identical strains and subtypes of B. bronchiseptica. There was no difference in the PFGE patterns of isolates made from carrier cats and those from cats with respiratory disease. Isolates from pigs and dogs were in general similar to the feline isolates and there was no great evidence for species specificity. The PFGE pattern of feline and canine isolates were more related to whether the animals were housed together rather than whether they came from dogs or cats. PMID:9593491

  16. Antibody response to epitopes of chlamydial major outer membrane proteins on infectious elementary bodies and of the reduced polyacrylamide gel electrophoresis-separated form.

    PubMed Central

    Baghian, A; Shaffer, L; Storz, J

    1990-01-01

    Approximately 60% of the outer membrane of chlamydial elementary bodies (EBs) consists of the major outer membrane protein (MOMP) that has structural and metabolic functions. The antigenic properties of MOMPs from mammalian strains of serovars 1 and 2 and an avian strain of Chlamydia psittaci were analyzed. Polyclonal-monospecific antisera (PMAs), one monoclonal antibody (MAb), and polyclonal antisera (PAs) were produced against reduced polyacrylamide gel electrophoresis-separated MOMPs and against infectious EBs. Three PMAs and the MAb, which were induced by reduced polyacrylamide gel electrophoresis-separated MOMPs, reacted strongly in Western blot (immunoblot) assays with MOMPs of serovar 1 and 2 strains as well as with that of the avian strain 6BC, and two of these PMAs reacted weakly (dilution, 1:20) with the MOMP of strain LGV-2. The third PMA and the MAb against the MOMP of the serovar 2 strain did not react with the MOMP of LGV-2. Four PAs were produced against infectious EBs of the serovar 1 strain. One of these PAs reacted with the homologous MOMP and that of the avian strain 6BC but did not recognize MOMPs of other chlamydial strains. Three of the PAs reacted with MOMPs of homologous strains only and failed to recognize MOMPs of avian, serovar 2, and LGV-2 strains. Five PAs induced against infectious EBs of the serovar strain 2 reacted only with the MOMPs of the homologous strains and failed to recognize MOMPs of other strains of chlamydiae. Consequently, MOMPs of C. psittaci strains possess genus-, species-, and serovar-specific epitopes whereby the immune response to serovar-specific epitopes of MOMP predominate when infectious EBs are used for immunization. Images PMID:1691145

  17. Development of a low-cost, high-throughput native polyacrylamide gel electrophoresis (N-PAGE) protocol for lipoprotein sub-fractionation using Quality by Design approach.

    PubMed

    Pathak, Mili; Chaudhary, Neha; Rathore, Anurag S

    2014-04-01

    Ratio of low density to high density lipoprotein concentration is critical for normal functioning of human body. Deviation in this ratio has been linked to various diseases, many of which are fatal if not diagnosed at early stages. For example, cardiovascular diseases (CVD) have been linked to the level of low density lipoprotein (LDL). Henceforth, detection of the lipoprotein subtractions is crucial for health of an individual. To date, methods like ultracentrifugation, nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC) and gradient gel electrophoresis (GGE) have been used for separation and identification of lipoprotein types and subtypes. However, these methods are expensive, time consuming and require specialized equipments and expertise. This paper aims to propose a low-cost, high-throughput native polyacrylamide gel electrophoresis (N-PAGE) based protocol for analysis of lipoproteins. Quality by Design (QbD) based approach has been utilized. The initial screening of parameters was followed by a fractional factorial design to optimize the protocol. The lipoprotein subtractions obtained by the optimized protocol were compared with the commercially available and commonly used Lipoprint() Lipoprotein Subfractions Testing System from Quantimetrix. The proposed method gave comparable results to those obtained with the commercial system. The proposed method is capable of analysis of up to forty different samples in two hours at a cost of approximately 2$/sample. This is an order of magnitude better than the present cost of 265$/sample when using the commercial system. We think that the proposed method would be of particular interest to the developing and under-developed economies of the world, where this cost differential would be deemed quite significant and would make testing affordable to the majority of the population. PMID:24518131

  18. Polyclonal infections due to Mycobacterium avium complex in patients with AIDS detected by pulsed-field gel electrophoresis of sequential clinical isolates.

    PubMed Central

    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. Comparison of restriction enzyme analysis versus pulsed-field gradient gel electrophoresis as a typing system for Torulopsis glabrata and Candida species other than C. albicans.

    PubMed Central

    Vazquez, J A; Beckley, A; Donabedian, S; Sobel, J D; Zervos, M J

    1993-01-01

    Candida species have recently emerged as important nosocomial pathogens. Because of the lack of a reliable system for detecting differences within the same species, little is known about the epidemiology of infection with Candida species. We describe a typing system for Torulopsis glabrata and the non-C. albicans Candida species that uses contour-clamped homogeneous electric field electrophoresis (CHEF), a version of pulsed-field gradient gel electrophoresis, and compared it with restriction enzyme analysis (REA) of genomic DNA. One hundred seventeen clinical isolates from 40 patients were evaluated. CHEF and REA were performed on each of the isolates, and the results of the two procedures were compared. The REA procedure revealed 8 different types of Candida lusitaniae, 20 of Torulopsis glabrata, 5 of Candida tropicalis, 3 of Candida parapsilosis, and 7 of Candida kefyr, whereas the CHEF method revealed 14 different types of C. lusitaniae, 16 of T. glabrata, 10 of C. tropicalis, 10 of C. parapsilosis, and 7 of C. kefyr. The CHEF technique yielded unique patterns of electrophoretic karyotypes that could be used to distinguish intraspecies variations. When compared with REA, CHEF demonstrated greater sensitivity in recognizing subtle strain-to-strain variations in most isolates and will be a useful epidemiologic tool for studying non-C. albicans Candida species and T. glabrata. Images PMID:8396585

  20. Denaturing Gradient Gel Electrophoresis Analysis of the 16S rRNA Gene V1 Region To Monitor Dynamic Changes in the Bacterial Population during Fermentation of Italian Sausages

    Microsoft Academic Search

    LUCA COCOLIN; MARISA MANZANO; CARLO CANTONI; GIUSEPPE COMI

    2001-01-01

    In this study, a PCR-denaturing gradient gel electrophoresis (DGGE) protocol was used to monitor the dynamic changes in the microbial population during ripening of natural fermented sausages. The method was first optimized by using control strains from international collections, and a natural sausage fermentation was studied by PCR-DGGE and traditional methods. Total microbial DNA and RNA were extracted directly from

  1. Two potential clinical applications of the alkaline single-cell gel electrophoresis assay: (1) human bladder washings and transitional cell carcinoma of the bladder; and (2) human sperm and male infertility

    Microsoft Academic Search

    V. J McKelvey-Martin; N Melia; I. K Walsh; S. R Johnston; C. M Hughes; S. E. M Lewis; W Thompson

    1997-01-01

    Part 1: The alkaline single-cell gel electrophoresis (comet) assay was used to analyse the integrity and DNA content of exfoliated cells extracted from bladder washing specimens from 9 transitional cell carcinoma patients and 15 control patients. DNA damage, as expressed by % tail DNA and tail moment values, was observed to occur in cells from both control and bladder cancer

  2. Comparative evaluation of the in vitro micronucleus test and the alkaline single cell gel electrophoresis assay for the detection of DNA damaging agents: genotoxic effects of cobalt powder, tungsten carbide and cobalttungsten carbide

    Microsoft Academic Search

    Freddy Van Goethem; Dominique Lison; Micheline Kirsch-Volders

    1997-01-01

    Although it is well known that micronuclei may arise from either DNA breakage leading to acentric chromosome fragments or from chromosome\\/chromatid lagging in anaphase, the ratio between the amount of DNA breakage induced and the frequency of micronuclei expressed in the following interphase is unclear. With the development of the alkaline single cell gel electrophoresis assay, which measures single strand

  3. Protein composition of wheat gluten polymer fractions determined by quantitative two-dimensional gel electrophoresis and tandem mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  4. Application of a label-free, gel-free quantitative proteomics method for ecotoxicological studies of small fish species

    EPA Science Inventory

    Although two-dimensional electrophoresis (2D-GE) remains the basis for many ecotoxicoproteomic analyses, new, non gel-based methods are beginning to be applied to overcome throughput and coverage limitations of 2D-GE. The overall objective of our research was to apply a comprehe...

  5. Exposures of Sus scrofa to a TASER() conducted electrical weapon: no effects on 2-dimensional gel electrophoresis patterns of plasma proteins.

    PubMed

    Jauchem, James R; Cerna, Cesario Z; Lim, Tiffany Y; Seaman, Ronald L

    2014-12-01

    In an earlier study, we found significant changes in red-blood-cell, leukocyte, and platelet counts, and in red-blood-cell membrane proteins, following exposures of anesthetized pigs to a conducted electrical weapon. In the current study, we examined potential changes in plasma proteins [analyzed via two-dimensional gel electrophoresis (2-DGE)] following two 30s exposures of anesthetized pigs (Sus scrofa) to a TASER () C2 conducted electrical weapon. Patterns of proteins, separated by 2-DGE, were consistent and reproducible between animals and between times of sampling. We determined that the blood plasma collection, handling, storage, and processing techniques we used are suitable for swine blood. There were no statistically significant changes in plasma proteins following the conducted-electrical-weapon exposures. Overall gel patterns of fibrinogen were similar to results of other studies of both pigs and humans (in control settings, not exposed to conducted electrical weapons). The lack of significant changes in plasma proteins may be added to the body of evidence regarding relative safety of TASER C2 device exposures. PMID:25319243

  6. Bacterial community structure in Apis florea larvae analyzed by denaturing gradient gel electrophoresis and 16S rRNA gene sequencing.

    PubMed

    Saraithong, Prakaimuk; Li, Yihong; Saenphet, Kanokporn; Chen, Zhou; Chantawannakul, Panuwan

    2014-11-13

    This study characterizes the colonization and composition of bacterial flora in dwarf Asian honeybee (Apis florea) larvae and compares bacterial diversity and distribution among different sampling locations. A. florea larvae were collected from 3 locations in Chiang Mai province, Thailand. Bacterial DNA was extracted from each larva using the phenol-chloroform method. Denaturing gradient gel electrophoresis was performed, and the dominant bands were excised from the gels, cloned, and sequenced for bacterial species identification. The result revealed similarities of bacterial community profiles in each individual colony, but differences between colonies from the same and different locations. A. florea larvae harbor bacteria belonging to 2 phyla (Firmicutes and Proteobacteria), 5 classes (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacilli, and Clostridia), 6 genera (Clostridium, Gilliamella, Melissococcus, Lactobacillus, Saccharibacter, and Snodgrassella), and an unknown genus from uncultured bacterial species. The classes with the highest abundance of bacteria were Alphaproteobacteria (34%), Bacilli (25%), Betaproteobacteria (11%), Gammaproteobacteria (10%), and Clostridia (8%), respectively. Similarly, uncultured bacterial species were identified (12%). Environmental bacterial species, such as Saccharibacter floricola, were also found. This is the first study in which sequences closely related to Melissococcus plutonius, the causal pathogen responsible for European foulbrood, have been identified in Thai A. florea larvae. PMID:25393530

  7. Capillary Electrophoresis for the Analysis of Biopolymers

    E-print Network

    Krylov, Sergey

    becoming available for some parts of proteomic research, but manually intensive slab-gel electrophoresis instruments will displace cumbersome slab-gel electrophoresis for protein analysis. We also believeCapillary Electrophoresis for the Analysis of Biopolymers Sergey N. Krylov and Norman J. Dovichi

  8. Identification of increased amounts of eppin protein complex components in sperm cells of diabetic and obese individuals by difference gel electrophoresis.

