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1

Silver Staining of 2D Electrophoresis Gels Thierry Rabilloud  

E-print Network

and 10 times higher than colloidal Coomassie Blue. However, the first silver staining protocols wereSilver Staining of 2D Electrophoresis Gels Thierry Rabilloud CEA-DSV-iRTSV/LCBM and UMR CNRS Head: Silver staining #12;i. Abstract Silver staining is used to detect proteins after electrophoretic

Paris-Sud XI, Université de

2

Protein profiling of human postmortem brain using 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE)  

Microsoft Academic Search

Two-dimensional gel electrophoresis (2-D GE) is a key tool for comparative proteomics research. With its ability to separate complex protein mixtures with high resolution, 2-D GE is a technique commonly employed for protein profiling studies. Significant improvements have been made in 2-D GE technology with the development of two-dimensional fluorescence difference gel electrophoresis (2-D DIGE), where proteins are first labelled

J E Swatton; S Prabakaran; N A Karp; K S Lilley; S Bahn

2004-01-01

3

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

E-print Network

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 with Coomassie Blue. However, the first silver staining protocols were not trouble-free. High

Paris-Sud XI, Université de

4

Gel Electrophoresis  

NSDL National Science Digital Library

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.

Foundation, Wgbh E.

2007-04-19

5

Total protein extraction and 2-D gel electrophoresis methods for Burkholderia species.  

PubMed

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining. PMID:24192802

Velapatio, Billie; Zlosnik, James E A; Hird, Trevor J; Speert, David P

2013-01-01

6

Gel Electrophoresis  

NSDL National Science Digital Library

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.

Yu, Julie

2007-01-01

7

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

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.

2008-10-06

8

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

PubMed Central

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

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

2009-01-01

9

Wheat quality related differential expressions of albumins and globulins revealed by two-dimensional difference gel electrophoresis (2-D DIGE)  

Microsoft Academic Search

Comparative proteomics analysis offers a new approach to identify differential proteins among different wheat genotypes and developmental stages. In this study, the non-prolamin expression profiles during grain development of two common or bread wheat cultivars (Triticum aestivum L.), Jing 411 and Sunstate, with different quality properties were analyzed using two-dimensional difference gel electrophoresis (2-D DIGE). Five grain developmental stages during

Liyan Gao; Aili Wang; Xiaohui Li; Kun Dong; Ke Wang; Rudi Appels; Wujun Ma; Yueming Yan

2009-01-01

10

A 2-D gel electrophoresis DNA image analysis algorithm with automatic thresholding  

NASA Astrophysics Data System (ADS)

Polymerase chain reaction (PCR) and gel electrophoresis are two widely used techniques for genetic studies that require the bench scientist to perform many tedious manual steps. Advances in automation are making these techniques more accessible, but detection and image analysis still remain labor-intensive. Although several commercial software packages are now available, DNA image analysis still requires some intervention by the user, and thus a certain level of image processing expertise. To allow researchers to speed up their analyses and obtain more repeatable results, we present a fully automated image analysis system for DNA or protein studies with high accuracy. The proposed system is based mainly on four steps: automatic thresholding, shifting, filtering, and processing. The automatic thresholding that is used to equalize the gray values of the gel electrophoreses image background is one of the key and novel operations in this algorithm. An enhancement is also used to improve poor quality images that have faint DNA bands. Experimental results show that the proposed method eliminates defects due to noise for good and average quality gel electrophoresis images, while it also improves the appearance of poor quality images.

Kaabouch, Naima; Schultz, Richard R.

2007-01-01

11

Protein expression dynamics during replicative senescence of endothelial cells studied by 2-D difference in-gel electrophoresis.  

PubMed

Endothelial senescence contributes to endothelium dysfunctionality and is thereby linked to vascular aging. A dynamic proteomic study on human umbilical vein endothelial cells, isolated from three umbilical cords, was performed. The cells were cultured towards replicative senescence and whole cell lysates were subjected to 2-D difference gel electrophoresis (DIGE). Despite the biological variability of the three independent isolations, a set of proteins was found that showed senescence-dependent expression patterns in all isolations. We focused on those proteins that showed significant changes, with a paired analysis of variance (RM-ANOVA) p-value of < or =0.05. Thirty-five proteins were identified with LC-Fourier transform MS, and functional annotation revealed that endothelial replicative senescence is accompanied by increased cellular stress, protein biosynthesis and reduction in DNA repair and maintenance. Nuclear integrity becomes affected and cytoskeletal structure is also changed. Such important changes in the cell infrastructure might accelerate endothelium dysfunctionality. This study provides biological information that will initiate studies to further unravel endothelial senescence and gain more knowledge about the consequences of this process in the in vivo situation. PMID:16609940

Eman, Michael R; Regan-Klapisz, Elsa; Pinkse, Martijn W H; Koop, Inge M; Haverkamp, Johan; Heck, Albert J R; Verkleij, Arie J; Post, Jan A

2006-04-01

12

Electrophoresis and Gel Analysis  

NSDL National Science Digital Library

In this animation produced by WGBH and Digizyme, Inc., see how molecules of DNA are separated using gel electrophoresis, and how this process enables scientists to compare the molecular variations of two or more DNA samples.

2011-08-05

13

Gel Electrophoresis of Dyes  

NSDL National Science Digital Library

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).

Stephens, Janice; Leach, Jan

2011-01-01

14

Light-triggered conversion of non-ionic into ionic surfactants: towards chameleon detergents for 2-D gel electrophoresis.  

PubMed

Caged non-ionic detergents, comprised of polar oligo(ethylene glycol) and non-polar alkyl chains joined by a photocleavable ortho-nitrobenzyl sulfonate linker have been synthesized and characterized. The light-triggered transformation of such chameleon surfactant from a charge-neutral into a charged form offers great potential to improve 2-D gel electrophoretic separation of complex protein mixtures. PMID:22222955

Bogen, Oliver; Milosevic, Petar; Daroszi, Hans; Dreger, Mathias; Hecht, Stefan; Hucho, Ferdinand

2012-03-01

15

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

PubMed Central

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

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

2010-01-01

16

Interactions by 2D Gel Electrophoresis Overlap (iGEO): a novel high fidelity approach to identify constituents of protein complexes  

PubMed Central

Background Here we describe a novel approach used to identify the constituents of protein complexes with high fidelity, using the integrin-associated scaffolding protein PINCH as a test case. PINCH is comprised of five LIM domains, zinc-finger protein interaction modules. In Drosophila melanogaster, PINCH has two known high-affinity binding partnersIntegrin-linked kinase (ILK) that binds to LIM1 and Ras Suppressor 1 (RSU1) that binds to LIM5but has been postulated to bind additional proteins as well. Results To purify PINCH complexes, in parallel we fused different affinity tags (Protein A and Flag) to different locations within the PINCH sequence (N- and C-terminus). We expressed these tagged versions of PINCH both in cell culture (overexpressed in Drosophila S2 cell culture in the presence of endogenous PINCH) and in vivo (at native levels in Drosophila lacking endogenous PINCH). After affinity purification, we analyzed PINCH complexes by a novel 2D-gel electrophoresis analysis, iGEO (interactions by 2D Gel Electrophoresis Overlap), with mass spectrometric identification of individual spots of interest. iGEO allowed the identification of protein partners that associate with PINCH under two independent purification strategies, providing confidence in the significance of the interaction. Proteins identified by iGEO were validated against a highly inclusive list of candidate PINCH interacting proteins identified in previous analyses by MuDPIT mass spectrometry. Conclusions The iGEO strategy confirmed a core complex comprised of PINCH, RSU1, ILK, and ILK binding partner Parvin. Our iGEO method also identified five novel protein partners that specifically interacted with PINCH in Drosophila S2 cell culture. Because of the improved reproducibility of 2D-GE methodology and the increasing affordability of the required labeling reagents, iGEO is a method that is accessible to most moderately well-equipped biological laboratories. The biochemical co-purifications inherent in iGEO allow for rapid and unambiguous identification of the constituents of protein complexes, without the need for extensive follow-up experiments. PMID:23663728

2013-01-01

17

Copolymers For Capillary Gel Electrophoresis  

DOEpatents

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.

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

2005-08-09

18

2D difference gel electrophoresis analysis of different time points during the course of neoplastic transformation of human mammary epithelial cells.  

PubMed

Cell culture models of oncogenesis that use cellular reprogramming to generate a neoplastic cell from a normal cell provide one of the few opportunities to study the early stages of breast cancer development. Human mammary epithelial cells (HMECs) were induced to undergo a neoplastic transformation using defined genetic elements to generate transformed HMECs (THMECs). To identify proteins that displayed significantly different levels of abundance at three consecutive time points in oncogenesis over an 80 day period, protein extracts were analyzed by two-dimensional difference gel electrophoresis (2D-DIGE). Nine proteins were found to be significantly different in abundance: keratin 1, keratin 7, heat shock protein 4A-like, t-complex protein 1, stathmin, gelsolin, FK506 binding protein 5, ribosomal protein P0, and maspin. Keratin 7 and maspin displayed a linear down-regulation over 80 days. All of these proteins have been shown to be involved in the maintenance of a metastatic state including cytoskeletal modifications and motility. We conclude that, following neoplastic induction, THMECs display an early and progressive increase in metastatic potential. Further investigations into the function and regulatory mechanisms of these proteins will provide an unparalleled understanding of the initial states through which a breast cancer cell transitions following acquisition of the genetic abnormalities required for oncogenesis. PMID:21105747

DeAngelis, J Tyson; Li, Yuanyuan; Mitchell, Natalie; Wilson, Landon; Kim, Helen; Tollefsbol, Trygve O

2011-02-01

19

2D Difference Gel Electrophoresis Analysis of Different Time Points during the Course of Neoplastic Transformation of Human Mammary Epithelial Cells  

PubMed Central

Cell culture models of oncogenesis that use cellular reprogramming to generate a neoplastic cell from a normal cell provide one of the few opportunities to study the early stages of breast cancer development. Human mammary epithelial cells (HMECs) were induced to undergo a neoplastic transformation using defined genetic elements to generate transformed HMECs (THMECs). To identify proteins that displayed significantly different levels of abundance at three consecutive time points in oncogenesis over an 80 day period, protein extracts were analyzed by two-dimensional difference gel electrophoresis (2D-DIGE). Nine proteins were found to be significantly different in abundance: keratin 1, keratin 7, heat shock protein 4A-like, t-complex protein 1, stathmin, gelsolin, FK506 binding protein 5, ribosomal protein P0, and maspin. Keratin 7 and maspin displayed a linear down-regulation over 80 days. All of these proteins have been shown to be involved in the maintenance of a metastatic state including cytoskeletal modifications and motility. We conclude that, following neoplastic induction, THMECs display an early and progressive increase in metastatic potential. Further investigations into the function and regulatory mechanisms of these proteins will provide an unparalleled understanding of the initial states through which a breast cancer cell transitions following acquisition of the genetic abnormalities required for oncogenesis. PMID:21105747

DeAngelis, J. Tyson; Li, Yuanyuan; Mitchell, Natalie; Wilson, Landon; Kim, Helen; Tollefsbol, Trygve O.

2012-01-01

20

Agarose Gel Electrophoresis of RNA  

Microsoft Academic Search

\\u000a Gel electrophoresis is one of the most important techniques currently available for the fractionation of RNA. The experimental\\u000a procedure is relatively simple, but nevertheless achieves very reproducible results and high resolution. RNA is a polyanion\\u000a and will therefore migrate toward the positive electrode in an electric field. If the migration occurs through a gel matrix\\u000a of carefully chosen pore size,

Robert J. Slater

21

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.

2013-07-05

22

Unified Theory for Gel Electrophoresis and Gel Filtration  

Microsoft Academic Search

Unified theory for gel electrophoresis and gel filtration: The behavior of macromolecules in gel filtration and gel electrophoresis may be predicted from Ogston's model for a random meshwork of fibers. This model has been generalized to apply to nonspherical molecules and to several gel types. The model provides equations for inter-relationships between mobility, partition coefficient, gel concentration, and molecular radius;

David Rodbard; Andreas Chrambach

1970-01-01

23

Pre-Cast Gel Electrophoresis Guide  

E-print Network

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.....................................................................................................................1 Novex® Pre-Cast Gels

Kirschner, Marc W.

24

Conducting Polymer Electrodes for Gel Electrophoresis  

PubMed Central

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

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

2014-01-01

25

Application of both high-performance liquid chromatography combined with tandem mass spectrometry shotgun and 2-D polyacrylamide gel electrophoresis for streptococcal exoproteins gave reliable proteomic data.  