    PubMed

    Paasch, Uwe; Heidenreich, Falk; Pursche, Theresia; Kuhlisch, Eberhard; Kettner, Karina; Grunewald, Sonja; Kratzsch, Jrgen; Dittmar, Gunnar; Glander, Hans-Jrgen; Hoflack, Bernard; Kriegel, Thomas M

    2011-08-01

    Metabolic disorders like diabetes mellitus and obesity may compromise the fertility of men and women. To unveil disease-associated proteomic changes potentially affecting male fertility, the proteomes of sperm cells from type-1 diabetic, type-2 diabetic, non-diabetic obese and clinically healthy individuals were comparatively analyzed by difference gel electrophoresis. The adaptation of a general protein extraction procedure to the solubilization of proteins from sperm cells allowed for the resolution of 3187 fluorescent spots in the difference gel electrophoresis image of the master gel, which contained the entirety of solubilized sperm proteins. Comparison of the pathological and reference proteomes by applying an average abundance ratio setting of 1.6 and a p ? 0.05 criterion resulted in the identification of 79 fluorescent spots containing proteins that were present at significantly changed levels in the sperm cells. Biometric evaluation of the fluorescence data followed by mass spectrometric protein identification revealed altered levels of 12, 71, and 13 protein species in the proteomes of the type-1 diabetic, type-2 diabetic, and non-diabetic obese patients, respectively, with considerably enhanced amounts of the same set of one molecular form of semenogelin-1, one form of clusterin, and two forms of lactotransferrin in each group of pathologic samples. Remarkably, ?-galactosidase-1-like protein was the only protein that was detected at decreased levels in all three pathologic situations. The former three proteins are part of the eppin (epididymal proteinase inhibitor) protein complex, which is thought to fulfill fertilization-related functions, such as ejaculate sperm protection, motility regulation and gain of competence for acrosome reaction, whereas the putative role of the latter protein to function as a glycosyl hydrolase during sperm maturation remains to be explored at the protein/enzyme level. The strikingly similar differences detected in the three groups of pathological sperm proteomes reflect a disease-associated enhanced formation of predominantly proteolytically modified forms of three eppin protein complex components, possibly as a response to enduring hyperglycemia and enhanced oxidative stress. PMID:21525168

  9. Identification of Increased Amounts of Eppin Protein Complex Components in Sperm Cells of Diabetic and Obese Individuals by Difference Gel Electrophoresis*

    PubMed Central

    Paasch, Uwe; Heidenreich, Falk; Pursche, Theresia; Kuhlisch, Eberhard; Kettner, Karina; Grunewald, Sonja; Kratzsch, Jrgen; Dittmar, Gunnar; Glander, Hans-Jrgen; Hoflack, Bernard; Kriegel, Thomas M.

    2011-01-01

    Metabolic disorders like diabetes mellitus and obesity may compromise the fertility of men and women. To unveil disease-associated proteomic changes potentially affecting male fertility, the proteomes of sperm cells from type-1 diabetic, type-2 diabetic, non-diabetic obese and clinically healthy individuals were comparatively analyzed by difference gel electrophoresis. The adaptation of a general protein extraction procedure to the solubilization of proteins from sperm cells allowed for the resolution of 3187 fluorescent spots in the difference gel electrophoresis image of the master gel, which contained the entirety of solubilized sperm proteins. Comparison of the pathological and reference proteomes by applying an average abundance ratio setting of 1.6 and a p ? 0.05 criterion resulted in the identification of 79 fluorescent spots containing proteins that were present at significantly changed levels in the sperm cells. Biometric evaluation of the fluorescence data followed by mass spectrometric protein identification revealed altered levels of 12, 71, and 13 protein species in the proteomes of the type-1 diabetic, type-2 diabetic, and non-diabetic obese patients, respectively, with considerably enhanced amounts of the same set of one molecular form of semenogelin-1, one form of clusterin, and two forms of lactotransferrin in each group of pathologic samples. Remarkably, ?-galactosidase-1-like protein was the only protein that was detected at decreased levels in all three pathologic situations. The former three proteins are part of the eppin (epididymal proteinase inhibitor) protein complex, which is thought to fulfill fertilization-related functions, such as ejaculate sperm protection, motility regulation and gain of competence for acrosome reaction, whereas the putative role of the latter protein to function as a glycosyl hydrolase during sperm maturation remains to be explored at the protein/enzyme level. The strikingly similar differences detected in the three groups of pathological sperm proteomes reflect a disease-associated enhanced formation of predominantly proteolytically modified forms of three eppin protein complex components, possibly as a response to enduring hyperglycemia and enhanced oxidative stress. PMID:21525168

  10. Detection of Salmonellae in Chicken Feces by a Combination of Tetrathionate Broth Enrichment, Capillary PCR, and Capillary Gel Electrophoresis

    Microsoft Academic Search

    K. TAYFUN CARLI; CAN BORA UNAL; VILDAN CANER; AYSEGUL EYIGOR

    2001-01-01

    This report describes a rapid detection procedure for salmonellae from chicken feces by the combination of tetrathionate primary enrichment (preenrichment (PE))-bacterial lysis-capillary PCR and capillary gel elec- trophoresis. Pure Salmonella enterica serovar Enteritidis 64K was reisolated and detected by capillary PCR after buffered peptone water and nutrient broth, tetrathionate broth base Hajna (TTBH), and tetrathionate broth (TTB) preenrichments. When the

  11. Groove-binding unsymmetrical cyanine dyes for staining of DNA: dissociation rates in free solution and electrophoresis gels

    Microsoft Academic Search

    Maja Eriksson; H. Jonas Karlsson; Gunnar Westman; Bjorn Akerman

    2003-01-01

    The rates of dissociation of three non-intercalative unsymmetrical cyanine dyes, BEBO, BETO and BOXTO from mixed-sequence DNA have been studied with the DNA either free in solution or in confining porous agarose gels. The properties of the new dyes were compared to the related inter- calating dyes BO, BO-PRO, TO-PRO and YO-PRO. With DNA in solution, BEBO dissociates more slowly

  12. Direct detection of an antimicrobial peptide of Pediococcus acidilactici in sodium dodecyl sulfate-polyacrylamide gel electrophoresis

    Microsoft Academic Search

    Arun K. Bhunia; M. C. Johnson; Bibek Ray

    1987-01-01

    Summary An SDS-PAGE technique is described that allows identification of the antimicrobial activity of a peptide secreted by a strain ofPediococcus acidilactici. This peptide has an antimicrobial property against several baeteria associated with food. This technique enables detection of the specific peptide (or protein) band(s) associated with the inhibitory effect which can then be eluted from the gel for further

  13. The XbaI-BlnI-CeuI genomic cleavage map of Salmonella typhimurium LT2 determined by double digestion, end labelling, and pulsed-field gel electrophoresis.

    PubMed Central

    Liu, S L; Hessel, A; Sanderson, K E

    1993-01-01

    Endonuclease digestion of the 4,800-kb chromosome of Salmonella typhimurium LT2 yielded 24 XbaI fragments, 12 BlnI fragments, and 7 CeuI fragments, which were separated by pulsed-field gel electrophoresis. The 90-kb plasmid pSLT has one XbaI site and one BlnI site. The locations of the fragments around the circular chromosome and of the digestion sites of the different endonucleases with respect to each other were determined by excision of agarose blocks containing fragments from single digestion, redigestion with a second enzyme, end labelling with 32P by using T7 DNA polymerase, reelectrophoresis, and autoradiography. Forty-three cleavage sites were established on the chromosome, and the fragments and cleavage sites were designated in alphabetical order and numerical order, respectively, around the chromosome. One hundred nine independent Tn10 insertions previously mapped by genetic means were located by pulsed-field gel electrophoresis on the basis of the presence of XbaI and BlnI sites in Tn10. The genomic cleavage map was divided into 100 units called centisomes; the endonuclease cleavage sites and the genes defined by the positions of Tn10 insertions were located by centisome around the map. There is very good agreement between the genomic cleavage map, defined in centisomes, and the linkage map, defined in minutes. All seven rRNA genes were located on the map; all have the CeuI digestion site, all four with the tRNA gene for glutamate have the XbaI and the BlnI sites, but only four of the seven have the BlnI site in the 16S rRNA (rrs) gene. Their inferred orientation of transcription is the same as in Escherichia coli. A rearrangement of the rrnB and rrnD genes with respect to the arrangement in E. coli, observed earlier by others, has been confirmed. The sites for all three enzymes in the rrn genes are strongly conserved compared with those in E. coli, but the XbaI and BlnI sites outside the rrn genes show very little conservation. Images PMID:8320226

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

    PubMed

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

    2014-12-01

    An inverse-shifting polymerase chain reaction (IS-PCR) combined with short-end capillary gel electrophoresis (CGE) was developed for genotyping of intron 22 inversion Type 1 (Inv22-1) and Type 2 (Inv22-2) of hemophilia A (HA). Severe HA cases are affected by intron 22 inversion around 45-50%. Inv22-1 has higher frequency than Inv22-2. The aim of this study is to distinguish them by genotyping. In order to improve Inv22 genotyping efficiency, five primers were designed and applied to differentiate the wild type, Inv22-1, Inv22-2 and carrier. Three amplicons of 405, 457 and 512 bp were recognized for wild type; 333, 457 and 584 bp for Inv22-1; 385, 405 and 584 bp for Inv22-2. The Inv22-1 carrier has 5 amplicons including 333, 405, 457, 512, 584 bp and Inv22-2 carrier is differentiated by 385, 405, 457, 512 and 584 bp. The amplicons between Inv22-1 and Inv22-2 carriers are only different in 333 bp for Inv22-1 carrier and 385 bp for Inv22-2 carrier. Capillary gel electrophoresis (CGE) was used for separation within 5 min. The separation voltage was set at 8 kV (cathode at detector), and the temperature was kept at 25C. The sieving matrix was 89 mM Tris, 89 mM boric acid, 2mM EDTA containing 0.4% (w/v) HPMC and 1 ?M of YO-PRO()-1 Iodide. Total of 50 HA patients (including 35 non-Inv22, 14 Inv22-1, and one Inv22-2 patients) and 7 HA carriers were diagnosed in the application. Seven random samples (5 patients and 2 carriers) were subjected to comparison and gave identical results of DNA sequencing and this modified IS-PCR. PMID:25159417

  15. Rapid, simple method of preparing rotaviral double-stranded ribonucleic acid for analysis by polyacrylamide gel electrophoresis.