PubMed

Streptococci secrete a large number of exoproteins including virulence-associated toxins and enzymes. To construct a reliable database of streptococcal exoproteins, we integrated the results that were derived from two approaches: LC-based shotgun proteomic analysis and 2-D PAGE-based proteomic analysis. We identified 74 and 82 proteins by LC-based and gel-based analysis, respectively. Forty-five proteins were identified by both methods. In addition, two proteins, one identified by both methods and the other only by LC-based shotgun analysis, were newly annotated. We therefore found the importance of combinational analysis by the two methods for the construction of a more reliable database. PMID:21204954

Okamoto, Akira; Hasegawa, Tadao; Yamada, Keiko; Ohta, Michio

2011-02-01

26

Separation of basic proteins from Leishmania using a combination of Free flow electrophoresis (FFE) and 2D electrophoresis (2-DE) under basic conditions.  

PubMed

Basic proteins, an important class of proteins in intracellular organisms such as Leishmania, are usually underrepresented on 2D gels. This chapter describes a method combining basic proteins fractionation using Free flow electrophoresis in isoelectric focusing mode (IEF-FFE) followed by protein separation using two-dimensional gel electrophoresis (2-DE) in basic conditions. The combination of these two techniques represents a great improvement for the visualization of Leishmania proteins with basic pI using 2D gels. PMID:25388119

Brotherton, Marie-Christine; Racine, Gina; Ouellette, Marc

2015-01-01

27

Proteomic analysis of Acinetobacter lwoffii K24 by 2-D gel electrophoresis and electrospray ionization quadrupole-time of flight mass spectrometry.  

PubMed

The MS/MS analysis by Electrospray ionization quadrupole-time of flight mass spectrometry (ESI-Q-TOF MS) was applied to identify proteins in proteome analysis of bacteria whose genomes are not known. The protein identification by ESI-Q-TOF MS was performed sequentially by database search and then de novo sequencing using MS/MS spectra. Soil bacteria having unanalyzed genome, Acinetobacter lwoffii K24 is an aniline degrading bacterium. In this report, we present the results of a comparison between the proteome profile of A. lwoffii K24 cultured in aniline- or succinate-containing media. Protein analysis was performed using two-dimensional gel electrophoresis (2-DE) with pH 3-10 immobilized pH gradient (IPG) strips followed by ESI-Q-TOF MS. More than 780 protein spots were detected by 2-DE from the soluble proteome. Forty-eight of these proteins were expressed exclusively in aniline cultured bacteria, and 81 proteins increased and 162 proteins decreased in aniline-cultured versus succinate cultured A. lwoffii K24. Internal amino acid sequences of 43 major protein spots were successfully determined by ESI-Q-TOF MS to try to identify the bacterial proteins responding to aniline culture condition. Since the A. lwoffii K24 genome is not yet sequenced, many proteins were found to be hypothetical. Comparative proteome analysis of the insoluble protein fractions showed that one novel protein that was strongly induced by succinate-cultured A. lwoffii K24 was repressed under aniline culture conditions. These results suggest that comprehensive analysis of bacterial proteomes by 2-DE and amino acid sequence analysis by ESI-Q-TOF MS is useful for understanding induced novel proteins of biodegrading bacteria. PMID:15134882

Kim, Eun-A; Kim, Jin Young; Kim, Soo-Jung; Park, Kyeong Ryang; Chung, Hae-Jin; Leem, Sun-Hee; Kim, Seung Il

2004-06-01

28

A Simple Vertical Slab Gel Electrophoresis Apparatus.  

ERIC Educational Resources Information Center

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

Carter, J. B.; And Others

1983-01-01

29

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

Microsoft Academic Search

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

Gerard Muyzer; Kornelia Smalla

1998-01-01

30

Abbreviations: IEF, isoelectric focussing; NEPHGE, non-equilibrium pH gradient electrophoresis; PAGE, polyacrylamide gel electrophoresis; pI, isoelectric point; SDS, sodium dodecyl sulphate; SI, self-incompatibility; 1-D, 1 dimensional; 2-D, 2 dimensional  

E-print Network

-incompatibility; 1-D, 1 dimensional; 2-D, 2 dimensional 6 A Proteomics Approach to the Study of Distyly in Turnera of the proteins involved in distyly. Distyly is a genetic polymorphism in which there are two self of reproductive organs. Little is known of the molecular basis of distyly in any species. Previous molecular

Shore, Joel S.

31

Gel Electrophoresis on a Budget to Dye For  

NSDL National Science Digital Library

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

Yu, Julie H.

2010-01-01

32

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 âprecursor to DNA separationsâ 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.

2008-12-05

33

RNA purification by preparative polyacrylamide gel electrophoresis.  

PubMed

Preparative polyacrylamide gel electrophoresis (PAGE) is a powerful tool for purifying RNA samples. Denaturing PAGE allows separation of nucleic acids that differ by a single nucleotide in length. It is commonly used to separate and purify RNA species after in vitro transcription, to purify naturally occurring RNA variants such as tRNAs, to remove degradation products, and to purify labeled RNA species. To preserve RNA integrity following purification, RNA is usually visualized by UV shadowing or stained with ethidium bromide or SYBR green dyes. PMID:24034329

Petrov, Alexey; Wu, Tinghe; Puglisi, Elisabetta Viani; Puglisi, Joseph D

2013-01-01

34

Hybrid slab-microchannel gel electrophoresis system  

DOEpatents

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.

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

1998-01-01

35

Micro-size polyacrylamide gel electrophoresis system  

NASA Astrophysics Data System (ADS)

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

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

1987-09-01

36

Inexpensive and Safe DNA Gel Electrophoresis Using Household Materials  

ERIC Educational Resources Information Center

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

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

2012-01-01

37

Hybrid slab-microchannel gel electrophoresis system  

DOEpatents

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.

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

1998-05-05

38

Starch-gel Electrophoresis of Anterior Pituitary Hormones  

Microsoft Academic Search

SEPARATION of pituitary hormones by zone electrophoresis on paper has not proved very successful, possibly due to complex formation between proteins1. Starch-gel zone electrophoresis introduced by Smithies2,3 separates proteins not only by their net charge but also by their molecular size. This procedure has superior resolving power to paper or agar-gel zone electrophoresis and has been successfully applied to the

K. A. Ferguson; A. L. C. Wallace

1961-01-01

39

Improvements to polar 2-D electrophoresis for proteomic applications.  

PubMed

Recently, we reported a new way of performing 2-DE, called P-dimensional electrophoresis (2-PE). In this approach, the second dimension is achieved in a radial gel which can accommodate up to six 7cm long IPG strips simultaneously, improving reproducibility and throughput power in respect to 2-DE. Nevertheless, 2-PE was up to now limited to the use of only short strips because of technical difficulties. Here, we describe how to load longer strips (e.g., 18-24cm) on 2-PE and report some representative images for a qualitative assessment. PMID:24459004

Polati, Rita; Cecconi, Daniela; Dosselli, Ryan; Zaccarin, Mattia; Millioni, Renato

2014-04-01

40

Two-dimensional gel electrophoresis image registration using block-matching techniques and deformation models.  

PubMed

Block-matching techniques have been widely used in the task of estimating displacement in medical images, and they represent the best approach in scenes with deformable structures such as tissues, fluids, and gels. In this article, a new iterative block-matching technique-based on successive deformation, search, fitting, filtering, and interpolation stages-is proposed to measure elastic displacements in two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) images. The proposed technique uses different deformation models in the task of correlating proteins in real 2D electrophoresis gel images, obtaining an accuracy of 96.6% and improving the results obtained with other techniques. This technique represents a general solution, being easy to adapt to different 2D deformable cases and providing an experimental reference for block-matching algorithms. PMID:24613260

Rodriguez, Alvaro; Fernandez-Lozano, Carlos; Dorado, Julian; Rabual, Juan R

2014-06-01

41

Dna electrophoresis in photopolymerized polyacrylamide gels on a microfluidic device  

E-print Network

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...

Lo, Chih-Cheng

2009-05-15

42

Gel Electrophoresis on a Budget to Dye for  

ERIC Educational Resources Information Center

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

Yu, Julie H.

2010-01-01

43

Functional Annotation of 2D Protein Maps: The GelMap Portal  

PubMed Central

In classical proteome analyses, final experimental data are (a) images of 2D protein separations obtained by gel electrophoresis and (b) corresponding lists of proteins which were identified by mass spectrometry (MS). For data annotation, software tools were developed which allow the linking of protein identity data directly to 2D gels (clickable gels). GelMap is a new online software tool to annotate 2D protein maps. It allows (i) functional annotation of all identified proteins according to biological categories defined by the user, e.g., subcellular localization, metabolic pathway, or assignment to a protein complex and (ii) annotation of several proteins per analyzed protein spot according to MS primary data. Options to differentially display proteins of functional categories offer new opportunities for data evaluation. For instance, if used for the annotation of 2D Blue native/SDS gels, GelMap allows the identification of protein complexes of low abundance. A web portal has been established for presentation and evaluation of protein identity data related to 2D gels and is freely accessible at http://www.gelmap.de/. PMID:22639671

Senkler, Michael; Braun, Hans-Peter

2012-01-01

44

Gel Electrophoresis--The Easy Way for Students  

ERIC Educational Resources Information Center

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

VanRooy, Wilhelmina; Sultana, Khalida

2010-01-01

45

Gel Electrophoresis of Gold-DNA Nano-Conjugates  

SciTech Connect

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.

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

2006-01-10

46

Nanoparticle gel electrophoresis: bare charged spheres in polyelectrolyte hydrogels.  

PubMed

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

Li, Fei; Hill, Reghan J

2013-03-15

47

THERMAL DETECTION OF DNA AND PROTEINS DURING GEL ELECTROPHORESIS  

SciTech Connect

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.

R. JOHNSTON

2000-08-01

48

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

PubMed

The present study accents on the privileges of microchip-based electrophoresis to the conventional gel electrophoresis in separation of telomerase repeat amplification protocol/polymerase chain reaction (PCR) ladder products obtained in telomerase-catalyzed reaction in cancer cells. We try to clarify the interpretation of the results obtained by both electrophoretic procedures and to avoid misinterpretation as a result of PCR-dependent artefacts. PMID:16078194

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

2005-08-01

49

Zone Electrophoresis of Human Parotid Saliva in Acrylamide Gel  

Microsoft Academic Search

THE zone electrophoresis of human parotid saliva proteins has been carried out with a variety of supporting media, including filter paper, cellulose acetate, agar gel and starch gel, with separations usually of from five to twelve fractions1-8. In a comparative investigation of these media, D'Silva and Ferguson7 obtained their most satisfactory results with a combination of cellulose acetate and starch

T. S. Meyer; B. L. Lamberts

1965-01-01

50

Solid friction in gel electrophoresis S. F. Burlatskya)  

E-print Network

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

Deutch, John

51

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.

52

Statistical analysis of denaturing gel electrophoresis (DGE) fingerprinting patterns  

Microsoft Academic Search

Summary Technical developments in molecular biology have found extensive applications in the field of microbial ecology. Among these techniques, fingerprinting methods such as denaturing gel electrophoresis (DGE, including the three options: DGGE, TGGE and TTGE) has been applied to environmental samples over this last decade. Microbial ecologists took advantage of this technique, originally developed for the detection of single mutations,

N. Fromin; J. Hamelin; S. Tarnawski; D. Roesti; F. Gillet; M. Aragno; P. Rossi

2002-01-01

53

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

E-print Network

-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

Sherman, Tim

54

Protocols of two-dimensional difference gel electrophoresis to investigate mechanisms of toxicity.  

PubMed

In recent years, several global omics technologies have been increasingly used to better understand the molecular mechanisms of drug toxicity. Two-dimensional difference gel electrophoresis (2D-DIGE) is a large-scale proteomics high-resolution gel-based quantitative method widely used to detect protein expression alterations after drug treatment. The 2D-DIGE technology is based on the labeling of proteins with different fluorescent dyes, allowing the separation of different samples on the same gel with the use of an internal standard, thus reducing the complexity of spot pattern comparison and providing a reliable method applied to toxicology studies for the detection of modulated proteins in targeted organs. PMID:20972754

Com, Emmanuelle; Gruhler, Albrecht; Courcol, Martine; Gautier, Jean-Charles

2011-01-01

55

Microfluidic 2-D PAGE using multifunctional in situ polyacrylamide gels and discontinuous buffers.  

PubMed

A two-dimensional microfluidic system is presented for intact protein separations combining isoelectric focusing (IEF) and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) employing in situ photopolymerized polyacrylamide (PAAm) gels. The PAAm gels are used for multiple functions. In addition to serving as a highly-resolving separation medium for gel electrophoresis, discrete polyacrylamide gel plugs are used to enable the efficient isolation of different on-chip media including anolyte, catholyte, and sample/ampholyte solutions for IEF. The gel plugs are demonstrated as on-chip reagent containers, holding defined quantities of SDS for on-chip SDS-protein complexation, and enabling the use of a discontinuous buffer system for sample band sharpening during SDS-PAGE. The 2-D chip also employs several unique design features including an angled isoelectric focusing channel to minimize sample tailing, and backbiasing channels designed to achieve uniform interdimensional sample transfer. Separation results using E. coli cell lysate are presented using a 10-channel chip with and without the discontinuous buffer system, with resolving power more than doubled in the former case. Further improvements in separation resolution are demonstrated using a 20-channel chip design. PMID:19190795

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

2009-02-21

56

Analysis of RNA by analytical polyacrylamide gel electrophoresis.  

PubMed

Polyacrylamide gel electrophoresis (PAGE) is a powerful tool for analyzing RNA samples. Denaturing PAGE provides information on the sample composition and structural integrity of the individual RNA species. Nondenaturing gel electrophoresis allows separation of the conformers and alternatively folded RNA species. It also can be used to resolve RNA protein complexes and to detect RNA complex formation by analyzing changes in the electrophoretic mobility of the RNA. RNA can be visualized within gels by different methods depending on the nature of the detection reagent. RNA molecules can be stained with various dyes, including toluidine blue, SYBR green, and ethidium bromide. Radioactively labeled RNA molecules are visualized by autoradiography, and fluorescently labeled RNA molecules can be observed with a fluorescence scanner. Generally, gels between 0.4 and 1.5mm thick are used for analytical PAGE. Gels thinner than 1mm are fragile and thus usually are not stained but rather are used for radiolabeled RNA. The gels are dried and the radiolabeled RNA is visualized by autoradiography. PMID:24034328

Petrov, Alexey; Tsa, Albet; Puglisi, Joseph D

2013-01-01

57

Thermally reversible gels in electrophoresis. I - Matrix characterization  

NASA Technical Reports Server (NTRS)

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.

Righetti, Pier Giorgio; Snyder, Robert S.

1988-01-01

58

SDS capillary gel electrophoresis of proteins in microfabricated channels  

PubMed Central

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

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

1999-01-01

59

Comparative gel electrophoresis analysis of helical junctions in RNA.  

PubMed

Comparative gel electrophoresis provides information on the relative angles subtended between helical arms at a branchpoint in RNA. It is based upon the comparison of electrophoretic mobility in polyacrylamide gels of species containing two long arms, with the remaining one(s) being significantly shorter. Although the method currently lacks a really well-established basis of physical theory, it is very powerful, yet simple to apply. It has had a number of significant successes in RNA, DNA and DNA-protein complexes, and in all cases to date the results have stood the test of time and eventual comparison with crystallographic analysis. PMID:20946788

Lilley, David M J

2009-01-01

60

High resolution two-dimensional gel electrophoresis of human erythrocyte membrane proteins.  

PubMed Central

Three different two-dimensional (2-D) gel electrophoretic techniques have been modified to provide high resolution of human erythrocyte membrane proteins. The resulting gels were referenced to the established one-dimensional (1-D) sodium dodecylsulfate (SDS) gel electrophoretic profile, and the effects of endogenous proteolysis and cytosolic contamination were studied. It is concluded that in vitro proteolysis and cytosolic contamination do not contribute significantly to the patterns observed on the 2-D gels, under the conditions used for erythrocyte ghost preparation. The procedures require only small quantities of blood; as many as twenty 2-D gel profiles can be obtained from 5 ml of blood. The combination of nonequilibrium isoelectric focusing (IEF) in the first dimension, SDS electrophoresis in the second dimension, and very sensitive silver staining techniques resolves more than 250 individual protein spots. This appears to be the most useful single procedure for the analysis of red cell membrane proteins. Membrane protein profiles from patients with Duchenne muscular dystrophy, Wernicke-Korsakoff syndrome, and acanthocytosis with degeneration of the basal ganglia were compared with normal controls. The patterns for Duchenne muscular dystrophy and Wernicke-Korsakoff syndrome were not different from normal patterns. The pattern for the patient with acanthocytosis and degeneration of the basal ganglia consistently showed a high level for one protein in the 100,000 mol. wt. range. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7081216

Copeland, B R; Todd, S A; Furlong, C E

1982-01-01

61

The electrophoresis of transferrins in urea/polyacrylamide gels.  

PubMed Central

The denaturation of transferrin by urea has been studied by (a) electrophoresis in polyacrylamide gels incorporating a urea gradient, (b) measurements of the loss of iron-binding capacity and (c) u.v. difference spectrometry. In human serum transferrin and hen ovotransferrin the N-terminal and C-terminal domains of the iron-free protein were found to denature at different urea concentrations. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 7. PMID:7213345

Evans, R W; Williams, J

1980-01-01

62

Conformation Sensitive Gel Electrophoresis for Simple and Accurate Detection of Mutations: Comparison with Denaturing Gradient Gel Electrophoresis and Nucleotide Sequencing  

Microsoft Academic Search

Previously, an assay called conformation sensitive gel electrophoresis (CSGE) was developed for scanning PCR products for the presence of single-base and larger base mismatches in DNA. The assay was based on the assumption that mildly denaturing solvents in an appropriate buffer can accentuate the conformational changes produced by single-base mismatches in double-stranded DNA and thereby increase the differential migration in

Jarmo Korkko; Susanna Annunen; Tero Pihlajamaa; Darwin J. Prockop; Leena Ala-Kokko

1998-01-01

63

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...

64

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...

65

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

PubMed

Analysis of proteins of the banana and citrus race of Radopholus similis was carried out by several different types of polyacrylamide gel electrophoresis. These included standard slab gel, SDS slab gel, gradient slab gel, and two-ditnensional slab gel electrophoresis. A major band difference was detected between the two races by slab gel electrophoresis. However, several other poorly resolved but consistent hands of high molecular weight proteins near the gel origin also were considered as diagnostic. Resolution of protein bands was greatly improved by SDS and gradient slab gel electrophoresis, but no differences could be detected among the proteins resolved between the two rares with these techniques. Two-dimensional gels revealed a large number of proteins, but background staining obscured them hindering interpretation. When nematode races were reared on three different host plants, no differences in protein patterns were detected between them, indicating host preferences does not play a role in determining the types proteins occurring in these nematodes. PMID:19295815

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

1983-07-01

66

Electrophoresis for genotyping: temporal thermal gradient gel electrophoresis for profiling of oligonucleotide dissociation.  

PubMed Central

Traditional use of an oligonucleotide probe to determine genotype depends on perfect base pairing to a single-stranded target which is stable to a higher temperature than when imperfect binding occurs due to a mismatch in the target sequence. Bound oligonucleotide is detected at a predetermined single temperature 'snapshot' of the melting profile, allowing the distinction of perfect from imperfect base pairing. In heterozygotes, the presence of the alternative sequence must be verified with a second oligonucleotide complementary to the variant. Here we describe a system of real-time variable temperature electrophoresis during which the oligonucleotide dissociates from its target. In 20% polyacrylamide the target strand has minimal mobility and released oligonucleotide migrates extremely quickly so that the 'freed' rather than the 'bound' is displayed. The full profile of oligonucleotide dissociation during gel electrophoresis is represented along the gel track, and a single oligonucleotide is sufficient to confirm heterozygosity, since the profile displays two separate peaks. Resolution is great, with use of short track lengths enabling analysis of dense arrays of samples. Each gel track can contain a different target or oligonucleotide and the temperature gradient can accommodate oligonucleotides of different melting temperatures. This provides a convenient system to examine the interaction of many different oligonucleotides and target sequences simultaneously and requires no prior knowledge of the mutant sequence(s) nor of oligonucleotide melting temperatures. The application of the technique is described for screening of a hotspot for mutations in the LDL receptor gene in patients with familial hypercholesterolaemia. Images PMID:7630718

Day, I N; O'Dell, S D; Cash, I D; Humphries, S E; Weavind, G P

1995-01-01

67

Electrophoresis  

NSDL National Science Digital Library

Electrophoresis involves the movement of electrically charged substances under the influence of an electric field. This website demonstrates electrophoresis by providing a java applet which virtually applies an electric field across an agarose or polyacrylamide electrophoresis gel in which biological macromolecules are placed. The applet shows how the molecules move and at the end of the experiment plots the logarithm of the molecular weight versus the logarithm of the distance moved. This tutorial is one of a large collection of tutorials on electricity and magnetism available from Molecular Expressions.

Davidson, Michael

2010-12-29

68

Preparative electrophoresis on linear polyacrylamide-agarose composite gels.  

PubMed

A preparative method for isolating centigram quantities of high molecular weight polypeptide chains with high resolution and recovery uses linear polyacrylamide/agarose composite (LPAC) gels as electrophoretic media from which the polypeptides can be easily extracted. The composites are prepared in a manner yielding linear copolymers of acrylamide and 1-allyloxy-2,3-propanediol within 2% agarose gels. After electrophoresis in sodium dodecyl sulfate (SDS), protein bands were rapidly visualized for excision by briefly immersing the gel in cold 0.1 M KCl which precipitates the protein-associated SDS. The gel slices are then freeze-thawed to disrupt the agarose matrix and promote syneresis of fluid upon centrifugation. The polypeptides are then separated from the polyacrylamide in the supernatant solution by precipitating with either acidic isopropanol, trichloroacetic acid, ammonium sulfate or other general protein precipitants. As determined with polypeptide chains of fibrinogen and its cross-linked derivatives, recoveries were virtually complete (95.4% +/- 2.2%), and were independent of molecular weights over the range tested (10(4) --10(6)). PMID:8907537

Shainoff, J R; Smejkal, G B; Mitkevich, O; DiBello, P M

1996-01-01

69

Microscopic Dynamics of Quasi-2D Dense Colloidal Gels  

NASA Astrophysics Data System (ADS)

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.

Lohr, Matthew; Yodh, Arjun

2011-03-01

70

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

Microsoft Academic Search

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

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

1989-01-01

71

Protein Separation by Capillary Gel Electrophoresis: A Review  

PubMed Central

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

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

2011-01-01

72

Monitoring Piscirickettsia salmonis by denaturant gel electrophoresis and competitive PCR.  

PubMed

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

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

2000-05-25

73

Renaturation of enzymes after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate  

SciTech Connect

A number of enzymes, including amylases, dehydrogenases, and proteases, were shown to be renaturable after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Enzyme activity was detected in situ by action on substrates introduced into the gel and subsequent staining of either the product or unreacted substrate. Enzymes appeared to recover activity as soon as the detergent diffused out of the gel. Renatured enzymes were retained in gels after electrophoresis longer than native enzymes which had been subjected to electrophoresis in the absence of detergent. Re-electrophoresis of the renatured enzymes showed that part of the retained activity was physically anchored to the gel, possibly by the folding of polypeptides around the gel matrix as the enzymes were renatured.

Lacks, S.A.; Springhorn, S.S.

1980-08-10

74

Novel detection schemes and automated image analysis algorithms for planar chromatography and gel electrophoresis  

SciTech Connect

After a discussion of charge coupled devices and personal computer capabilities, examples of their applications involving novel analytical techniques are presented: laser-based indirect fluorometric detection in thin-layer chromatography; on-line detection of DNA and proteins in gel electrophoresis by uv absorption; automated image analysis for distortion compensation in sequencing gel electrophoresis; and expert systems for data acquisition to achieve constant signal-to-noise, with application to DNA sequencing slab gels.

Koutney, L.B.