    PubMed Central

    Theil, K W; McCloskey, C M; Saif, L J; Redman, D R; Bohl, E H; Hancock, D D; Kohler, E M; Moorhead, P D

    1981-01-01

    A procedure for extracting rotaviral double-stranded ribonucleic acid (RNA) directly from fecal and intestinal specimens collected from calves and pigs is described. This procedure provides a rapid, simple, reproducible method of obtaining rotaviral double-stranded RNA preparations suitable for electrophoretic analysis in polyacrylamide-agarose composite gels. The rotaviral genome electrophoretic migration pattern produced by double-stranded RNA extracted directly from a specimen by this procedure was qualitatively identical to the electrophoretic migration pattern obtained with double-stranded RNA extracted from purified rotavirus derived from the same specimen. Direct extraction of specimens containing porcine rotavirus-like virus by this procedure gave preparations that had electrophoretic migration patterns similar, but not identical, to the characteristic electrophoretic migration pattern of the rotaviral genome. Sufficient rotaviral double-stranded RNA could be extracted from 6 ml of fecal or intestinal specimen by this procedure to permit 15 or more electrophoretic assays. Images PMID:6270190

  16. Assessment of Resolution and Intercenter Reproducibility of Results of Genotyping Staphylococcus aureus by Pulsed-Field Gel Electrophoresis of SmaI Macrorestriction Fragments: a Multicenter Study

    PubMed Central

    van Belkum, Alex; van Leeuwen, Willem; Kaufmann, Mary Elizabeth; Cookson, Barry; Forey, Franoise; Etienne, Jerome; Goering, Richard; Tenover, Fred; Steward, Christine; OBrien, Frances; Grubb, Warren; Tassios, Panayotis; Legakis, Nicholas; Morvan, Anne; El Solh, Nvine; de Ryck, Raf; Struelens, Marc; Salmenlinna, Saara; Vuopio-Varkila, Jaana; Kooistra, Mirjam; Talens, Adriaan; Witte, Wolfgang; Verbrugh, Henri

    1998-01-01

    Twenty well-characterized isolates of methicillin-resistant Staphylococcus aureus were used to study the optimal resolution and interlaboratory reproducibility of pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. Five identical isolates (one PFGE type), 5 isolates that produced related PFGE subtypes, and 10 isolates with unique PFGE patterns were analyzed blindly in 12 different laboratories by in-house protocols. In several laboratories a standardized PFGE protocol with a commercial kit was applied successfully as well. Eight of the centers correctly identified the genetic homogeneity of the identical isolates by both the in-house and standard protocols. Four of 12 laboratories failed to produce interpretable data by the standardized protocol, due to technical problems (primarily plug preparation). With the five related isolates, five of eight participants identified the same subtype interrelationships with both in-house and standard protocols. However, two participants identified multiple strain types in this group or classified some of the isolates as unrelated isolates rather than as subtypes. The remaining laboratory failed to distinguish differences between some of the related isolates by utilizing both the in-house and standardized protocols. There were large differences in the relative genome lengths of the isolates as calculated on the basis of the gel pictures. By visual inspection, the numbers of restriction fragments and overall banding pattern similarity in the three groups of isolates showed interlaboratory concordance, but centralized computer analysis of data from four laboratories yielded percent similarity values of only 85% for the group of identical isolates. The differences between the data sets obtained with in-house and standardized protocols could be the experimental parameters which differed with respect to the brand of equipment used, imaging software, running time (20 to 48 h), and pulsing conditions. In conclusion, it appears that the standardization of PFGE depends on controlling a variety of experimental intricacies, as is the case with other bacterial typing procedures. PMID:9620395

  17. Psoriasin, one of several new proteins identified in nasal lavage fluid from allergic and non-allergic individuals using 2-dimensional gel electrophoresis and mass spectrometry

    PubMed Central

    Bryborn, Malin; Adner, Mikael; Cardell, Lars-Olaf

    2005-01-01

    Background Extravasation and luminal entry of plasma occurs continuously in the nose. This process is markedly facilitated in patients with symptomatic allergic rhinitis, resulting in an increased secretion of proteins. Identification of these proteins is an important step in the understanding of the pathological mechanisms in allergic diseases. DNA microarrays have recently made it possible to compare mRNA profiles of lavage fluids from healthy and diseased patients, whereas information on the protein level is still lacking. Methods Nasal lavage fluid was collected from 11 patients with symptomatic allergic rhinitis and 11 healthy volunteers. 2-dimensional gel electrophoresis was used to separate proteins in the lavage fluids. Protein spots were picked from the gels and identified using mass spectrometry and database search. Selected proteins were confirmed with western blot. Results 61 spots were identified, of which 21 were separate proteins. 6 of these proteins (psoriasin, galectin-3, alpha enolase, intersectin-2, Wnt-2B and hypothetical protein MGC33648) had not previously been described in nasal lavage fluids. The levels of psoriasin were markedly down-regulated in allergic individuals. Prolactin-inducible protein was also found to be down-regulated, whereas different fragments of albumin together with Ig gamma 2 chain c region, transthyretin and splice isoform 1 of Wnt-2B were up-regulated among the allergic patients. Conclusion The identification of proteins in nasal lavage fluid with 2-dimensional gelelectrophoresis in combination with mass spectrometry is a novel tool to profile protein expression in allergic rhinitis and it might prove useful in the hunt for new therapeutic targets or diagnostic markers for allergic diseases. Psoriasin is a potent chemotactic factor and its down-regulation during inflammation might be of importance for the outcome of the disease. PMID:16236163

  18. Development and Validation of an Internationally-Standardized, High-Resolution Capillary Gel-Based Electrophoresis PCR-Ribotyping Protocol for Clostridium difficile

    PubMed Central

    Fawley, Warren N.; Knetsch, C. W.; MacCannell, Duncan R.; Harmanus, Celine; Du, Tim; Mulvey, Michael R.; Paulick, Ashley; Anderson, Lydia; Kuijper, E. J.; Wilcox, Mark H.

    2015-01-01

    PCR-ribotyping has been adopted in many laboratories as the method of choice for C. difficile typing and surveillance. However, issues with the conventional agarose gel-based technique, including inter-laboratory variation and interpretation of banding patterns have impeded progress. The method has recently been adapted to incorporate high-resolution capillary gel-based electrophoresis (CE-ribotyping), so improving discrimination, accuracy and reproducibility. However, reports to date have all represented single-centre studies and inter-laboratory variability has not been formally measured or assessed. Here, we achieved in a multi-centre setting a high level of reproducibility, accuracy and portability associated with a consensus CE-ribotyping protocol. Local databases were built at four participating laboratories using a distributed set of 70 known PCR-ribotypes. A panel of 50 isolates and 60 electronic profiles (blinded and randomized) were distributed to each testing centre for PCR-ribotype identification based on local databases generated using the standard set of 70 PCR-ribotypes, and the performance of the consensus protocol assessed. A maximum standard deviation of only 3.8bp was recorded in individual fragment sizes, and PCR-ribotypes from 98.2% of anonymised strains were successfully discriminated across four ribotyping centres spanning Europe and North America (98.8% after analysing discrepancies). Consensus CE-ribotyping increases comparability of typing data between centres and thereby facilitates the rapid and accurate transfer of standardized typing data to support future national and international C. difficile surveillance programs. PMID:25679978

  19. Development and Validation of an Internationally-Standardized, High-Resolution Capillary Gel-Based Electrophoresis PCR-Ribotyping Protocol for Clostridium difficile.

    PubMed

    Fawley, Warren N; Knetsch, C W; MacCannell, Duncan R; Harmanus, Celine; Du, Tim; Mulvey, Michael R; Paulick, Ashley; Anderson, Lydia; Kuijper, E J; Wilcox, Mark H

    2015-01-01

    PCR-ribotyping has been adopted in many laboratories as the method of choice for C. difficile typing and surveillance. However, issues with the conventional agarose gel-based technique, including inter-laboratory variation and interpretation of banding patterns have impeded progress. The method has recently been adapted to incorporate high-resolution capillary gel-based electrophoresis (CE-ribotyping), so improving discrimination, accuracy and reproducibility. However, reports to date have all represented single-centre studies and inter-laboratory variability has not been formally measured or assessed. Here, we achieved in a multi-centre setting a high level of reproducibility, accuracy and portability associated with a consensus CE-ribotyping protocol. Local databases were built at four participating laboratories using a distributed set of 70 known PCR-ribotypes. A panel of 50 isolates and 60 electronic profiles (blinded and randomized) were distributed to each testing centre for PCR-ribotype identification based on local databases generated using the standard set of 70 PCR-ribotypes, and the performance of the consensus protocol assessed. A maximum standard deviation of only 3.8bp was recorded in individual fragment sizes, and PCR-ribotypes from 98.2% of anonymised strains were successfully discriminated across four ribotyping centres spanning Europe and North America (98.8% after analysing discrepancies). Consensus CE-ribotyping increases comparability of typing data between centres and thereby facilitates the rapid and accurate transfer of standardized typing data to support future national and international C. difficile surveillance programs. PMID:25679978

  20. Vibrational spectroscopy and electrophoresis as a "golden means" in monitoring of polysaccharides in medical plant and gels

    NASA Astrophysics Data System (ADS)

    Pielesz, A.

    In recent years, some bioactive polysaccharides isolated from natural sources have attracted much attention in the field of biochemistry and pharmacology. Of them, polysaccharides or their glycoconjugates were shown to exhibit multiple biological activities including anticarcinogenic, anticoagulant, immunostimulating, antioxidant, etc. Pharmacotherapy using plant-derived substances can be currently regarded as a very promising future alternative to conventional therapy. The advanced biotechnologies available today enable chemical investigation of well-defined bioactive plant components as sources of novel drugs. The need for safer drugs without side effects has led to the use of natural ingredients with proven safety. Special interest is focused on plant polysaccharides. This article attempts to review the current structural and conformational characterization of some importantly bioactive monosaccharides isolated from following plant cell-wall: Symphytum officinale (comfrey), Thymus pulegioides (thyme), Trigonella foenum-graecum L. (fenugreek), Tussilago farfara L. (coltsfoot), Hyssopus officinalis (hyssop), Althaea officinalis L. (marshmallow) and Equisetum arvense L. (horsetail). The chemical structures of monosaccharides were analysed using FTIR and Raman spectroscopies as well as cellulose acetate membrane electrophoresis (CAE). The dried plant samples were gently hydrolysed with sulphuric acid. The presence of glucuronic acid, galacturonic acid, alginic acid, glucose, mannose and xylose in the hydrolysates of reference substances and non-defatted plant films was proved. The possibility of a taxonomic classification of plant cell walls based on infrared and Raman spectroscopies and the use of spectral fingerprinting for authentication and detection of adulteration of products rich in cell-wall materials are discussed. Individual bands were selected to monitor the sugar content in medical plant cell walls and to confirm the identity of the analysed plants.

  1. Comparison of Ribotyping, Randomly Amplified Polymorphic DNA Analysis, and Pulsed-Field Gel Electrophoresis in Typing of Lactobacillus rhamnosus and L. casei Strains

    PubMed Central

    Tynkkynen, Soile; Satokari, Reetta; Saarela, Maria; Mattila-Sandholm, Tiina; Saxelin, Maija

    1999-01-01

    A total of 24 strains, biochemically identified as members of the Lactobacillus casei group, were identified by PCR with species-specific primers. The same set of strains was typed by randomly amplified polymorphic DNA (RAPD) analysis, ribotyping, and pulsed-field gel electrophoresis (PFGE) in order to compare the discriminatory power of the methods. Species-specific primers for L. rhamnosus and L. casei identified the type strain L. rhamnosus ATCC 7469 and the neotype strain L. casei ATCC 334, respectively, but did not give any signal with the recently revived species L. zeae, which contains the type strain ATCC 15820 and the strain ATCC 393, which was previously classified as L. casei. Our results are in accordance with the suggested new classification of the L. casei group. Altogether, 21 of the 24 strains studied were identified with the species-specific primers. In strain typing, PFGE was the most discriminatory method, revealing 17 genotypes for the 24 strains studied. Ribotyping and RAPD analysis yielded 15 and 12 genotypes, respectively. PMID:10473394

  2. Comparison of ribotyping, randomly amplified polymorphic DNA analysis, and pulsed-field gel electrophoresis in typing of Lactobacillus rhamnosus and L. casei strains.