1992-09-09

75

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

PubMed Central

To understand the organization of fibers in an agarose gel, digitized electron micrographs are used here to determine the frequency distribution of interfiber distance (2Pc) in thin sections of agarose gels. For a preparation of underivatized agarose, a 1.5% gel has a Pc distribution that is indistinguishable from the Pc distribution of a computer-generated, random-fiber gel; the log of the occurrence frequency (F) decreases linearly as a function of Pc. As the agarose concentration decreases below 1.5%, the semilogarithmic F versus Pc plot becomes progressively less linear. Two straight lines represent the data; the plot is steeper at the lower Pc values. As the percentage of agarose increases above 1.5%, the semilogarithmic F versus Pc plot becomes steeper at the higher Pc values. This change in the shape of semilogarithmic F versus Pc plots is possibly explained by the existence in agarose gels of two zones, one whose Pc distribution is more sensitive to the average agarose concentration than the other. To compare the structure of agarose gels to their sieving during electrophoresis, the root mean square value of Pc (Pc) is compared to the sieving-based radius of the effective pore (PE; Griess et. al. (16)) for both underivatized agarose and a derivatized agarose that has a smaller PE at any given agarose percentage. For 0.8-2.0% gels of either underivatized or derivatized agarose, PE/Pc is a constant within experimental error. Deviations from this constant are observed at lower gel percentages. This relationship of PE to Pc constrains theoretical descriptions of the motion of spheres in fibrous networks. Images FIGURE 1 FIGURE 2 FIGURE 3 PMID:8369423

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

1993-01-01

76

Analysis of nephritogenic antigens in human glomerular basement membrane by two-dimensional gel electrophoresis.  

PubMed

Collagenase digests of GBM were partially purified by column chromatography and analyzed by 2-D gel electrophoresis. Silver staining of 2-D gels showed charge- and size-related heterogeneity of proteins in the 45 to 50 kDa and 25 to 27 kDa regions. These components were transferred to nitrocellulose sheets and reacted with 10 human anti-GBM autoantibodies. Detection of bound anti-GBM autoantibodies to blotted proteins was carried out with peroxidase-labeled goat anti-human IgG and revealed binding predominantly to the cationic (pI 8 to 9.0) 45 to 50 kDa and 25 to 27 kDa components. Positive-staining patterns of blotted proteins were similar with all anti-GBM autoantibodies except that three sera additionally identified neutral (pI 5.5 to 6.5) protein components. One anti-GBM autoantibody, which developed following renal transplantation, lacked reactivity with the most cationic components in the 25 to 27 kDa region. These findings suggest heterogeneity of nephritogenic GBM antigens. The cationic 45 to 50 kDa components were sensitive to reduction, while one neutral 45 to 50 kDa component was resistant; a complex array of 25 to 30 kDa proteins (pI 5.5 to 7.5) were observed by silver staining postreduction. None of the reduced protein components reacted with anti-GBM antibodies, suggesting that epitopes on nephritogenic GBM antigens may be related to disulfide-bonded regions. Although there is variable immunohistochemical reactivity of anti-GBM autoantibodies with the GBM of infant kidneys, 2-D gels of collagenase-digested human infant GBM blotted and reacted with anti-GBM autoantibodies and showed staining patterns similar to that of adult GBM. These studies demonstrate the presence of nephritogenic antigens in the GBM of immature human kidney which are not detectable by immunohistochemical analysis. PMID:2985699

Yoshioka, K; Kleppel, M; Fish, A J

1985-06-01

77

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

PubMed

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

Hanneken, Marina; Knig, Simone

2014-04-01

78

Two dimensional non-equilibrium pH gel electrophoresis mapping of cytosolic protein changes caused by dietary protein depletion in mouse liver  

Microsoft Academic Search

Two-dimensional non-equilibrium pH gel electrophoresis (2D-NEPHGE) analysis was used to evaluate the effects of dietary protein depletion on the protein composition of mouse liver cytosol. Analysing the cytosol from both normal and protein depleted liver, the position in gels of more than three hundred protein spots was determined. After 5 days of protein depletion, about 20% of the spots either

Pedro Mariano Sanllorenti; Jorge Rosenfeld; Virginia Paola Ronchi; Pascual Ferrara; Rubn Danilo Conde

2001-01-01

79

The latest advancements in proteomic two-dimensional gel electrophoresis analysis applied to biological samples.  

PubMed

Two-dimensional gel electrophoresis (2DE) is one of the fundamental approaches in proteomics for the separation and visualization of complex protein mixtures. Proteins can be analyzed by 2DE using isoelectric focusing (IEF) in the first dimension, combined to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension, gel staining (silver and Coomassie), image analysis, and 2DE gel database. High-resolution 2DE can resolve up to 5,000 different proteins simultaneously (?2,000 proteins routinely), and detect and quantify <1 ng of protein per spot. Here, we describe the latest developments for a more complete analysis of biological fluids. PMID:25384742

Santucci, Laura; Bruschi, Maurizio; Ghiggeri, Gian Marco; Candiano, Giovanni

2015-01-01

80

A Bayesian model for classifying all differentially expressed proteins simultaneously in 2D PAGE gels  

PubMed Central

Background Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) is commonly used to identify differentially expressed proteins under two or more experimental or observational conditions. Wu et al (2009) developed a univariate probabilistic model which was used to identify differential expression between Case and Control groups, by applying a Likelihood Ratio Test (LRT) to each protein on a 2D PAGE. In contrast to commonly used statistical approaches, this model takes into account the two possible causes of missing values in 2D PAGE: either (1) the non-expression of a protein; or (2) a level of expression that falls below the limit of detection. Results We develop a global Bayesian model which extends the previously described model. Unlike the univariate approach, the model reported here is able treat all differentially expressed proteins simultaneously. Whereas each protein is modelled by the univariate likelihood function previously described, several global distributions are used to model the underlying relationship between the parameters associated with individual proteins. These global distributions are able to combine information from each protein to give more accurate estimates of the true parameters. In our implementation of the procedure, all parameters are recovered by Markov chain Monte Carlo (MCMC) integration. The 95% highest posterior density (HPD) intervals for the marginal posterior distributions are used to determine whether differences in protein expression are due to differences in mean expression intensities, and/or differences in the probabilities of expression. Conclusions Simulation analyses showed that the global model is able to accurately recover the underlying global distributions, and identify more differentially expressed proteins than the simple application of a LRT. Additionally, simulations also indicate that the probability of incorrectly identifying a protein as differentially expressed (i.e., the False Discovery Rate) is very low. The source code is available at https://github.com/stevenhwu/BIDE-2D. PMID:22712439

2012-01-01

81

Two dimensional gel electrophoresis analysis of DNA replication in Tetrahymena pyriformis  

E-print Network

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...

Ma, Yue

2012-06-07

82

A Statistical Model to Identify Differentially Expressed Proteins in 2D PAGE Gels  

PubMed Central

Two dimensional polyacrylamide gel electrophoresis (2D PAGE) is used to identify differentially expressed proteins and may be applied to biomarker discovery. A limitation of this approach is the inability to detect a protein when its concentration falls below the limit of detection. Consequently, differential expression of proteins may be missed when the level of a protein in the cases or controls is below the limit of detection for 2D PAGE. Standard statistical techniques have difficulty dealing with undetected proteins. To address this issue, we propose a mixture model that takes into account both detected and non-detected proteins. Non-detected proteins are classified either as (a) proteins that are not expressed in at least one replicate, or (b) proteins that are expressed but are below the limit of detection. We obtain maximum likelihood estimates of the parameters of the mixture model, including the group-specific probability of expression and mean expression intensities. Differentially expressed proteins can be detected by using a Likelihood Ratio Test (LRT). Our simulation results, using data generated from biological experiments, show that the likelihood model has higher statistical power than standard statistical approaches to detect differentially expressed proteins. An R package, Slider (Statistical Likelihood model for Identifying Differential Expression in R), is freely available at http://www.cebl.auckland.ac.nz/slider.php. PMID:19763172

Wu, Steven H.; Black, Michael A.; North, Robyn A.; Atkinson, Kelly R.; Rodrigo, Allen G.

2009-01-01

83

Proteomic Profile in Glomeruli of Type-2 Diabetic KKAy Mice using 2-Dimensional Differential Gel Electrophoresis  

PubMed Central

Background Diabetic nephropathy (DN) is a leading cause of end-stage renal disease. To search for glomerular proteins associated with early-stage DN, glomeruli of spontaneous type 2 diabetic KKAy mice were analyzed by 2-dimensional differential gel electrophoresis (2D-DIGE). Material/Methods Glomeruli of 20-week spontaneous type 2 diabetic KKAy mice and age-matched C57BL/6 mice were isolated by kidney perfusion with magnetic beads. Proteomic profiles of glomeruli were investigated by using 2D-DIGE and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Western blot analysis was used to confirm the results of proteomics. Immunohistochemical and semi-quantitative analysis were used to confirm the differential expression of prohibitin and annexin A2 in glomeruli. Results We identified 19 differentially expressed proteins 17 proteins were significantly up-regulated and 2 proteins were significantly down-regulated in glomeruli of diabetic KKAy mice. Among them, prohibitin and annexin A2 were up-regulated and Western blot analysis validated the same result in proteomics. Immunohistochemical analysis also revealed up-regulation of prohibitin and annexin A2 in glomeruli of KKAy mice. Conclusions Our findings suggest that prohibitin and annexin A2 may be associated with early-stage DN. Further functional research might help to reveal the pathogenesis of DN. PMID:25515740

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

2014-01-01

84

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

SciTech Connect

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

Kovacs, E.; Langemann, H. (University Clinics, Kantonsspital, Basel (Switzerland))

1990-11-01

85

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

PubMed

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

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

2014-12-01

86

Rapid microwell polymerase chain reaction with subsequent ultrathin-layer gel electrophoresis of DNA.  

PubMed

Large-scale genotyping, mapping and expression profiling require affordable, fully automated high-throughput devices enabling rapid, high-performance analysis using minute quantities of reagents. In this paper, we describe a new combination of microwell polymerase chain reaction (PCR) based DNA amplification technique with automated ultrathin-layer gel electrophoresis analysis of the resulting products. This technique decreases the reagent consumption (total reaction volume 0.75-1 microL), the time requirement of the PCR (15-20 min) and subsequent ultrathin-layer gel electrophoresis based fragment analysis (5 min) by automating the current manual procedure and reducing the human intervention using sample loading robots and computerized real time data analysis. Small aliquots (0.2 microL) of the submicroliter size PCR reaction were transferred onto loading membranes and analyzed by ultrathin-layer gel electrophoresis which is a novel, high-performance and automated microseparation technique. This system employs integrated scanning laser-induced fluorescence-avalanche photodiode detection and combines the advantages of conventional slab and capillary gel electrophoresis. Visualization of the DNA fragments was accomplished by "in migratio" complexation with ethidium bromide during the electrophoresis process also enabling real time imaging and data analysis. PMID:11870770

Shandrick, Sarah; Ronai, Zsolt; Guttman, Andrs

2002-02-01

87

2D gel proteomics: an approach to study age-related differences in protein abundance or isoform complexity in biological samples.  

PubMed

This chapter describes protocols for two-dimensional (2D) gel electrophoresis (isoelectric focusing [IEF] followed by sodium-dodecyl sulfate (SDS)-polyacrylamide gel electro-phoresis [PAGE]), staining of gels with the fluorescent dye Sypro Ruby, 2D gel image analysis, peptide mass fingerprint (PMF) analysis using matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS), liquid chromatography (LC)-tandem mass spectrometry (MS/MS), Western blot analysis of protein oxidations, and mass spectrometric mapping of sites of protein oxidations. Many of these methods were used to identify proteins affected in rat brain following ingestion of grape seed extract (GSE), a dietary supplement touted for anti-oxidant activity. Although beneficial actions in cell and animal models of chronic disease have been described for GSE, it has not been shown whether specific proteins were affected, or the nature of the effects. Applying 2D gel proteomics technology allowed discovery of proteins targeted by GSE without a priori knowledge of which one(s) might be affected. The newer 2D blue native (BN) electrophoresis methodology, which resolves protein complexes in a nondenaturing first dimension and then the components of these complexes in a denaturing second dimension, is discussed as a complementary approach. Analysis of protein oxidations and protein-protein interactions have special relevance to aging-related research, since oxidative stress and altered protein interactions may be at the heart of aging-related diseases. Finally, quality control issues related to implementation of high throughput technologies are addressed, to underscore the importance of minimizing bias and randomizing human and technical error in generating large datasets that are expensive and time-consuming to repeat. PMID:17634592

Kim, Helen; Eliuk, Shannon; Deshane, Jessy; Meleth, Sreelatha; Sanderson, Todd; Pinner, Anita; Robinson, Gloria; Wilson, Landon; Kirk, Marion; Barnes, Stephen

2007-01-01

88

IEF and NEPHGE 2-D PAGE Basic tube gel recipe, 5 ml volume  

E-print Network

40 IEF and NEPHGE 2-D PAGE Basic tube gel recipe, 5 ml volume: Urea 2.75 g (ultrapure only) ddH2O 1 enough for ten 10.5 cm X 1.5 mm tube gels. Reserve a small portion to make a 75% overlay solution. Add 5 µl of TEMED and 3-5 µl of 25% APS, filter (0.45 mm), and pour gels. Glass tubes must be cleaned

Aris, John P.