    PubMed

    Tynkkynen, S; Satokari, R; Saarela, M; Mattila-Sandholm, T; Saxelin, M

    1999-09-01

    A total of 24 strains, biochemically identified as members of the Lactobacillus casei group, were identified by PCR with species-specific primers. The same set of strains was typed by randomly amplified polymorphic DNA (RAPD) analysis, ribotyping, and pulsed-field gel electrophoresis (PFGE) in order to compare the discriminatory power of the methods. Species-specific primers for L. rhamnosus and L. casei identified the type strain L. rhamnosus ATCC 7469 and the neotype strain L. casei ATCC 334, respectively, but did not give any signal with the recently revived species L. zeae, which contains the type strain ATCC 15820 and the strain ATCC 393, which was previously classified as L. casei. Our results are in accordance with the suggested new classification of the L. casei group. Altogether, 21 of the 24 strains studied were identified with the species-specific primers. In strain typing, PFGE was the most discriminatory method, revealing 17 genotypes for the 24 strains studied. Ribotyping and RAPD analysis yielded 15 and 12 genotypes, respectively. PMID:10473394

  3. Separation of Native Allophycocyanin and R-Phycocyanin from Marine Red Macroalga Polysiphonia urceolata by the Polyacrylamide Gel Electrophoresis Performed in Novel Buffer Systems

    PubMed Central

    Wang, Yu; Gong, Xueqin; Wang, Shumei; Chen, Lixue; Sun, Li

    2014-01-01

    Three buffer systems of Imidazole?Acetic acid, HEPES?Imidazole/Bis-tris and Bis-tris?HEPES?MES were designed based on the principle of discontinuous polyacrylamide gel electrophoresis (PAGE) for the native PAGE which could be performed in pH 7.0 and 6.5 in order to analyze and prepare the minor components of allophycocyanin (AP) and R-phycocyanin (R-PC) from marine red macroalga Polysiphonia urceolata. These AP and R-PC phycobiliproteins are easily denatured in alkaline environments. The obtained results demonstrated that the PAGE modes performed in the buffer systems of HEPES?Imidazole/Bis-tris and Bis-tris?HEPES?MES gave the satisfactory resolution and separation of AP and R-PC proteins. The absorption and fluorescence spectra of the AP and R-PC proteins which were prepared by the established PAGE modes proved that they maintained natural spectroscopic characteristics. The established PAGE modes may also provide useful references and selections for some other proteins that are sensitive to alkaline environments or are not effectively separated by the classical PAGE modes performed normally in alkaline buffer systems. PMID:25166028

  4. A study on the vertical profile of bacterial DNA structure in the Puruogangri (Tibetan Plateau) ice core using denaturing gradient gel electrophoresis

    NASA Astrophysics Data System (ADS)

    Zhang, Xinfang; Yao, Tandong; An, Lizhe; Tian, Lide; Xu, Shijian

    The bacterial DNA structures at different depths in the Puruogangri (Tibetan Plateau) ice core (83.45 m) were investigated by the denaturing gradient gel electrophoresis (DGGE) DNA fingerprinting technique. DGGE profiles indicated that the bacterial species diversity in glacial ice is high, and indigenous species represented by common bands in all samples may grow on the glacial surface. Bacterial diversity, as estimated by Shannon indices (mean 2.91; SD 0.25; n = 13), was comparable to that of soil habitats and had a positive correlation with Ca2+ concentration (R = 0.71; P < 0.01), a good proxy of dust. This suggested that the soil ecosystem was the main source of bacteria in this glacier. The low similarity indices (0-43%) were found between the ice-core samples, which corresponded to the episodic deposition under defined climatic conditions and low activity of microorganisms in glacial ice. The profiles of bacterial species composition in glacial ice may be a bioindicator of climatic changes or dating.

  5. Denaturing gradient gel electrophoresis fingerprinting of soil bacteria in the vicinity of the Chinese Great Wall Station, King George Island, Antarctica.

    PubMed

    Pan, Qi; Wang, Feng; Zhang, Yang; Cai, Minghong; He, Jianfeng; Yang, Haizhen

    2013-08-01

    Bacterial diversity was investigated in soil samples collected from 13 sites around the Great Wall Station, Fildes Peninsula, King George Island, Antarctica, using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes. The classes alpha-, beta-, and gamma-Proteobacteria, as well as the phylum Actinobacteria, were found to be the dominant bacteria in the soils around the Great Wall Station. Although the selected samples were not contaminated by oil, a relationship between soil parameters, microbial biodiversity, and human impact was still seen. Sample sites in human impacted areas showed lower bacterial biodiversity (average H' = 2.65) when compared to non-impacted sites (average H' = 3.05). There was no statistically significant correlation between soil bacterial diversity and total organic carbon (TOC), total nitrogen, or total phosphorus contents of the soil. Canonical correlation analysis showed that TOC content was the most important factor determining bacterial community profiles among the measured soil parameters. In conclusion, microbial biodiversity and community characteristics within relatively small scales (1.5 km) were determined as a function of local environment parameters and anthropogenic impact. PMID:24520704

  6. Molecular Characterization of Salmonella Paratyphi B dT+ and Salmonella Heidelberg from Poultry and Retail Chicken Meat in Colombia by Pulsed-Field Gel Electrophoresis.

    PubMed

    Donado-Godoy, Pilar; Byrne, Barbara A; Hume, Michael; Len, Maribel; Prez-Gutirrez, Enriqu; Vives Flores, Martha J; Clavijo, Viviana; Holguin, ngela; Romero-Zuiga, Juan J; Castellanos, Ricardo; Tafur, McAllister; Smith, Woutrina A

    2015-04-01

    Salmonella Paratyphi B dT+ variant (also termed Salmonella Java) and Salmonella Heidelberg are pathogens of public health importance that are frequently isolated from poultry. As a step toward implementing the Colombian Integrated Program for Antimicrobial Resistant Surveillance, this study characterized molecular patterns of Salmonella Paratyphi B dT+ and Salmonella Heidelberg isolated from poultry farms, fecal samples, and retail chicken meat using pulsed-field gel electrophoresis (PFGE). The objective of this study was to determine the genetic relationship among isolates and to determine potential geographically predominant genotypes. Based on PFGE analysis, both serovars exhibited high heterogeneity: the chromosomal DNA fingerprints of 82 Salmonella Paratyphi B dT+ isolates revealed 42 PFGE patterns, whereas the 21 isolates of Salmonella Heidelberg revealed 10 patterns. Similar genotypes of both serovars were demonstrated to be present on farms and in retail outlets. For Salmonella Paratyphi B dT+, closely genetically related strains were found among isolates coming from different farms and different integrated poultry companies within two departments (Santander and Cundinamarca) and also from farms located in the two geographically distant departments. For Salmonella Heidelberg, there were fewer farms with genetically related isolates than for Salmonella Paratyphi B dT+. A possible dissemination of similar genotypes of both serovars along the poultry production chain is hypothesized, and some facilitating factors existing in Colombia are reviewed. PMID:25836408

  7. Comparative horizontal starch gel isoenzyme electrophoresis of Lymnaea (Bullastra) cumingiana (Pulmonata: Lymnaeidae) and related taxa in the Indo-Pacific region.

    PubMed

    Monzon, R B; Thammapalerd, N; Kitikoon, V; Temcharoen, P; Sornmani, S; Viyanant, V

    1994-03-01

    Foot muscle tissue extracts from six lymnaeid species of the Indo-Pacific region [Lymnaea (Bullastra) cumingiana and L. (Radix) quadrasi from the Philippines, L. (R.) rubiginosa from Indonesia and Thailand, and L. (R.) viridis from Guam and Hong Kong] were subjected to horizontal starch gel isoenzyme electrophoresis and assayed for seven isoenzymes (AcP, AlP, CA, EST, LAP, CAT and GOT) to elucidate their taxonomic relationships. L. cumingiana exhibited banding patterns for EST, LAP and CAT uniquely different from the rest, thus supporting the hypothesis that it is a distinct species. Zymogram patterns for AlP, CA, EST and LAP attest to the close affinity between L. quadrasi and L. rubiginosa (Indonesia and Thailand). Minor differences suggest a closer relationship between the two geographical strains of L. rubiginosa than with L. quadrasi, lending support to the hypothesis that L. quadrasi is inseparable as a race or variety from the typical L. swinhoei Adams, which in turn is but a race of L. auricularia, which also encompasses L. rubiginosa. The two geographical strains of L. viridis from Guam and Hong Kong showed the greatest consistency with regards to similarity and congruence in banding patterns. Non-specific esterases (EST) were the most useful in distinguishing the six species from each other. PMID:7825010

  8. Improved microbiological techniques using the polymerase chain reaction and pulsed-field gel electrophoresis for diagnosis and follow-up of enterohaemorrhagic Escherichia coli infection.

    PubMed

    Welinder-Olsson, C; Kjellin, E; Badenfors, M; Kaijser, B

    2000-11-01

    The aims of the present investigation were to evaluate the microbiological diagnostic procedures, especially polymerase chain reaction (PCR) versus culture and seroagglutination, in relation to the clinical features of enterohaemorrhagic Escherichia coli (EHEC) infection and to study the status of EHEC in the western part of Sweden. During 1997 and 1998, stool specimens from 3,948 patients were analysed by PCR for the presence of EHEC with verotoxin (VT)1- and/or VT2-producing DNA sequences. The stool specimens were also cultured for Escherichia coli O157:H7, Salmonella, Campylobacter, Shigella and Yersinia. Fifty-five patients were positive by PCR. Thirty-nine patients were positive for EHEC by PCR and culture. Of these, 29 were infected with EHEC serogroup O157:H7 strains. All EHEC isolates were analysed by pulsed-field gel electrophoresis (PFGE); 17 different clones were identified. Studies on the duration of the presence of EHEC in the gut showed that EHEC often disappears rather quickly, i.e. within 2 weeks. In one patient, however, EHEC remained for several months. In conclusion, PCR, rather than culture and agglutination, should be the method of choice for microbiological diagnosis of EHEC infection. PCR is more sensitive than culture for detecting EHEC in the gut. PMID:11152309

  9. Characterisation of E. coli O157 isolates from bovine hide and beef trimming in Irish abattoirs by pulsed field gel electrophoresis.

    PubMed

    Duffy, G; O'Brien, S B; Carney, E; Sheridan, J J; McDowell, D A; Blair, I S

    2005-03-01

    Escherichia coli O157 isolates from bovine hide (n=117) and beef trimmings (n=32) from a single abattoir were examined by pulsed field gel electrophoresis (PFGE). Using BioNumerics software, dendrograms of isolates from each sample type (i.e. hide and beef trimming) were produced. In assessing the genetic relatedness of isolates, a similarity criterion of 80% was applied. The 117 E. coli O157 hide isolates were grouped into 14 clusters, comprising of 109 different PFGE profiles. Of the 109 different PFGE profiles, 8 were common to multiple isolates (i.e. shared 100% similarity by PFGE). The 32 E. coli O157 beef trimming isolates produced 28 different PFGE profiles and 2 clusters. Of the 28 PFGE profiles, 2 were common to multiple isolates and the remaining 26 were distinct. On a number of sampling occasions, isolates displaying identical PFGE patterns were recovered from multiple isolates collected from a single sample type (i.e. hides or trimmings), suggesting cross contamination from contaminated hides/animals to uncontaminated hides/animals and from contaminated beef trimmings to uncontaminated beef trimmings during abattoir operations. PMID:15649539

  10. Seasonal distributions of dominant 16S rRNA-defined populations in a hot spring microbial mat examined by denaturing gradient gel electrophoresis.

    PubMed Central

    Ferris, M J; Ward, D M

    1997-01-01

    Denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA gene segments was used to examine the distributions of bacterial populations within a hot spring microbial mat (Octopus Spring, Yellowstone National Park). Populations at sites along the thermal gradient of the spring's effluent channel were surveyed at seasonal intervals. No shift in the thermal gradient was detected, and populations at spatially or temperature-defined sites exhibited only slight changes over the annual sampling period. A new cyanobacterial 16S rRNA sequence type was detected at temperatures from 63 to 75 degrees C. A new green nonsulfur bacterium-like sequence type was also detected at temperatures from 53 to 62 degrees C. Genetically unique though closely related cyanobacterial and green nonsulfur bacterium-like populations were successively distributed along the thermal gradient of the Octopus Spring effluent channel. At least two cyanobacterial populations were detected at each site; however, a limited ability to detect some cyanobacterial populations suggests that only dominant populations were observed. PMID:9097434

  11. Use of Gel Electrophoresis and Raman Spectroscopy to Characterize the Effect of the Electronic Structure of Single-Walled Carbon Nanotubes on Cellular Uptake

    PubMed Central

    2015-01-01

    It is well-known that the uptake of single-walled carbon nanotubes (SWNTs) by living cells depends on factors such as SWNT length and surface chemistry. Surprisingly, little is known about whether the electronic structure of a SWNT influences uptake. One reason for this has been the lack of methods to measure the uptake of SWNTs by cell populations. Previously, we developed a rapid, sensitive, and label-free sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) method for measuring the amount of SWNTs in lysates prepared from cultured cells (Wang et al. Anal. Chem.2009, 81, 294419296592). Herein, we describe the use of SDS-PAGE and microprobe Raman spectroscopy to detect and distinguish the electronic structure of SWNTs internalized by mammalian cells. Using normal rat kidney (NRK) cells and SWNTs dispersed with bovine serum albumin (BSA), we demonstrate that the method can detect both metallic and semiconducting SWNTs in lysates of cells that had internalized BSA-SWNTs and that the uptake of BSA-SWNTs by NRK cells is not influenced by SWNT electronic structure. PMID:24564772

  12. Single-cell gel electrophoresis assay in the ten spotted live-bearer fish, Cnesterodon decemmaculatus (Jenyns, 1842), as bioassay for agrochemical-induced genotoxicity.

    PubMed

    Vera-Candioti, Josefina; Soloneski, Sonia; Larramendy, Marcelo L

    2013-12-01

    The ability of two 48 percent chlorpyrifos-based insecticides (Lorsban* 48E and CPF Zamba), two 50 percent pirimicarb-based insecticides (Aficida and Patton Flow), and two 48 percent glyphosate-based herbicides (Panzer and Credit) to induce DNA single-strand breaks in peripheral blood erythrocytes of Cnesterodon decemmaculatus (Jenyns, 1842) (Pisces, Poeciliidae) exposed under laboratory conditions was evaluated by the single-cell gel electrophoresis (SCGE) assay. In those fish exposed to Lorsban* 48E, CPF Zamba, Aficida, Patton Flow, Credit, and Panzer, a significant increase of the genetic damage was observed for all formulations regardless of the harvesting time. This genotoxic effect was achieved by an enhancement of Type II-IV comets and a concomitant decrease of Type 0-I comets over control values. A regression analysis revealed that the damage varied as a negative function of the exposure time in those Lorsban* 48E- and Aficida-treated fish. On the other hand, a positive correlation between damage increase and exposure time was achieved after Patton Flow and Credit treatment. Finally, no correlation was observed between increase in the genetic damage and exposure time after treatment with CPF Zamba or Panzer. These results highlight that all agrochemicals inflict primary genotoxic damage at the DNA level at sublethal concentrations, regardless of the exposure time of the aquatic organisms under study, at least within a period of 96 h of treatment. PMID:24011534

  13. Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

    NASA Technical Reports Server (NTRS)

    Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Said; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

    2002-01-01

    A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

  14. Parallel Characterization of Anaerobic Toluene- and Ethylbenzene-Degrading Microbial Consortia by PCR-Denaturing Gradient Gel Electrophoresis, RNA-DNA Membrane Hybridization, and DNA Microarray Technology

    PubMed Central

    Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Sad; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

    2002-01-01

    A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis. PMID:12088997

  15. 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.

    PubMed

    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 (<1.7% of false classification rate), with limit of detection values of 0.1% for each event, which were also achieved with simulated mixtures at different relative percentages of targets. The assay was further combined with a second multiplex PCR-CGE-SC assay to allow simultaneous detection of 6 cotton and 5 maize targets (two endogenous genes and 9 GM events) in two multiplex PCRs and a single CGE, making the approach more economic. Besides allowing simultaneous detection of many targets with adequate specificity and sensitivity, the multiplex PCR-CGE-SC approach has high throughput and automation capabilities, while keeping a very simple protocol, e.g., amplification and 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

  16. Comparison of pulsed-field gel electrophoresis & repetitive sequence-based PCR methods for molecular epidemiological studies of Escherichia coli clinical isolates

    PubMed Central

    Bae, Il Kwon; Kim, Juwon; Sun, Je Young Hannah; Jeong, Seok Hoon; Kim, Yong-Rok; Wang, Kang-Kyun; Lee, Kyungwon

    2014-01-01

    Background & objectives: PFGE, rep-PCR, and MLST are widely used to identify related bacterial isolates and determine epidemiologic associations during outbreaks. This study was performed to compare the ability of repetitive sequence-based PCR (rep-PCR) and pulsed-field gel electrophoresis (PFGE) to determine the genetic relationships among Escherichia coli isolates assigned to various sequence types (STs) by two multilocus sequence typing (MLST) schemes. Methods: A total of 41 extended-spectrum ?-lactamase- (ESBL-) and/or AmpC ?-lactamase-producing E. coli clinical isolates were included in this study. MLST experiments were performed following the Achtman's MLST scheme and the Whittam's MLST scheme, respectively. Rep-PCR experiments were performed using the DiversiLab system. PFGE experiments were also performed. Results: A comparison of the two MLST methods demonstrated that these two schemes yielded compatible results. PFGE correctly segregated E. coli isolates belonging to different STs as different types, but did not group E. coli isolates belonging to the same ST in the same group. Rep-PCR accurately grouped E. coli isolates belonging to the same ST together, but this method demonstrated limited ability to discriminate between E. coli isolates belonging to different STs. Interpretation & conclusions: These results suggest that PFGE would be more effective when investigating outbreaks in a limited space, such as a specialty hospital or an intensive care unit, whereas rep-PCR should be used for nationwide or worldwide epidemiology studies. PMID:25579152

  17. Variation and Genomic Localization of Genes Encoding DROSOPHILA MELANOGASTER Male Accessory Gland Proteins Separated by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

    PubMed Central

    Whalen, Michael; Wilson, Thomas G.

    1986-01-01

    Accessory gland proteins from Drosophila melanogaster males have been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into nine major bands. When individual males from 175 strains were examined, considerable polymorphism for nearly one-half of the major protein bands was seen, including null alleles for three bands. Variation was observed not only among long-established laboratory strains but also among stocks recently derived from natural populations. There was little difference in the amount of variation between P and M strains, indicating that P element mutagenesis is not a factor producing the variation. Codominant expression of variants for each of five bands was found in heterozygotes, suggesting structural gene variation and not posttranslational modification variation. Stocks carrying electrophoretic variants of four of the major proteins were used to map the presumed structural genes for these proteins; the loci were found to be dispersed on the second chromosome. Since males homozygous for variant proteins were fertile, the polymorphism seems to have little immediate effect on successful sperm transfer. We propose that a high degree of polymorphism can be tolerated because these proteins play a nutritive rather than enzymatic role in Drosophila reproduction. PMID:3095182

  18. Detection by denaturing gradient gel electrophoresis of pncA mutations associated with pyrazinamide resistance in Mycobacterium tuberculosis isolates from the United States-Mexico border region.

    PubMed

    McCammon, Mark T; Gillette, John S; Thomas, Derek P; Ramaswamy, Srinivas V; Rosas, Ishmael I; Graviss, Edward A; Vijg, Jan; Quitugua, Teresa N

    2005-06-01

    Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with pyrazinamide (PZA) resistance in the pncA gene of Mycobacterium tuberculosis. DGGE scans for mutations across large regions of DNA and rivals sequencing in its ability to detect DNA alterations. Specific mutations can often be recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint. Five PCR target fragments were designed to scan for DNA alterations across 600 bp of pncA in 181 M. tuberculosis isolates from patients residing in the U.S-Mexico border states of Texas and Tamaulipas, respectively. A region of pncA was observed with a high GC content and a melting temperature approaching 90 degrees C that was initially refractory to denaturation, and a DGGE target fragment was specifically designed to detect mutations in this region. DGGE detected pncA mutations in 82 of 83 PZA-resistant isolates. By contrast, only 1 of 98 PZA-susceptible isolates harbored a detectable DNA alteration. The pncA gene was sequenced from 41 isolates, and 32 DNA alterations in 32 PZA-resistant isolates were identified, including 11 new mutations. DGGE also detected nine isolates whose susceptibility to PZA appeared to be incorrect, and DNA sequencing confirmed these apparent errors in drug susceptibility testing. These results demonstrate the power and usefulness of DGGE in detecting mutations associated with PZA resistance in M. tuberculosis. PMID:15917514

  19. Apoptosis induced by mild hyperthermia in human and murine tumour cell lines: a study using electron microscopy and DNA gel electrophoresis.