89

Polymer gel dosimetry utilizing a 2D (SE) and a 2D (HASTE) multiple echo sequences  

NASA Astrophysics Data System (ADS)

Two pulse sequences were used for the estimation of dosimetric characteristics of VIPET polymer gels. The first one, multi- echo spin echo (MESE) is the well-established method for T2 measurements. The other method is a new multi-echo single shot turbo spin echo pulse sequence, MEHASTE that reduces the acquisition time significantly. Both techniques showed a linear R2-dose response. With MESE sequence, the dose sensitivity was slightly enhanced as compared to MEHASTE. The linear portion of the R2-dose curve was restricted using the MEHASTE sequence. For doses above 7 Gy both methods fulfill the 2% ICRU criterion limit for dose resolution estimations (95% confidence level). Finally, for a time period of one month the temporal stability of R2-dose response was maintained stable utilizing both MESE and MEHASTE pulse sequences. MEHASTE serves as an excellent means for fast 3D polymer gel dosimetry.

Papoutsaki, M.-V.; Pappas, E.; Papadakis, A. E.; Varveris, C.; Damilakis, J.; Maris, T. G.

2013-06-01

90

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

NASA Astrophysics Data System (ADS)

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.

Amirkhanian, Varoujan; Tsai, Shou-Kuan

2014-03-01

91

Comparison of restriction enzymes for pulsed-field gel electrophoresis typing of Moraxella catarrhalis.  

PubMed

NotI, the most prevalent restriction enzyme used for typing Moraxella catarrhalis, failed to digest genomic DNA from respiratory samples. An improved pulsed-field gel electrophoresis (PFGE) methodology determined SpeI as the best choice for typing this bacterial species, with a good restriction of clinical samples and a good clustering correlation with NotI. PMID:23678064

Marti, Sara; Puig, Carmen; Domenech, Arnau; Liares, Josefina; Ardanuy, Carmen

2013-07-01

92

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

ERIC Educational Resources Information Center

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

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

2006-01-01

93

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

PubMed Central

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

Rouli, Laetitia; Robert, Catherine; Yagupsky, Pablo

2014-01-01

94

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

Microsoft Academic Search

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

Hseyin BASIM

2001-01-01

95

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

PubMed Central

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

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

2011-01-01

96

Fractionation of large mammalian DNA restriction fragments using vertical pulsed-field gradient gel electrophoresis  

Microsoft Academic Search

A new design for pulsed field gradient (PFG) gel electrophoresis of large (>50 kb) DNA fragments is described. The method eliminates distortion of migration of DNA because of the geometry of the applied electric field, requires a single power supply and a simple switching device, and is extremely simple to use. Parameters investigated include variation in pulse time, conditions of

Katheleen Gardiner; William Laas; David Patterson

1986-01-01

97

Identification of frankia strains by two-dimensional polyacrylamide gel electrophoresis.  

PubMed

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. PMID:16346488

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

1984-03-01

98

Identification of Frankia Strains by Two-Dimensional Polyacrylamide Gel Electrophoresis  

PubMed Central

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

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

1984-01-01

99

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.

100

Optimization of pulsed-field gel electrophoresis protocols for Salmonella Paratyphi A subtyping.  

PubMed

Salmonella enterica serovar Paratyphi A infection has caused public health problems in some countries in recent years. Pulsed-field gel electrophoresis (PFGE) has been used for the subtyping and epidemiological investigations of some serotypes of Salmonella, mainly in outbreaks caused by non-typhoidal Salmonella. In this study, different restriction endonucleases and electrophoresis parameters were compared for the PFGE subtyping by using Salmonella Paratyphi A strain panels. Two protocols for the enzymes SpeI and XbaI showed higher discriminatory power, which may facilitate epidemiological analysis for more accurate case definition, and clonality study of Salmonella Paratyphi A. PMID:22443482

Chen, Chunxia; Zhao, Yingwei; Han, Hui; Pang, Bo; Zhang, Jingyun; Yan, Meiying; Diao, Baowei; Cui, Zhigang; Zhou, Haijian; Liang, Weili; Feng, Yanfang; Kan, Biao

2012-04-01

101

Secondary pulsed field gel electrophoresis: a new method for faster separation of larger DNA molecules.  

PubMed Central

A novel technique, which we call secondary pulsed field gel electrophoresis (SPFG) has been developed. In SPFG, short pulses are applied in the direction of net migration of the DNA in addition to the reorienting pulses used in conventional pulsed field electrophoresis (PFG). Experimental results show that SPFG extends and improves the electrophoretic resolution of DNA for molecules from 0.5 megabase pairs to over 10 megabase pairs in size. This improved resolution is obtained with dramatically shorter run times. Thus SPFG appears to circumvent a number of the key limitations in previous PFG protocols. Images PMID:2030945

Zhang, T Y; Smith, C L; Cantor, C R

1991-01-01

102

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

PubMed Central

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 done in lab. Here we report the development and implementation of novel exercises that integrate the biological concepts of DNA structure and replication with the techniques of PCR and gel electrophoresis. Learning goals were defined based on concepts taught throughout the cell biology lab course and learning objectives specific to the PCR and gel electrophoresis lab. Exercises developed to promote critical thinking and target the underlying concepts of PCR, primer design, gel analysis, and troubleshooting were incorporated into an existing lab unit based on the detection of genetically modified organisms. Evaluative assessments for each exercise were aligned with the learning goals and used to measure student learning achievements. Our analysis found that the exercises were effective in enhancing student understanding of these concepts as shown by student performance across all learning goals. The new materials were particularly helpful in acquiring relevant knowledge, fostering critical-thinking skills, and uncovering prevalent misconceptions. PMID:18316813

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

2008-01-01

103

Examination of the Protein Composition of the Cell Envelope of Escherichia coli by Polyacrylamide Gel Electrophoresis  

PubMed Central

An envelope preparation containing the cell wall and cytoplasmic membrane of Escherichia coli was obtained by breaking the cells with a French pressure cell and sedimentating the envelope fraction by ultracentrifugation. This fraction was prepared for polyacrylamide gel electrophoresis by dissolving the protein in an acidified N,N?-dimethylformamide, removing lipids by gel filtration in the same organic solvent and removing the solvent by dialysis against aqueous urea solutions. More than 80% of the total protein of the envelope fraction was recovered in soluble form. Electrophoresis on sodium dodecyl sulfate-containing gels yielded from 20 to 30 well-resolved bands of protein. One major protein band was observed on the gels. This protein had a molecular weight of 44,000 and accounted for as much as 40% of the total protein of the envelope fraction. A double-labeling technique was used to examine the protein composition of the envelope fraction from cells grown under different sets of conditions which result in large changes in the levels of membrane-bound oxidative enzymes. These changes in growth conditions resulted in only minor alterations in the protein profiles observed on the gels, suggesting that this organism is able to adapt to changes in growth environment with only minor modifications of the major proteins of the cell envelope. PMID:4923077

Schnaitman, Carl A.

1970-01-01

104

Dinoflagellate community analysis of a fish kill using denaturing gradient gel electrophoresis  

Microsoft Academic Search

A molecular method using the polymerase chain reaction (PCR) amplification of small subunit gene sequences (18S rDNA) and denaturing gradient gel electrophoresis (DGGE) was used to determine both the population complexity and species identification of organisms in harmful algal blooms. Eighteen laboratory cultures of dinoflagellates, including Akashiwo, Gymnodinium, Heterocapsa, Karenia, Karlodinium, Pfiesteria, and Pfiesteria-like species were analyzed using dinoflagellate-specific oligonucleotide

Qian Wang; Jonathan R. Deeds; Robert Belas

2005-01-01

105

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

E-print Network

SDS-PAGE (Sodium dodecyl sulphate Poly acrylamide gel electrophoresis) 1. Glass cassette (10 ml) using following reagents except 10% APS10 and TEMED15 . 2.4ml dH2O 5.0ml Acrylamide/Bis20 2 following reagents except 10% APS10 and TEMED15 . 5.7ml dH2O 1.7ml Acrylamide/Bis20 2.5ml stacking buffer40

Schmidt, Marius

106

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

Microsoft Academic Search

Denaturing gradient gel electrophoresis (DGGE) is commonly utilized to identify and quantify microbial diversity, but the\\u000a conditions required for different electrophoretic systems to yield equivalent results and optimal resolution have not been\\u000a assessed. Herein, the influence of different DGGE system configuration parameters on microbial diversity estimates was tested\\u000a using Symbiodinium, a group of marine eukaryotic microbes that are important constituents

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

2010-01-01

107

Residual Casein Fractions in Ripened Cheese Determined by Polyacrylamide-Gel Electrophoresis  

Microsoft Academic Search

Polyaerylamide-gel electrophoresis (PAE) of ripened-cheese caseins provides an ideal means of studying cheese-ripening by detecting small changes in the specific casein fractions. Properties, such as molecu- lar sieving, and the facility to use simulta- neously casein standards, apparently are important advantages of PAE. Rennet enzymes appear specifically to alter a~-casein after curd formation in Cheddar cheese manufacture, fl-Casein ev- idently

R. A. Ledford; A. C. O'Sullivan; K. R. Nath

1966-01-01

108

Survey of Oral Microbial Diversity using PCR-based Denaturing Gradient Gel Electrophoresis  

Microsoft Academic Search

Polymicrobial biofilms in the human oral cavity exhibit marked diversity. PCR-based denaturing gradient gel electrophoresis (PCR-DGGE) surveys microbial diversity by displaying PCR-generated 16S rDNA fragments that migrate at different distances, reflecting the differences in the base-pair (i.e., % G+C) composition of the fragment. This study examined DGGE-generated diversity profiles of cultivable bacteria from individuals with different caries status. Initially, we

Y. Li; C. Y. S. Ku; J. Xu; D. Saxena; P. W. Caufield

2005-01-01

109

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

Microsoft Academic Search

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

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

2001-01-01

110

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

Microsoft Academic Search

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

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

111

Use of pulsed-field gel electrophoresis for investigation of hospital outbreaks of Acinetobacter baumannii.  

PubMed Central

Genomic DNAs from taxonomically and epidemiologically well-defined strains of Acinetobacter baumannii were digested with restriction endonucleases that cleave with low frequency, and the fragments were separated by pulse-field gel electrophoresis. Restriction fragment length polymorphisms were observed. Restriction fragment length polymorphism analysis can be used as an epidemiological tool to delineate outbreaks of nosocomial infections caused by A. baumannii. Images PMID:1352519

Gouby, A; Carles-Nurit, M J; Bouziges, N; Bourg, G; Mesnard, R; Bouvet, P J

1992-01-01

112

Separation of Glutathione Transferase Subunits from Proteus vulgaris by Two-Dimensional Gel Electrophoresis  

Microsoft Academic Search

Cytosolic glutathione transferases of Proteus vulgaris were purified by affinity chromatography and characterized by two-dimensional gel electrophoresis. Four different subunits were identified, and each subunit contained a different molecular mass, ranging from 26.2 kDa to 28.5 kDa; a different pI value, ranging from 8.2 to 9.4; and a different amount of protein fraction, ranging from 10% to 56%. All four

Giaming Hong; Yi-Chih Chien; Cheng-I Chien

2003-01-01

113

Structure of Virioplankton in the Charente Estuary (France): Transmission Electron Microscopy versus Pulsed Field Gel Electrophoresis  

Microsoft Academic Search

Changes in the composition of viral communities were investigated along a salinity gradient and at different times by means\\u000a of transmission electron microscopy (TEM) and pulsed field gel electrophoresis (PFGE). Samples were collected in fresh (Charente\\u000a River), estuarine (Charente Estuary), and coastal (Pertuis d'Antioche, French Atlantic coast) waters. Both methods revealed\\u000a similar patterns in viral community structure with a dominance

J. C. Auguet; H. Montani; P. Lebaron

2006-01-01

114

Epidemiological Validation of Pulsed-Field Gel Electrophoresis Patterns for Methicillin-Resistant Staphylococcus aureus  

Microsoft Academic Search

To determine the stability of pulsed-field gel electrophoresis (PFGE) patterns of methicillin-resistant Staphylococcus aureus in the nosocomial setting, we analyzed isolates from long-term carriers (>1 month) and from patients involved in well-defined nosocomial epidemics. The number of fragment differences between the first isolate and subsequent isolates in long-term carriers showed a bimodal distribution, with one group having 0 to 6

D. S. Blanc; M. J. Struelens; A. Deplano; R. De Ryck; P. M. Hauser; C. Petignat; P. Francioli

2001-01-01

115

Two-dimensional gel electrophoresis mapping of proteins isolated from the hyperthermophile Pyrococcus furiosus  

Microsoft Academic Search

Two-dimensional gel electrophoresis (2DE) in polyacrylamide was used to map the proteins in lysates of the archaeon (formerly archaebacterium) Pyrococcus furiosus and to analyze enzymes purified from P. furiosus. The location of the enzymes in the 2DE maps was determined by comigration of lysate proteins with purified enzymes. A 2DE map of P. furiosus proteins with some identifications was produced,

Carol S. Giometti; Sandra L. Tollaksen; Swarnalatha Mukund; Zhi Hao Zhou; Keren Ma; Xuhong Mai; Michael W. W. Adams

1995-01-01

116

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

PubMed

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

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

2003-10-01

117

Beverage-agarose gel electrophoresis: an inquiry-based laboratory exercise with virtual adaptation.  