    PubMed

    Takano, Y S; Harmon, B V; Kerr, J F

    1991-04-01

    Mild hyperthermia is known to enhance apoptosis in a range of normal and neoplastic cell populations. Studies of tumours previously shown to respond to heating in this manner might be expected to provide insights not only into the mechanism of hyperthermic cell killing, but also into the apoptotic process in general. In the present study, cell death induced by 43 degrees C heating for 30 min in two human Burkitt's lymphoma lines, BM 13674 and WW1, and in murine mastocytoma P-815 x 2.1 was found to be exclusively apoptotic in type, identification being based on light and electron microscopic appearances and on the presence of internucleosomal cleavage of DNA into fragments that are multiples of 180-200 base pairs, which was demonstrated by agarose gel electrophoresis. The heat-induced apoptosis was prevented by the presence of zinc sulphate, an inhibitor of the endonuclease considered to be responsible for the DNA cleavage, but was not suppressed by the protein synthesis inhibitor cycloheximide. The findings question the validity of the widely held view that active protein synthesis is an invariable prerequisite for the execution of apoptosis. It is suggested that an inositol triphosphate-mediated increase in cytosolic Ca2+, resulting from limited membrane damage, might be the critical event responsible for activation of apoptosis by mild hyperthermia. PMID:1903443

  20. Approach to Analyze the Diversity of Myxobacteria in Soil by Semi-Nested PCR-Denaturing Gradient Gel Electrophoresis (DGGE) Based on Taxon-Specific Gene

    PubMed Central

    Li, Baiyuan; Yao, Qing; Zhu, Honghui

    2014-01-01

    The genotypic diversity of insoluble macromolecules degraded myxobacteria, provided an opportunity to discover new bacterial resources and find new ecological functions. In this study, we developed a semi-nested-PCR-denaturing gradient gel electrophoresis (DGGE) strategy to determine the presence and genotypic diversity of myxobacteria in soil. After two rounds of PCR with myxobacteria-specific primers, an 194 bp fragment of mglA, a key gene involved in gliding motility, suitable for DGGE was obtained. A large number of bands were observed in DGGE patterns, indicating diverse myxobacteria inhabiting in soils. Furthermore, sequencing and BLAST revealed that most of the bands belonged to the myxobacteria-group, and only three of the twenty-eight bands belonged to other group, i.e., Deinococcus maricopensis. The results verified that myxobacterial strains with discrepant sequence compositions of gene mglA could be discriminated by DGGE with myxobacteria-specific primers. Collectively, the developed semi-nested-PCR-DGGE strategy is a useful tool for studying the diversity of myxobacteria. PMID:25280065

  1. Improved Identification of Rapidly Growing Mycobacteria by a 16S23S Internal Transcribed Spacer Region PCR and Capillary Gel Electrophoresis

    PubMed Central

    Gray, Timothy J.; Kong, Fanrong; Jelfs, Peter; Sintchenko, Vitali; Chen, Sharon C-A.

    2014-01-01

    The identification of rapidly growing mycobacteria (RGM) remains problematic because of evolving taxonomy, limitations of current phenotypic methods and absence of a universal gene target for reliable speciation. This study evaluated a novel method of identification of RGM by amplification of the mycobacterial 16S23S rRNA internal transcribed spacer (ITS) followed by resolution of amplified fragments by capillary gel electrophoresis (CGE). Nineteen American Type Culture Collection (ATCC) Mycobacterium strains and 178 clinical isolates of RGM (12 species) were studied. All RGM ATCC strains generated unique electropherograms with no overlap with slowly growing mycobacteria species, including M. tuberculosis. A total of 47 electropherograms for the 178 clinical isolates were observed allowing the speciation of 175/178 (98.3%) isolates, including the differentiation of the closely related species, M. massiliense (M. abscessus subspecies bolletii) and M. abscessus (M. abscessus sensu stricto). ITS fragment size ranged from 332 to 534 bp and 33.7% of clinical isolates generated electropherograms with two distinct peaks, while the remainder where characterized with a single peak. Unique peaks (fragment lengths) were identified for 11/12 (92%) RGM species with only M. moriokaense having an indistinguishable electropherogram from a rarely encountered CGE subtype of M. fortuitum. We conclude that amplification of the 16S23S ITS gene region followed by resolution of fragments by CGE is a simple, rapid, accurate and reproducible method for species identification and characterization of the RGM. PMID:25013955

  2. Molecular characterization of mild-to-moderate hemophilia A: Detection of the mutation in 25 of 29 patients by denaturing gradient gel electrophoresis

    SciTech Connect

    Higuchi, Miyoko; Antonarakis, S.E.; Kasch, L. Economou-Petersen, E.; Kazazian, H.H. Jr. (Johns Hopkins Univ., Baltimore, MD (United States)); Oldenburg, J.; Olek, K. (Univ. of Bonn (West Germany)); Arai, Morio; Inaba, Hiroshi (Tokyo Medical College (Japan))

    1991-10-01

    To date it has been difficult to characterize completely a genetic disorder, such as hemophilia A, in which the involved gene is large and unrelated affected individuals have different mutations, most of which are point mutations. Toward this end, the authors analyzed the DNA of 29 patients with mild-to-moderate hemophilia A in which the causative mutation is likely to be a missense mutation. Using computer analysis, they determined the melting properties of factor VIII gene sequences to design primer sets for PCR amplification and subsequent denaturing gradient gel electrophoresis (DGGE). A total of 45 primer sets was chosen to amplify 99% of the coding region of the gene and 41 of 50 splice junctions. To facilitate detection of point mutations, they mixed DNA from two male patients, and both homoduplexes and heteroduplexes were analyzed. With these 45 primer sets, 26 DNAs containing previously identified point mutations were easily distinguishable from normal. After analyzing the 29 patient with unknown mutations, they identified the disease-producing mutation in 25 (86%). Two polymorphisms and two rare normal variants were also found. Therefore, DGGE after computer analysis is a powerful method for nearly complete characterization of disease-producing mutations and polymorphisms in large genes such as that for factor VIII.

  3. A difference gel electrophoresis study on thylakoids isolated from poplar leaves reveals a negative impact of ozone exposure on membrane proteins.

    PubMed

    Bohler, Sacha; Sergeant, Kjell; Hoffmann, Lucien; Dizengremel, Pierre; Hausman, Jean-Francois; Renaut, Jenny; Jolivet, Yves

    2011-07-01

    Populus tremula L. x P. alba L. (Populus x canescens (Aiton) Smith), clone INRA 717-1-B4, saplings were subjected to 120 ppb ozone exposure for 28 days. Chloroplasts were isolated, and the membrane proteins, solubilized using the detergent 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), were analyzed in a difference gel electrophoresis (DiGE) experiment comparing control versus ozone-exposed plants. Extrinsic photosystem (PS) proteins and adenosine triphosphatase (ATPase) subunits were detected to vary in abundance. The general trend was a decrease in abundance, except for ferredoxin-NADP(+) oxidoreductase (FNR), which increased after the first 7 days of exposure. The up-regulation of FNR would increase NAPDH production for reducing power and detoxification inside and outside of the chloroplast. Later on, FNR and a number of PS and ATPase subunits decrease in abundance. This could be the result of oxidative processes on chloroplast proteins but could also be a way to down-regulate photochemical reactions in response to an inhibition in Calvin cycle activity. PMID:21520910

  4. Coagulase-negative staphylococci from non-mastitic bovine mammary gland: characterization of Staphylococcus chromogenes and Staphylococcus haemolyticus by antibiotic susceptibility testing and pulsed-field gel electrophoresis.

    PubMed

    Pate, Mateja; Zdovc, Irena; Avberek, Jana; Ocepek, Matja; Pengov, Andrej; Podpe?an, Obalt

    2012-05-01

    During routine microbiological examination of milk samples from dairy cows without clinical signs of mastitis, quarter milk samples of 231 dairy cows from 12 herds were investigated for the presence of coagulase-negative staphylococci (CNS). The isolates were identified on the basis of colony morphology, Gram staining, catalase and coagulase test and the commercial kit, API Staph. CNS was detected in 29% (67/231) of the cows. A total of seven CNS species were identified with the most prevalent being Staphylococcus (Staph.) chromogenes (30%) and Staph. haemolyticus (288%), followed by Staph. simulans (112%), Staph. xylosus (112%), Staph. epidermidis (75%), Staph. hyicus (63%) and Staph. sciuri (5%). The predominant species, Staph. chromogenes and Staph. haemolyticus, were further characterized by antibiotic susceptibility testing using the agar disc diffusion method (Kirby-Bauer) and by pulsed-field gel electrophoresis (PFGE). Considerable resistance to ampicillin and penicillin was observed in both species. Isolates with identical or highly similar PFGE profiles were detected at the herd level despite a marked heterogeneity seen for both species. On the basis of somatic cell count, absence of clinical signs of inflammation and heterogeneity of genotypes, we assume that CNS isolated in this study could not be considered as important causative agents of the bovine mammary gland inflammation. PMID:22067091

  5. Two-dimensional gel electrophoresis analysis of the leiomyoma interstitial fluid reveals altered protein expression with a possible involvement in pathogenesis.

    PubMed

    Ura, Blendi; Scrimin, Federica; Zanconati, Fabrizio; Arrigoni, Giorgio; Monasta, Lorenzo; Romano, Andrea; Banco, Rubina; Zweyer, Marina; Milani, Daniela; Ricci, Giuseppe

    2015-05-01

    Uterine leiomyoma is the most common smooth benign neoplasm. In the present study, we analyzed the global interstitial fluid (IF) profile of leiomyoma vs. normal myometrium to identify protein dysregulation involved in leiomyoma pathogenesis. Two-dimensional gel electrophoresis and mass spectrometry were used to generate and compare the global interstitial fluid profiles of the leiomyoma and of the normal tissue. Two proteins were validated by immunohistochemistry. By comparing the interstitial fluid profile of the leiomyoma with that of the normal myometrium, the levels of seven proteins were found to be significantly different: four structural organization proteins (desmin, prelamin-A/C, transgelin and ?-actinin-1), an inflammatory response (?1-antitrypsin), a response to oxidative stress (peroxiredoxin-2), and a folding protein (heat shock 70kDa protein 1A/1B). Desmin, ?1-antitrypsin and peroxiredoxin-2 were upregulated in the leiomyoma, whereas heat shock 70kDa protein 1A/1B, ?-actinin-1, prelamin-A/C and transgelin were downregulated. Desmin and ?1-antitrypsin were further validated by immunohistochemistry. By identifying proteins with altered expression levels compared to the myometrium from several pathways of the leiomyoma pathogenesis, we found the leiomyoma interstitial fluid to have a characteristic proteomic profile. A better appreciation of the pathophysiology of the disease can be useful in the development of conservative treatments that serve as viable alternatives to hysterectomy. PMID:25738828

  6. Exploitation of detergent thermodynamics in the direct solubilization of myelin membrane proteins for two-dimensional gel electrophoresis for proteomic analysis.

    PubMed

    Nair, Sreepriya; Xavier, Tessy; Kumar, Madathiparambil Kumaran Satheesh; Saha, Sharmistha; Menon, Krishnakumar N

    2011-12-01

    Performing 2-DE of lipid-rich multilamellar membranes like myelin is a cumbersome task. However, for understanding its molecular organization and changes during diseases, identification of proteins of myelin is essential. Although the 2-D-proteomic approach of myelin has been employed to understand the myelin proteome, representation of myelin proteins in its entirety is still a challenge. 2-DE profiling of myelin proteins is very important for the detection of immuno-reactivity to myelin proteins from various biological fluids following Western blotting in diseases like multiple sclerosis. Here we developed a novel approach by exploiting the thermodynamic principles behind detergent-mediated solubilization of myelin membranes without any conventional processing of myelin involving precipitation of myelin proteins. We show that the addition of myelin to ASB-14-4 resulted in significant increase in protein representation of myelin in 2-DE compared with the addition of ASB-14-4 to myelin. Moreover, the number and resolution of spots are significantly higher in myelin to ASB-14-4 strategy than other strategies of myelin sample processing such as ASB-14-4 to myelin or ethanol or acetone or methanol-ammonium acetate precipitation of myelin proteins. In addition, the step involves no precipitation that selective removal of any proteins as a result of precipitation is nil and a qualitative representation of myelin proteins in a 2-D gel is achieved. PMID:22102008

  7. Comparative evaluation of an automated ribotyping system versus pulsed-field gel electrophoresis for epidemiological typing of clinical isolates of Escherichia coli and Pseudomonas aeruginosa from patients with recurrent gram-negative bacteremia

    Microsoft Academic Search

    M. A. Pfaller; C. Wendt; R. J. Hollis; R. P. Wenzel; S. J. Fritschel; J. J. Neubauer; L. A. Herwaldt

    1996-01-01

    Ribotyping and macrorestriction analysis of chromosomal DNA using pulsed-field gel electrophoresis (PFGE) are among the more useful molecular epidemiologic typing methods. Because these techniques are labor intensive, automation of one or more steps may allow clinical laboratories to apply molecular typing methods. We compared the recently developed automated ribotyping system, the RiboPrinter Microbial Characterization System (DuPont), with PFGE as a

  8. Analysis of Ammonia-Oxidizing Bacteria of the bSubdivision of the ClassProteobacteriain Coastal Sand Dunes by Denaturing Gradient Gel Electrophoresis and Sequencing of PCR-Amplified 16S Ribosomal DNA Fragments

    Microsoft Academic Search

    GEORGE A. KOWALCHUK; JOHN R. STEPHEN; WIETSE DE BOER; JIM I. PROSSER; T. MARTIN EMBLEY; ANDJAN W. WOLDENDORP

    1997-01-01

    Denaturing gradient gel electrophoresis (DGGE) is a powerful and convenient tool for analyzing the se- quence diversity of complex natural microbial populations. DGGE was evaluated for the identification of am- monia oxidizers of the bsubdivision of theProteobacteriabased on the mobility of PCR-amplified 16S rDNA fragmentsandfortheanalysisofmixturesofPCRproductsfromthisgroupgeneratedbyselectivePCRofDNA extracted from coastal sand dunes. Degenerate PCR primers, CTO189f-GC and CTO654r, incorporating a 5* GC

  9. Comprehensive quantitative proteome analysis of 20S proteasome subtypes from rat liver by isotope coded affinity tag and 2-D gel-based approaches.

    PubMed

    Schmidt, Frank; Dahlmann, Burkhardt; Janek, Katharina; Kloss, Alexander; Wacker, Maik; Ackermann, Renate; Thiede, Bernd; Jungblut, Peter R

    2006-08-01

    Quantitative protein profiling is an essential part of proteomics and requires technologies that accurately, reproducibly, and comprehensively identify and quantify proteins. Over the past years, many quantitative proteomic methods have been developed. Here, 20S proteasome subtypes isolated from rat were compared by four approaches based on the combination of isotope-coded affinity tag (ICAT), 2-DE, LC and ESI and MALDI MS: (i) 2-DE, (ii) ICAT/2-DE MALDI-MS, (iii) ICAT/LC-ESI-MS, (iv) ICAT/LC-MALDI-MS. A definite qualitative advantage of 2-DE gels was the separation of all known protein species, the identification of cysteine sulfoxide of alpha-4 (RC6-IS) and N-terminal acetylation of several subunits. Furthermore, quantitative differences between the standard subunits beta-2, and beta-5 and their immunosubunits were only detected by 2-DE image analysis revealing a higher replacement of standard- by immuno-beta-subunits in subtype IV. It was obvious that for relative quantification only protein spot and mass peaks with a certain level of intensity displayed acceptable values of SD. However, ICAT in conjunction with LC/MALDI-MS was the most accurate method for quantification. The experimental data of this investigation are accessible via http://www.mpiib-berlin.mpg.de/2D-PAGE/. PMID:16858736

  10. Analysis of Molecular Epidemiology of Chilean Salmonella enterica Serotype Enteritidis Isolates by Pulsed-Field Gel Electrophoresis and Bacteriophage Typing

    PubMed Central

    Fernandez, Jorge; Fica, Alberto; Ebensperger, German; Calfullan, Hector; Prat, Soledad; Fernandez, Alda; Alexandre, Marcela; Heitmann, Ingrid

    2003-01-01

    Human Salmonella enterica serotype Enteritidis infections emerged in Chile in 1994. S. enterica serotype Enteritidis phage type 1 isolates predominated in the north, and phage type 4 isolates predominated in the central and southern regions. A study was planned to characterize this epidemic using the best discriminatory typing technique. Research involved 441 S. enterica serotype Enteritidis isolates, including clinical preepidemic samples (n = 74; 1975 to 1993) and epidemic (n = 199), food (n = 72), poultry (n = 57), and some Latin American (n = 39) isolates. The best method was selected based on a sample of preepidemic isolates, analyzing the discriminatory power (DP) obtained by phage typing and randomly amplified polymorphic DNA and pulsed-field gel electophoresis (PFGE) analysis. The highest DP was associated with BlnI PFGE-bacteriophage typing analysis (0.993). A total of 38 BlnI patterns (B patterns) were identified before the epidemic period, 19 since 1994, and only 4 in both periods. Two major clusters were identified by phylogenetic analysis, and the predominant B patterns clustered in the same branch. Combined analysis revealed that specific B pattern-phage type combinations (subtypes) disappeared before 1994, that different genotypes associated with S. enterica serotype Enteritidis phage type 4 had been observed since 1988, and that strain diversity increased before the expansion of S. enterica serotype Enteritidis in 1994. Predominant subtype B3-phage type 4 was associated with the central and southern regions, and subtype B38-phage type 1 was associated with the north (P < 0.0001). Food and poultry isolates matched the predominant S. enterica serotype Enteritidis subtypes, but isolates identified in neighboring countries (Peru and Bolivia) did not match S. enterica serotype Enteritidis subtypes identified in the north of Chile. The results of this work demonstrate that genetic diversity, replacement, and expansion of specific S. enterica serotype Enteritidis subtypes were associated with epidemic changes. PMID:12682153

  11. Determination of optimal protein quantity required to identify abundant and less abundant soybean seed proteins by 2D-PAGE and MS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Optimizing the amounts of proteins required to separate and characterize both abundant and less abundant proteins by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is critical for conducting proteomic research. In this study, we tested five different levels of soybean seed proteins (7...

  12. Use of Pulsed-Field Gel Electrophoresis To Characterize the Heterogeneity and Clonality of Salmonella Isolates Obtained from the Carcasses and Feces of Swine at Slaughter

    PubMed Central

    Wonderling, Laura; Pearce, Rachel; Wallace, F. Morgan; Call, Jeffrey E.; Feder, Ingrid; Tamplin, Mark; Luchansky, John B.

    2003-01-01

    Salmonella enterica isolates were recovered from swine at a collaborating processing plant over a 2-month period in the spring of 2000. In the present study, molecular subtyping by pulsed-field gel electrophoresis (PFGE) was performed on the 581 confirmed Salmonella isolates from the 84 Salmonella-positive samples obtained from the previous study. A total of 32 different PFGE pulsotypes were observed visually, and a BioNumerics software analysis clustered those pulsotypes into 12 PFGE groups. The B, F, and G groups predominated throughout the sampling period and were isolated from 39, 22, and 13% of the swine, respectively. In addition, multiple isolates were obtained from 67 of the 84 Salmonella-positive samples, and subtyping revealed multiple PFGE profiles in 35 of these 67 (62%) samples. Both carcass and fecal isolates of Salmonella were recovered from 13 swine, resulting in matched samples. Molecular typing of the 252 isolates recovered from the matched samples revealed that 7 (54%) of the 13 carcasses were contaminated with Salmonella pulsotypes that were not isolated from the feces of the same animal. Conversely, from 6 of the 13 (46%) matched animals, Salmonella clonal types were isolated from the feces that were not isolated from the carcass of the same animal. These data establish that each lot of swine introduces new contaminants into the plant environment and that swine feces from one animal can contaminate many carcasses. In addition, these results indicate that the examination of multiple Salmonella isolates from positive samples is necessary to determine the variety of potential contaminants of swine carcasses during slaughter and processing. PMID:12839796

  13. Boron bridging of rhamnogalacturonan-II, monitored by gel electrophoresis, occurs during polysaccharide synthesis and secretion but not post-secretion

    PubMed Central

    Chormova, Dimitra; Messenger, David J; Fry, Stephen C

    2014-01-01

    The cell-wall pectic domain rhamnogalacturonan-II (RG-II) is cross-linked via borate diester bridges, which influence the expansion, thickness and porosity of the wall. Previously, little was known about the mechanism or subcellular site of this cross-linking. Using polyacrylamide gel electrophoresis (PAGE) to separate monomeric from dimeric (boron-bridged) RG-II, we confirmed that Pb2+ promotes H3BO3-dependent dimerisation in vitro. H3BO3 concentrations as high as 50 mm did not prevent cross-linking. For in-vivo experiments, we successfully cultured Paul's Scarlet rose (Rosa sp.) cells in boron-free medium: their wall-bound pectin contained monomeric RG-II domains but no detectable dimers. Thus pectins containing RG-II domains can be held in the wall other than via boron bridges. Re-addition of H3BO3 to 3.3 ?m triggered a gradual appearance of RG-II dimer over 24 h but without detectable loss of existing monomers, suggesting that only newly synthesised RG-II was amenable to boron bridging. In agreement with this, Rosa cultures whose polysaccharide biosynthetic machinery had been compromised (by carbon starvation, respiratory inhibitors, anaerobiosis, freezing or boiling) lost the ability to generate RG-II dimers. We conclude that RG-II normally becomes boron-bridged during synthesis or secretion but not post-secretion. Supporting this conclusion, exogenous [3H]RG-II was neither dimerised in the medium nor cross-linked to existing wall-associated RG-II domains when added to Rosa cultures. In conclusion, in cultured Rosa cells RG-II domains have a brief window of opportunity for boron-bridging intraprotoplasmically or during secretion, but secretion into the apoplast is a point of no return beyond which additional boron-bridging does not readily occur. PMID:24320597

  14. Bacterial Community Composition in Central European Running Waters Examined by Temperature Gradient Gel Electrophoresis and Sequence Analysis of 16S rRNA Genes?

    PubMed Central

    Beier, Sara; Witzel, Karl-Paul; Marxsen, Jrgen

    2008-01-01

    The bacterial community composition in small streams and a river in central Germany was examined by temperature gradient gel electrophoresis (TGGE) with PCR products of 16S rRNA gene fragments and sequence analysis. Complex TGGE band patterns suggested high levels of diversity of bacterial species in all habitats of these environments. Cluster analyses demonstrated distinct differences among the communities in stream and spring water, sandy sediments, biofilms on stones, degrading leaves, and soil. The differences between stream water and sediment were more significant than those between sites within the same habitat along the stretch from the stream source to the mouth. TGGE data from an entire stream course suggest that, in the upper reach of the stream, a special suspended bacterial community is already established and changes only slightly downstream. The bacterial communities in water and sediment in an acidic headwater with a pH below 5 were highly similar to each other but deviated distinctly from the communities at the other sites. As ascertained by nucleotide sequence analysis, stream water communities were dominated by Betaproteobacteria (one-third of the total bacteria), whereas sediment communities were composed mainly of Betaproteobacteria and members of the Fibrobacteres/Acidobacteria group (each accounting for about 25% of bacteria). Sequences obtained from bacteria from water samples indicated the presence of typical cosmopolitan freshwater organisms. TGGE bands shared between stream and soil samples, as well as sequences found in bacteria from stream samples that were related to those of soil bacteria, demonstrated the occurrence of some species in both stream and soil habitats. Changes in bacterial community composition were correlated with geographic distance along a stream, but in comparisons of different streams and rivers, community composition was correlated only with environmental conditions. PMID:18024682

  15. Bacterial community composition in Central European running waters examined by temperature gradient gel electrophoresis and sequence analysis of 16S rRNA genes.