PubMed

A wide range of literature and experience has shown that teaching methods that promote active learning, such as inquiry-based approaches, are more effective than those that rely on passive learning. Gel electrophoresis, one of the most common laboratory techniques in molecular biology, has a wide range of applications in the life sciences. As such, we chose it as a platform to expose high school and undergraduate students to the active process of scientific inquiry in general, while specifically teaching electrophoresis. First, we optimized DNA electrophoresis in the laboratory by using common beverages instead of standard media (e.g., Tris-based media). Second, we adapted this laboratory process of progressive optimization to a Web-based format in which students had to achieve all the same steps of optimization by performing serial electrophoreses. And third, we evaluated the use of this entirely Web-based virtual laboratory exercise in high school and undergraduate biology courses. Students learned fundamental and practical principles of electrophoresis, while experiencing the essential inquiry-based process of optimizing a technique, and they also enjoyed it. Our findings provide a readily accessible, inexpensive, and intriguing technique for teaching electrophoresis and the progressive optimization of a laboratory technique. PMID:17012220

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

2006-01-01

118

Demonstration of chlorobenzene-induced DNA damage in mouse lymphocytes using the single cell gel electrophoresis assay  

Microsoft Academic Search

The DNA damaging effect of chlorobenzene was investigated in peripheral lymphocytes and bone marrow cells from C57BL\\/6 female mice using a gel electrophoresis assay for DNA from single cells (the single cell gel electrophoresis assay) under alkaline conditions. The effect of chlorobenzene was studied both after single and repeated intraperitoneal injections of 750 mg\\/kg body weight. The cytostatic agent cyclophosphamide

Hamid Vaghef; Bjrn Hellman

1995-01-01

119

Cytoplasmic polyhedrosis virus classification by electropherotype; validation by serological analyses and agarose gel electrophoresis.  

PubMed

Serological analyses of several different cytoplasmic polyhedrosis viruses (CPVs), including two type 1 CPVs from Bombyx mori, type 1 CPV from Dendrolimus spectabilis, type 12 CPV from Autographa gamma, type 2 CPV from Inachis io, type 5 CPV from Orgyia pseudotsugata and type 5 CPV from Heliothis armigera, demonstrated a close correlation between the antigenic properties of the polyhedrin or virus particle structural proteins and the genomic dsRNA electropherotypes. The dsRNAs of these viruses were analysed by electrophoresis in 3% and 10% polyacrylamide gels with a discontinuous Tris-HCl/Tris-glycine buffer system or by 1% agarose gel electrophoresis using a continuous Tris-acetate-EDTA buffer system. Electrophoretic analysis in agarose gels was found to be the most suitable for the classification of CPV isolates into electropherotypes, and the results obtained showed a close correlation with the observed antigenic relationships between different virus isolates. However, electrophoretic analysis in 10% polyacrylamide gels was most sensitive for the detection of intra-type variation and the presence of mixed virus isolates. PMID:2499658

Mertens, P P; Crook, N E; Rubinstein, R; Pedley, S; Payne, C C

1989-01-01

120

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...

121

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

SciTech Connect

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.

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

122

Analysis of Estrogen Receptor Dinucleotide Polymorphism by Capillary Gel Electrophoresis with a Population Genetic Study in 180 Finns  

Microsoft Academic Search

We developed a suitable method for analysing dinucleotide repeats found in the upstream of human alpha-estrogen receptor (ER) gene by applying capillary electrophoresis and automatic analysis. This method omits the gel-casting step as well as difficult handling of long polyacrylamide sequencing gels. Use of radioactive materials is also avoided. Using this method, the frequency distribution of ER alleles, determined in

Tarja A. Kunnas; Doris Holmberg-Marttila; Pekka J. Karhunen

1999-01-01

123

A wavelet relational fuzzy C-means algorithm for 2D gel image segmentation.  

PubMed

One of the most famous algorithms that appeared in the area of image segmentation is the Fuzzy C-Means (FCM) algorithm. This algorithm has been used in many applications such as data analysis, pattern recognition, and image segmentation. It has the advantages of producing high quality segmentation compared to the other available algorithms. Many modifications have been made to the algorithm to improve its segmentation quality. The proposed segmentation algorithm in this paper is based on the Fuzzy C-Means algorithm adding the relational fuzzy notion and the wavelet transform to it so as to enhance its performance especially in the area of 2D gel images. Both proposed modifications aim to minimize the oversegmentation error incurred by previous algorithms. The experimental results of comparing both the Fuzzy C-Means (FCM) and the Wavelet Fuzzy C-Means (WFCM) to the proposed algorithm on real 2D gel images acquired from human leukemias, HL-60 cell lines, and fetal alcohol syndrome (FAS) demonstrate the improvement achieved by the proposed algorithm in overcoming the segmentation error. In addition, we investigate the effect of denoising on the three algorithms. This investigation proves that denoising the 2D gel image before segmentation can improve (in most of the cases) the quality of the segmentation. PMID:24174990

Rashwan, Shaheera; Faheem, Mohamed Talaat; Sarhan, Amany; Youssef, Bayumy A B

2013-01-01

124

A Wavelet Relational Fuzzy C-Means Algorithm for 2D Gel Image Segmentation  

PubMed Central

One of the most famous algorithms that appeared in the area of image segmentation is the Fuzzy C-Means (FCM) algorithm. This algorithm has been used in many applications such as data analysis, pattern recognition, and image segmentation. It has the advantages of producing high quality segmentation compared to the other available algorithms. Many modifications have been made to the algorithm to improve its segmentation quality. The proposed segmentation algorithm in this paper is based on the Fuzzy C-Means algorithm adding the relational fuzzy notion and the wavelet transform to it so as to enhance its performance especially in the area of 2D gel images. Both proposed modifications aim to minimize the oversegmentation error incurred by previous algorithms. The experimental results of comparing both the Fuzzy C-Means (FCM) and the Wavelet Fuzzy C-Means (WFCM) to the proposed algorithm on real 2D gel images acquired from human leukemias, HL-60 cell lines, and fetal alcohol syndrome (FAS) demonstrate the improvement achieved by the proposed algorithm in overcoming the segmentation error. In addition, we investigate the effect of denoising on the three algorithms. This investigation proves that denoising the 2D gel image before segmentation can improve (in most of the cases) the quality of the segmentation. PMID:24174990

Rashwan, Shaheera; Faheem, Mohamed Talaat; Sarhan, Amany; Youssef, Bayumy A. B.

2013-01-01

125

Proposal for a Standard Representation of Two-Dimensional Gel Electrophoresis Data  

PubMed Central

The global analysis of proteins is now feasible due to improvements in techniques such as two-dimensional gel electrophoresis (2-DE), mass spectrometry, yeast two-hybrid systems and the development of bioinformatics applications. The experiments form the basis of proteomics, and present significant challenges in data analysis, storage and querying. We argue that a standard format for proteome data is required to enable the storage, exchange and subsequent re-analysis of large datasets. We describe the criteria that must be met for the development of a standard for proteomics. We have developed a model to represent data from 2-DE experiments, including difference gel electrophoresis along with image analysis and statistical analysis across multiple gels. This part of proteomics analysis is not represented in current proposals for proteomics standards. We are working with the Proteomics Standards Initiative to develop a model encompassing biological sample origin, experimental protocols, a number of separation techniques and mass spectrometry. The standard format will facilitate the development of central repositories of data, enabling results to be verified or re-analysed, and the correlation of results produced by different research groups using a variety of laboratory techniques. PMID:18628860

Wastling, Jonathan; Hunt, Ela

2003-01-01

126

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

NSDL National Science Digital Library

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.

Hsu, Tien Theresa

2005-08-17

127

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

PubMed Central

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

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

2013-01-01

128

Analysis of the microflora in Tibetan kefir grains using denaturing gradient gel electrophoresis.  

PubMed

The microflora of Tibetan kefir grains was investigated by culture- independent methods. Denaturing gradient gel electrophoresis (DGGE) of partially amplified 16S rRNA for bacteria and 26S rRNA for yeasts, followed by sequencing of the most intense bands, showed that the dominant microorganisms were Pseudomonas sp., Leuconostoc mesenteroides, Lactobacillus helveticus, Lactobacillus kefiranofaciens, Lactococcus lactis, Lactobacillus kefiri, Lactobacillus casei, Kazachstania unispora, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Kazachstania exigua. The bacterial communities between three kinds of Tibetan kefir grains showed 78-84% similarity, and yeasts 80-92%. The microflora is held together in the matrix of fibrillar material composed largely of a water-insoluble polysaccharide. PMID:19835760

Zhou, Jianzhong; Liu, Xiaoli; Jiang, Hanhu; Dong, Mingsheng

2009-12-01

129

The defect of Mk erythrocytes as revealed by sodium dodecylsulphate--polyacrylamide gel electrophoresis.  

PubMed

The membrane glycoprotein defect of Mk red cells was studied using discontinuous sodium dodecylsulphate--polyacrylamide gel electrophoresis, carbohydrate analytical procedures and lectins. The results suggest that Mk erythrocyte membranes contain only about half of the normal amount of the MN and Ss sialoglycoproteins, both molecules being not qualitatively different from normal. The major membrane protein exhibits an increased molecular weight in the above red cells. Some data on Mg and Miltenberger class five cells are presented for comparison. The results on Mk erythrocytes support the operon concept of the MNSs blood group system proposed previously. PMID:894053

Dahr, W; Uhlenbruck, G; Knott, H

1977-06-01

130

Use of Two-Dimensional Polyacrylamide Gel Electrophoresis to Identify and Classify Rhizobium Strains  

PubMed Central

Fifty-seven strains of various Rhizobium species were analyzed by two-dimensional gel electrophoresis. Since the protein pattern on such gels is a reflection of the genetic background of the tested strains, similarities in pattern allowed us to estimate the relatedness between these strains. All group II rhizobia (slow growing) were closely related and were very distinct from group I rhizobia (fast growing). Rhizobium meliloti strains formed a distinct group. The collection of R. leguminosarum and R. trifolii strains together formed another distinct group. Although there were some similarities within the R. phaseoli, sesbania rhizobia, and lotus rhizobia, the members within these seemed much more diverse than the members of the above groups. The technique also is useful to determine whether two unknown strains are identical. Images PMID:16345514

Roberts, Gary P.; Leps, Walter T.; Silver, Lin E.; Brill, Winston J.

1980-01-01

131

Interlaboratory Agreement of Pulsed-Field Gel Electrophoresis Identification of Leptospira Serovars  

PubMed Central

Leptospirosis may be caused by > 250 Leptospira serovars. Serovar classification is a complex task that most laboratories cannot perform. We assessed the interlaboratory reproducibility of a pulsed-field gel electrophoresis (PFGE) identification technique developed by the Centers for Disease Control and Prevention (CDC). Blinded exchange of 93 Leptospiraceae strains occurred between San Antonio Military Medical Center (SAMMC) and the CDC. PFGE was performed and gel images were analyzed and compared with patterns present in each laboratory's database (CDC database: > 800 strain patterns; SAMMC database: > 300 strain patterns). Overall, 93.7% (74 of 79) of strains present in each receiving laboratory's database were correctly identified. Five isolates were misidentified, and two isolates did not match serovar PFGE patterns in the receiving laboratory's database. Patterns for these seven isolates were identical between laboratories; four serovars represented misidentified reference strains. The PFGE methodology studied showed excellent interlaboratory reproducibility, enabling standardization and data sharing between laboratories. PMID:23817329

Mende, Katrin; Galloway, Renee L.; Becker, Sara J.; Beckius, Miriam L.; Murray, Clinton K.; Hospenthal, Duane R.

2013-01-01

132

Strategies to optimize two-dimensional gel electrophoresis analysis of the human joint proteome.  

PubMed

Due to the complex structure of the articular joint, it requires great effort to fully understand joint disease pathogenesis. The proteomic analysis of articular joint tissues could contribute greatly to our insight into the endogenous control mechanisms of matrix turnover and the unravelling of the molecular and cellular mechanisms involved in the progression of the arthritides. To date, most proteome analysis strategies use the two-dimensional gel electrophoresis (2-DE) technique to separate proteins according to their isoelectric point, molecular mass, solubility and relative abundance. In this work, we describe optimization of human joint sample preparation techniques to obtain high quality 2-DE maps of human joint tissues (cartilage and synovium), cells (chondrocytes and synoviocytes) and synovial fluid. These techniques improve the performance of gel-based differential proteomic analysis, and facilitate the application of proteomics to rheumatology studies. PMID:20082814

Ruiz-Romero, Cristina; Calamia, Valentina; Carreira, Vanessa; Mateos, Jess; Fernndez, Patricia; Blanco, Francisco J

2010-02-15

133

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

PubMed

The aims of the current study were to assess whether sodium dodecyl sulphate-agarose gel electrophoresis (SDS-AGE) and high-resolution electrophoresis (HRE) can identify dogs with a urinary protein-to-creatinine ratio (UPC ratio) >0.2 and whether HRE can provide preliminary information about the type of proteinuria, using SDS-AGE as a reference method. HRE and SDS-AGE were conducted on 87 urine samples classified according to the International Renal Interest Society as non-proteinuric (NP; UPC ratio: <0.20; 32/87), borderline proteinuric (BP; UPC ratio: 0.21-0.50; 15/87), or proteinuric (P; UPC ratio: >0.51; 40/87). SDS-AGE and HRE were positive in 14 out of 32 and 3 out of 32 NP samples and in 52 out of 55 and 40 out of 55 samples with a UPC ratio >0.20, respectively. The concordance between HRE or SDS and UPC ratio was comparable (? = 0.59; ? = 0.55). However, specificity (90%) and positive likelihood ratio (7.76) were higher for HRE than for SDS-AGE (56% and 2.16) while sensitivity was lower (73% vs. 94%). The analysis of HRE results revealed that a percentage of albumin >41.4% and an albumin/?(1)-globulin ratio (alb/?(1) ratio) >1.46 can identify samples classified by SDS-AGE as affected by glomerular proteinuria while a percentage of ?(1)-globulin >40.8% and an alb/?(1) ratio <0.84 can identify samples classified by SDS-AGE as affected by tubular proteinuria. In conclusion, both SDS-AGE and HRE could misclassify samples with a UPC ratio higher or lower than 0.20. Therefore, UPC ratio must always be determined before conducting these tests. The percentage of albumin and ?(1)-globulin or the alb/?(1) ratio determined by HRE can provide preliminary information about the origin of proteinuria. PMID:21908309

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

2011-07-01

134

Separation performance of single-stranded DNA electrophoresis in photopolymerized cross-linked polyacrylamide gels.  

PubMed

Considerable effort has been directed toward optimizing performance and maximizing throughput in ssDNA electrophoresis because it is a critical analytical step in a variety of genomic assays. Ultimately, it would be desirable to quantitatively determine the achievable level of separation resolution directly from measurements of fundamental physical properties associated with the gel matrix rather than by the trial and error process often employed. Unfortunately, this predictive capability is currently lacking, due in large part to the need for a more detailed understanding of the fundamental parameters governing separation performance (mobility, diffusion, and dispersion). We seek to address this issue by systematically characterizing electrophoretic mobility, diffusion, and dispersion behavior of ssDNA fragments in the 70-1,000 base range in a photopolymerized cross-linked polyacrylamide matrix using a slab gel DNA sequencer. Data are collected for gel concentrations of 6, 9, and 12%T at electric fields ranging from 15 to 40 V/cm, and resolution predictions are compared with corresponding experimentally measured values. The data exhibit a transition from behavior consistent with the Ogston model for small fragments to behavior in agreement with the biased reptation model at larger fragment sizes. Mobility data are also used to estimate the mean gel pore size and compare the predictions of several models. PMID:16331587

Lo, Roger C; Ugaz, Victor M

2006-02-01

135

Dog pancreatic microsomal-membrane polypeptides analysed by two-dimensional gel electrophoresis.  

PubMed Central

The two-dimensional polyacrylamide-gel electrophoresis technique of O'Farrell [(1975) J. Biol. Chem 250, 4007-4021] was applied to resolve and analyse the polypeptide composition of dog pancreatic rough microsomal membranes, which were shown to be active in co-translational processing of preprolactin synthesized from pituitary mRNA in a translation system in vitro. About 100 polypeptides are resolved. Treatment of rough microsomal membranes with EDTA and high KCl concentration yielded membranes stripped of their ribosomes with retention of activity for translocation and processing. Stripped microsomal membranes showed a selective concentration of approximately 25 polypeptides in the membranes when analysed by two-dimensional polyacrylamide-gel electrophoresis. The two-dimensional electrophoretic profile was catalogued into polypeptides that are glycoproteins, those that contain free thiol groups disposed at the cytosolic surface of microsomal vesicles and those that are of secretory origin but have been entrapped in the microsomal preparation. Several secretory components, including amylase, procarboxypeptidases, lipase and anionic trypsinogen, were tentatively identified among the microsomal polypeptides. The rough and stripped microsomal membranes from dog pancreas show a characteristic set of seven major acidic polypeptides, which are also identifiable in microsomal-membrane preparations isolated from dog liver and rat liver. One of these polypeptides was identified as protein disulphide-isomerase (EC 5.3.4.1). Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:6696719

Kaderbhai, M A; Austen, B M

1984-01-01

136

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

PubMed Central

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

Chen, Xiaojia; Ugaz, Victor M.

2010-01-01

137

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

PubMed Central

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

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

2012-01-01

138

Differential activity-based gel electrophoresis for comparative analysis of lipolytic and esterolytic activities  

PubMed Central

We established a novel technique for differential activity-based gel electrophoresis (DABGE) of lipolytic enzymes from two different biological samples. For this purpose, a set of three fluorescent suicide inhibitors was developed. These probes possess the same substrate analogous structures but carry different cyanine dyes (Cy2b, Cy3, and Cy5) as reporter fluorophores. For comparison of enzyme profiles, two samples are individually labeled with a different probe followed by mixing, gel electrophoresis, fluorescence imaging, and identification of the tagged proteins by MS/MS. Protocols for quantitative determination of active enzymes were developed on the basis of lipolytic proteomes that had been admixed with defined amounts of known lipases and esterases. A detailed analysis of the fluorescence intensities showed that the found enzyme ratios very closely reflected the relative amounts of the labeled enzymes that were used for spiking. The DABGE method was used to compare the lipolytic proteomes of brown and white adipose tissue showing specific enzyme patterns of both samples. This study represents the first application of this technology for comparative analysis of lipases and esterases. Further applications of this technique can be expected to provide entirely new information on lipid enzymology in health and disease with high precision. PMID:19282273

Morak, Maria; Schmidinger, Hannes; Krempl, Peter; Rechberger, Gerald; Kollroser, Manfred; Birner-Gruenberger, Ruth; Hermetter, Albin

2009-01-01

139

Single-strand conformational polymorphism and denaturing gradient gel electrophoresis in screening for variegate porphyria: identification of two new mutations.  

PubMed

Single-strand conformational polymorphism and denaturing gel electrophoresis were used to screen for mutations in the protoporphyrinogen oxidase gene (PPOX) of three patients with clinically and biochemically proven variegate porphyria in order to select genomic regions for specific DNA sequence analysis. Two previously undescribed mutations were identified: PPOX1423-1426-delATCT and PPOX2272insG. Denaturing gel electrophoresis was able to discern the point mutation in exon 5 (PPOX2272insG) of the PPOX gene. Once an index individual has been identified, single-strand conformational polymorphism and denaturing gel electrophoresis techniques are useful to identify family members who may be unaffected carriers. Such identification can help potential cases to avoid medications and other triggers that could precipitate acute porphyric attacks. PMID:12017191

Donnelly, James G; Detombe, Sarah; Hindmarsh, J Thomas

2002-01-01

140

2-D gel-based proteomic approaches to antibiotic drug discovery.  

PubMed

The global analysis of changes in the protein composition of bacterial cells in response to treatment with antibiotic agents grants insights into the physiological response of cells to inhibition of vital cellular functions. This unit gives an overview of how global proteomic studies can impact antibacterial drug discovery by identifying or validating compound mechanism of action and by increasing the confidence in the value of genes with unknown function as potential new targets. It describes the design and function of a reference compendium of proteomic responses to inhibition of vital cellular functions through antibacterial agents or genetic down-regulation of potential target genes. An overview of the workflow for two-dimensional gel electrophoresis-based experiments is also presented. PMID:22875564

Raatschen, Nadja; Bandow, Julia Elisabeth

2012-08-01

141

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

PubMed

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

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

2012-05-21

142

Pluronic copolymer liquid crystals: unique, replaceable media for capillary gel electrophoresis.  

PubMed

Liquid crystalline solutions of Pluronic copolymers are versatile alternatives to solutions of entangled, random coil polymers as replaceable media for capillary gel electrophoresis (CGE). Pluronic copolymers are tri-block polymers of poly(ethylene oxide) [(EO)x] and poly(propylene oxide) [(PO)y] with the general formula (EO)x(PO)y(EO)x. Large micelles form in aqueous solutions in which central, hydrophobic cores of (PO)y segments are surrounded by "brushes" of hydrated (EO)x tails. Solutions of Pluronic F127 (BASF Performance Chemicals) in a concentration range of about 18-30% are liquids at refrigerator temperatures (< or = 5 degrees C) and are easily introduced into capillaries. A self-supporting, gel-like liquid crystalline phase is formed as the temperature is raised to > or = 20 degrees C. This liquid crystalline phase consists of spherical micelles with diameters of 17-18 nm which pack with local cubic symmetry. CGE in Pluronic F127 liquid crystals separates species within several chemical classes as varied as nucleoside monophosphates and organic dyes, oligonucleotides of 4-60 nucleotides, DNA fragments of 50-3000 base pairs (bp), and supercoiled plasmid DNAs of 2000-10,000 bp. Mechanisms of molecular sieving in polymer liquid crystals must differ in fundamental ways from separations in random polymer gels because molecules move around uncrosslinked obstacles that are larger than the smallest dimensions of typical analytes. Molecular sieving in Pluronic liquid crystals is envisioned to occur as molecules squeeze between hydrated (EO)x strands of micelle brushes, or through brushtips and interstitial spaces between micelles. Small molecules such as nucleotides appear to separate by a different mechanism involving partitioning between hydrophilic and hydrophobic environments. This process is termed "hydrophobic interaction electrophoresis". The unique structures of Pluronic copolymers and their liquid crystalline phases provide new challenges and opportunities in separations science. PMID:9764501

Rill, R L; Liu, Y; Van Winkle, D H; Locke, B R

1998-08-21

143

Pseudomonas aeruginosa exotoxin: purification by preparative polyacrylamide gel electrophoresis and the development of a highly specific antitoxin serum.  

PubMed Central

Pseudomonas aeruginosa exotoxin has been purified to a specific activity of 12,000 to 16,000 mouse median lethal doses/mg of protein. Total recovery was about 25%, and the degree of purification was approximately 3,000-fold. Preparative polyacrylamide gel electrophoresis greatly facilitated purification. As judged by analytical disc gel electrophoresis, the purified toxin contained one major band of protein and only a negligible amount of contamination. Antiserum prepared against the purified toxin neutralized the lethal activity of crude toxin preprations and reacted by double immunodiffusion with a single component of concentrated broth cultures of P. aeruginosa isolates obtained from a clinical source. Images PMID:820647

Callahan, L T

1976-01-01

144

Electrophoresis of bacteriophage T7 and T7 capsids in agarose gels.  