    PubMed

    Beier, Sara; Witzel, Karl-Paul; Marxsen, Jrgen

    2008-01-01

    The bacterial community composition in small streams and a river in central Germany was examined by temperature gradient gel electrophoresis (TGGE) with PCR products of 16S rRNA gene fragments and sequence analysis. Complex TGGE band patterns suggested high levels of diversity of bacterial species in all habitats of these environments. Cluster analyses demonstrated distinct differences among the communities in stream and spring water, sandy sediments, biofilms on stones, degrading leaves, and soil. The differences between stream water and sediment were more significant than those between sites within the same habitat along the stretch from the stream source to the mouth. TGGE data from an entire stream course suggest that, in the upper reach of the stream, a special suspended bacterial community is already established and changes only slightly downstream. The bacterial communities in water and sediment in an acidic headwater with a pH below 5 were highly similar to each other but deviated distinctly from the communities at the other sites. As ascertained by nucleotide sequence analysis, stream water communities were dominated by Betaproteobacteria (one-third of the total bacteria), whereas sediment communities were composed mainly of Betaproteobacteria and members of the Fibrobacteres/Acidobacteria group (each accounting for about 25% of bacteria). Sequences obtained from bacteria from water samples indicated the presence of typical cosmopolitan freshwater organisms. TGGE bands shared between stream and soil samples, as well as sequences found in bacteria from stream samples that were related to those of soil bacteria, demonstrated the occurrence of some species in both stream and soil habitats. Changes in bacterial community composition were correlated with geographic distance along a stream, but in comparisons of different streams and rivers, community composition was correlated only with environmental conditions. PMID:18024682

  16. Boron bridging of rhamnogalacturonan-II, monitored by gel electrophoresis, occurs during polysaccharide synthesis and secretion but not post-secretion.

    PubMed

    Chormova, Dimitra; Messenger, David J; Fry, Stephen C

    2014-02-01

    The cell-wall pectic domain rhamnogalacturonan-II (RG-II) is cross-linked via borate diester bridges, which influence the expansion, thickness and porosity of the wall. Previously, little was known about the mechanism or subcellular site of this cross-linking. Using polyacrylamide gel electrophoresis (PAGE) to separate monomeric from dimeric (boron-bridged) RG-II, we confirmed that Pb(2+) promotes H3 BO3 -dependent dimerisation in vitro. H3 BO3 concentrations as high as 50 mm did not prevent cross-linking. For in-vivo experiments, we successfully cultured 'Paul's Scarlet' rose (Rosa sp.) cells in boron-free medium: their wall-bound pectin contained monomeric RG-II domains but no detectable dimers. Thus pectins containing RG-II domains can be held in the wall other than via boron bridges. Re-addition of H3 BO3 to 3.3 ?m triggered a gradual appearance of RG-II dimer over 24 h but without detectable loss of existing monomers, suggesting that only newly synthesised RG-II was amenable to boron bridging. In agreement with this, Rosa cultures whose polysaccharide biosynthetic machinery had been compromised (by carbon starvation, respiratory inhibitors, anaerobiosis, freezing or boiling) lost the ability to generate RG-II dimers. We conclude that RG-II normally becomes boron-bridged during synthesis or secretion but not post-secretion. Supporting this conclusion, exogenous [(3) H]RG-II was neither dimerised in the medium nor cross-linked to existing wall-associated RG-II domains when added to Rosa cultures. In conclusion, in cultured Rosa cells RG-II domains have a brief window of opportunity for boron-bridging intraprotoplasmically or during secretion, but secretion into the apoplast is a point of no return beyond which additional boron-bridging does not readily occur. PMID:24320597

  17. Genotyping of Francisella tularensis Strains by Pulsed-Field Gel Electrophoresis, Amplified Fragment Length Polymorphism Fingerprinting, and 16S rRNA Gene Sequencing

    PubMed Central

    Garca Del Blanco, N.; Dobson, M. E.; Vela, A. I.; De La Puente, V. A.; Gutirrez, C. B.; Hadfield, T. L.; Kuhnert, P.; Frey, J.; Domnguez, L.; Rodrguez Ferri, E. F.

    2002-01-01

    We evaluated three molecular methods for identification of Francisella strains: pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and 16S rRNA gene sequencing. The analysis was performed with 54 Francisella tularensis subsp. holarctica, 5 F. tularensis subsp. tularensis, 2 F. tularensis subsp. novicida, and 1 F. philomiragia strains. On the basis of the combination of results obtained by PFGE with the restriction enzymes XhoI and BamHI, PFGE revealed seven pulsotypes, which allowed us to discriminate the strains to the subspecies level and which even allowed us to discriminate among some isolates of F. tularensis subsp. holarctica. The AFLP analysis technique produced some degree of discrimination among F. tularensis subsp. holarctica strains (one primary cluster with three major subclusters and minor variations within subclusters) when EcoRI-C and MseI-A, EcoRI-T and MseI-T, EcoRI-A and MseI-C, and EcoRI-0 and MseI-CA were used as primers. The degree of similarity among the strains was about 94%. The percent similarities of the AFLP profiles of this subspecies compared to those of F. tularensis subsp. tularensis, F. tularensis subsp. novicida, and F. philomiragia were less than 90%, about 72%, and less than 24%, respectively, thus permitting easy differentiation of this subspecies. 16S rRNA gene sequencing revealed 100% similarity for all F. tularensis subsp. holarctica isolates compared in this study. These results suggest that although limited genetic heterogeneity among F. tularensis subsp. holarctica isolates was observed, PFGE and AFLP analysis appear to be promising tools for the diagnosis of infections caused by different subspecies of F. tularensis and suitable techniques for the differentiation of individual strains. PMID:12149360

  18. Genetic Diversity of nifH Gene Sequences in Paenibacillus azotofixans Strains and Soil Samples Analyzed by Denaturing Gradient Gel Electrophoresis of PCR-Amplified Gene Fragments

    PubMed Central

    Rosado, Alexandre S.; Duarte, Gabriela F.; Seldin, Lucy; Van Elsas, Jan Dirk

    1998-01-01

    The diversity of dinitrogenase reductase gene (nifH) fragments in Paenibacillus azotofixans strains was investigated by using molecular methods. The partial nifH gene sequences of eight P. azotofixans strains, as well as one strain each of the close relatives Paenibacillus durum, Paenibacillus polymyxa, and Paenibacillus macerans, were amplified by PCR by using degenerate primers and were characterized by DNA sequencing. We found that there are two nifH sequence clusters, designated clusters I and II, in P. azotofixans. The data further indicated that there was sequence divergence among the nifH genes of P. azotofixans strains at the DNA level. However, the gene products were more conserved at the protein level. Phylogenetic analysis showed that all nifH cluster II sequences were similar to the alternative (anf) nitrogenase sequence. A nested PCR assay for the detection of nifH (cluster I) of P. azotofixans was developed by using the degenerate primers as outer primers and two specific primers, designed on the basis of the sequence information obtained, as inner primers. The specificity of the inner primers was tested with several diazotrophic bacteria, and PCR revealed that these primers are specific for the P. azotofixans nifH gene. A GC clamp was attached to one inner primer, and a denaturing gradient gel electrophoresis (DGGE) protocol was developed to study the genetic diversity of this region of nifH in P. azotofixans strains, as well as in soil and rhizosphere samples. The results revealed sequence heterogeneity among different nifH genes. Moreover, nifH is probably a multicopy gene in P. azotofixans. Both similarities and differences were detected in the P. azotofixans nifH DGGE profiles generated with soil and rhizosphere DNAs. The DGGE assay developed here is reproducible and provides a rapid way to assess the intraspecific genetic diversity of an important functional gene in pure cultures, as well as in environmental samples. PMID:9687429

  19. Identification of bacteria in a biodegraded wall painting by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA.

    PubMed Central

    Rlleke, S; Muyzer, G; Wawer, C; Wanner, G; Lubitz, W

    1996-01-01

    Medieval wall paintings are often affected by biodecay. An inventory of the existing microorganisms associated with the damage to the paintings is not yet an integral part of the restoration process. This stems from the lack of effective means for such a stocktaking. Nevertheless, fungi and bacteria cause severe damage through mechanical processes from growth into the painting and its grounding and through their metabolism. Detailed information on the bacterial colonization of ancient wall paintings is essential for the protection of the paintings. We used a molecular approach based on the detection and identification of DNA sequences encoding rRNA (rDNA) to identify bacteria present on an ancient wall painting without prior cultivation of the organisms, since it has been shown that most of these bacteria cannot be cultivated under laboratory conditions. To trace the noncultivated fraction of bacteria, total DNA from a biodegraded wall painting sample from a 13th century fresco was extracted and 194-bp fragments of the 16S rDNA were amplified with eubacterial primers. The 16S rDNA fragments of uniform length obtained from the different bacterial species were separated according to their sequence differences by denaturing gradient gel electrophoresis (DGGE). By sequencing excised and reamplified individual DNA bands, we characterized the phylogenetic affiliation of the corresponding bacteria. Using this approach, we identified members or close relatives of the genera Halomonas, Clostridium, and Frankia. To our knowledge, these groups of bacteria have not yet been isolated and implicated by conventional microbiological techniques as contributing to the biodegradation of wall paintings. PMID:8787403

  20. Diversity and seasonal variability of beta-Proteobacteria in biofilms of polluted rivers: analysis by temperature gradient gel electrophoresis and cloning.

    PubMed

    Brmmer, I H M; Felske, A; Wagner-Dbler, I

    2003-08-01

    The beta-subgroup of the Proteobacteria has been shown to be important in aquatic habitats and was investigated in depth here by molecular 16S rRNA techniques in biofilms of the Elbe River and its polluted tributary, the Spittelwasser River. The bacterial 16S rRNA genes were cloned from each site, screened for beta-proteobacterial clones and sequenced. River biofilm clones from both rivers grouped into 9 clusters (RBFs). RBFs 1, 2, and 3 fell into the recently described betaI cluster of cosmopolitan freshwater bacteria, where they represented new species related to Rhodoferax, Aquaspirillum, and Hydrogenophaga: RBFs 4 to 7 affiliated with Aquabacterium commune, Ideonella dechloratans, and Sphaerotilus natans, respectively. The two remaining RBFs were uncultivated clusters, one of them being distantly related to Gallionella ferruginea. Seasonal changes in the relative intensity of the beta-proteobacterial 16S rRNA genes of biofilms harvested monthly for 1 year were determined by specific amplification and separation by temperature gradient gel electrophoresis (TGGE). Bands were identified by comparison of clones to community fingerprints by TGGE. Eight of 13 identified bands were shared by both habitats but showed different relative abundance and seasonal variability in the two rivers, probably caused by differences in temperature and pollutants. The data indicate new not-yet-cultivated clusters of river biofilm organisms, some of them probably distributed globally. They confirm the importance of certain known freshwater genera in river biofilms. The high phylogenetic resolution obtained by clone library analysis combined with the high temporal resolution obtained by TGGE suggest that the observed microdiversity in the river biofilm clone libraries might be caused by phylogenetically closely related microbial populations which are adapted to ecological parameters. PMID:12902230