PubMed Central

Agarose gel electrophoresis of the following was performed in 0.05 M sodium phosphate-0.001 M MgCl2 (pH 7.4): (i) bacteriophage T7; (ii) a T7 precursor capsid (capsid I), isolated from T7-infected Escherichia coli, which has a thicker and less angular envelope than bacteriophage T7; (iii) a second capsid (capsid II), isolated from T7-infected E. coli, which has a bacteriophage-like envelope; and (iv) capsids (capsid IV) produced by temperature shock of bacteriophage T7. Bacteriophage T7 and all of the above capsids migrated towards the anode. In a 0.9% agarose gel, capsid I had an electrophoretic mobility of 9.1 +/- 0.4 X 10(-5) cm2/V.s; bacteriophage T7 migrated 0.31 +/- 0.02 times as fast as capsid I. The mobilities of different preparations of capsid II varied in such gels: the fastest-migrating capsid II preparation was 0.51 +/- 0.03 times as fast as capsid I and the slowest was 0.37 +/- 0.02 times as fast as capsid I. Capsid IV with and without the phage tail migrated 0.29 +/- 0.02 and 0.42 +/- 0.02 times as fast as capsid I. The results of the extrapolation of bacteriophage and capsid mobilities to 0% agarose concentration indicated that the above differences in mobility are caused by differences in average surface charge density. To increase the accuracy of mobility comparisons and to increase the number of samples that could be simultaneously analyzed, multisample horizontal slab gels were used. Treatment with the ionic detergent sodium dodecyl sulfate converted capsid I to a capsid that migated in the capsid II region during electrophoresis through agarose gels. In the electron microscope, most of the envelopes of these latter capsids resembled the capsid II envelope, but some envelope regions were thicker than the capsid II envelope. Images PMID:731798

Serwer, P; Pichler, M E

1978-01-01

145

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

PubMed Central

Dehalococcoides mccartyi strains are obligate organohalide-respiring bacteria harboring multiple distinct reductive dehalogenase (RDase) genes within their genomes. A major challenge is to identify substrates for the enzymes encoded by these RDase genes. We demonstrate an approach that involves blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by enzyme activity assays with gel slices and subsequent identification of proteins in gel slices using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). RDase expression was investigated in cultures of Dehalococcoides mccartyi strain BAV1 and in the KB-1 consortium growing on chlorinated ethenes and 1,2-dichloroethane. In cultures of strain BAV1, BvcA was the only RDase detected, revealing that this enzyme catalyzes the dechlorination not only of vinyl chloride, but also of all dichloroethene isomers and 1,2-dichloroethane. In cultures of consortium KB-1, five distinct Dehalococcoides RDases and one Geobacter RDase were expressed under the conditions tested. Three of the five RDases included orthologs to the previously identified chlorinated ethene-dechlorinating enzymes VcrA, BvcA, and TceA. This study revealed substrate promiscuity for these three enzymes and provides a path forward to further explore the largely unknown RDase protein family. PMID:23204411

Tang, Shuiquan; Chan, Winnie W. M.; Fletcher, Kelly E.; Seifert, Jana; Liang, Xiaoming; Lffler, Frank E.; Adrian, Lorenz

2013-01-01

146

Colorful Electrophoresis  

NSDL National Science Digital Library

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.

Utah, University O.

2012-01-01

147

Cu(2+)-assisted two dimensional charge-mass double focusing gel electrophoresis and mass spectrometric analysis of histone variants.  

PubMed

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

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

2014-12-10

148

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

Microsoft Academic Search

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

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

1996-01-01

149

Pulsed-field gel electrophoresis analysis of multicellular DNA double-strand break damage and repair.  

PubMed

This assay quantifies the extent of double-strand break (DSB) DNA damage in cell populations embedded in agarose and analyzed for migratory DNA using pulsed-field gel electrophoresis with ethidium bromide staining. The assay can measure preexisting damage as well as induction of DSB by chemical (e.g., bleomycin), physical (e.g., X-irradiation), or biological (e.g., restriction enzymes) agents. By incubating the cells under physiological conditions prior to processing, the cells can be allowed to repair DSB, primarily via the process of nonhomologous end joining. The amount of repair, corresponding to the repair capacity of the treated cells, is then quantified by determining the ratio of the fractions of activity released in the lanes in comparison to the total amount of DNA fragmentation following determination of an optimal exposure for maximum initial fragmentation. Repair kinetics can also be analyzed through a time-course regimen. PMID:24623230

Joshi, Nina; Grant, Stephen G

2014-01-01

150

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

SciTech Connect

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.

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

1987-06-05

151

Comparison of Different Denaturing Gradient Gel Electrophoresis Primer Sets for the Study of Marine Bacterioplankton Communities?  

PubMed Central

An annual seasonal cycle of composition of a bacterioplankton community in an oligotrophic coastal system was studied by denaturing gradient gel electrophoresis (DGGE) using five different primer sets. Analysis of DGGE fingerprints showed that primer set 357fGC-907rM grouped samples according to seasons. Additionally, we used the set of 16S rRNA genes archived in the RDPII database to check the percentage of perfect matches of each primer for the most abundant bacterial groups inhabiting coastal plankton communities. Overall, primer set 357fGC-907rM was the most suitable for the routine use of PCR-DGGE analyses in this environment. PMID:17660308

Snchez, Olga; Gasol, Josep M.; Massana, Ramon; Mas, Jordi; Pedrs-Ali, Carlos

2007-01-01

152

The identification of rat intestinal membrane enzymes after electrophoresis on polyacrylamide gels containing sodium dodecyl sulphate.  

PubMed Central

Brush-border membranes were isolated from the rat small intestine and then treated with sodium dodecyl sulphate under non-reducing conditions at room temperature. Analysis of the solubilized components by polyacrylamide-gel electrophoresis identified three major glycoproteins that co-migrate with glucoamylase-maltase-sucrase, lactase and isomaltase-maltase-sucrase activities. High activities of alkaline phosphatase and trehalase were detectable, but they could not be attributed to distinct co-migrating protein bands. Analysis of mucosa from the distal small intestine by the same methods showed a pattern of bands different from that obtained with the proximal intestine, which appeared to correlate with the relative deficiency of some of the enzymes in the distal region. PMID:697763

Hauri, H P; Green, J R

1978-01-01

153

Tissue proteomics by one-dimensional gel electrophoresis combined with label-free protein quantification.  

PubMed

Label-free methods streamline quantitative proteomics of tissues by alleviating the need for metabolic labeling of proteins with stable isotopes. Here we detail and implement solutions to common problems in label-free data processing geared toward tissue proteomics by one-dimensional gel electrophoresis followed by liquid chromatography tandem mass spectrometry (geLC MS/MS). Our quantification pipeline showed high levels of performance in terms of duplicate reproducibility, linear dynamic range, and number of proteins identified and quantified. When applied to the liver of an adenomatous polyposis coli (APC) knockout mouse, we demonstrated an 8-fold increase in the number of statistically significant changing proteins compared to alternative approaches, including many more previously unidentified hydrophobic proteins. Better proteome coverage and quantification accuracy revealed molecular details of the perturbed energy metabolism. PMID:22671763

Vasilj, Andrej; Gentzel, Marc; Ueberham, Elke; Gebhardt, Rolf; Shevchenko, Andrej

2012-07-01

154

Cosmid mapping of the human chorionic gonadotropin beta subunit genes by field-inversion gel electrophoresis.  

PubMed Central

A cosmid clone containing the entire hCG beta gene cluster has been isolated. The restriction map of this clone has been determined by an indirect-end-label FIGE (field inversion gel electrophoresis) method. Analysis of this cosmid clone shows that there are 6 hCG beta genes in human genomic DNA. A previously uncloned portion of the hCG beta cluster, termed the "gap" region, has been shown not to contain any sequences homologous to the hCG beta cDNA. The restriction mapping method employed in this study takes advantage of the superior resolution of FIGE for high molecular weight DNA fragments in the size range 15-50 kb. This method is broadly applicable and permits rapid and accurate restriction mapping for extended regions of genomic DNA that have been cloned into cosmid or lambda vectors. Images PMID:3035494

Graham, M Y; Otani, T; Boime, I; Olson, M V; Carle, G F; Chaplin, D D

1987-01-01

155

Sodium dodecyl sulfate-polyacrylamide gel protein electrophoresis of freshwater photosynthetic sulfur bacteria.  

PubMed

Sodium dodecyl sulfate-polyacrylamide gel protein electrophoresis (SDS-PAGE) was carried out using different bacterial strains of the photosynthetic sulfur bacteria Chlorobium, Thiocapsa, Thiocystis, and Chromatium cultured in the laboratory, and the natural blooms in two karstic lakes (Lake Cis and Lake Vilar, NE Spain) where planktonic photosynthetic bacteria (purple and green sulfur bacteria) massively developed accounting for most of the microbial biomass. Several extraction, solubilization, and electrophoresis methods were tested to develop an optimal protocol for the best resolution of the SDS-PAGE. Protein composition from different water depths and at different times of the year was visualized within a molecular mass range between 100 and 15 kDa yielding up to 20 different protein bands. Protein banding patterns were reproducible and changed in time and with depth in agreement with changes in photosynthetic bacteria composition. When a taxonomically stable community was followed in time, differences were observed in the intensity but not in the composition of the SDS-PAGE banding pattern. Three environmental variables directly related to the activity of sulfur bacteria (light, oxygen, and sulfide concentrations) had a significant effect on protein banding patterns and explained 33% of the variance. Changes in natural protein profiles of the bacterial blooms agreed with changes in species composition and in the in situ metabolic state of the populations. PMID:20524118

Osuna, M Begoa; Casamayor, Emilio O

2011-01-01

156

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

PubMed Central

Background The analysis of hydrophobic membrane proteins by two-dimensional gel electrophoresis has long been hampered by the concept of inherent difficulty due to solubility issues. We have optimized extraction protocols by varying the detergent composition of the solubilization buffer with a variety of commercially available non-ionic and zwitterionic detergents and detergent-like phospholipids. Results After initial analyses by one-dimensional SDS-PAGE, quantitative two-dimensional analyses of human erythrocyte membranes, mouse liver membranes, and mouse brain membranes, extracted with buffers that included the zwitterionic detergent MEGA 10 (decanoyl-N-methylglucamide) and the zwitterionic lipid LPC (1-lauroyl lysophosphatidylcholine), showed selective improvement over extraction with the common 2-DE detergent CHAPS (3 [(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate). Mixtures of the three detergents showed additive improvements in spot number, density, and resolution. Substantial improvements in the analysis of a brain membrane proteome were observed. Conclusion This study demonstrates that an optimized detergent mix, coupled with rigorous sample handling and electrophoretic protocols, enables simple and effective analysis of membrane proteomes using two-dimensional electrophoresis. PMID:15941475

Churchward, Matthew A; Butt, R Hussain; Lang, John C; Hsu, Kimberly K; Coorssen, Jens R

2005-01-01

157

Optimization of Pulse-Field Gel Electrophoresis for Subtyping of Klebsiella pneumoniae  

PubMed Central

A total of 110 strains of Klebsiella pneumoniae were used to optimize pulsed-field gel electrophoresis (PFGE) for subtyping of K. pneumoniae. For optimization of electrophoresis parameters (EPs) of XbaI-PFGE, 11 isolates were analyzed with XbaI digestion using three EPs. The EP of a switch time of 6 to 36 s for 18.5 h gave clearest patterns and was declared the optimal EP for XbaI PFGE of K. pneumoniae. By software analysis and pilot study, AvrII was chosen as another PFGE enzyme. Both XbaI- and AvrII-PFGE gave D-values higher than 0.99 for 69 K. pneumoniae isolated from different sources. Our results also showed good typeability, reproducibility of both XbaI- and AvrII-PFGE for K. pneumoniae subtyping. Furthermore, the established PFGE method also had good discriminatory power to distinguish outbreak K. pneumoniae strains and a high degree of consistency with multilocus sequence typing method. A rapid PFGE protocol was established here, which could be used for genotyping and other researches of K. pneumoniae. PMID:23880721

Han, Hui; Zhou, Haijian; Li, Haishan; Gao, Yuan; Lu, Zhi; Hu, Kongxin; Xu, Baoliang

2013-01-01

158

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

Microsoft Academic Search

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

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

2003-01-01

159

Characterization of Listeria monocytogenes strains isolated from food and humans in Italy by pulsed-field gel electrophoresis  

Microsoft Academic Search

Pulsed-field gel electrophoresis (PFGE) was used to type 90 strains of Listeria monocytogenes isolated from clinical cases and from meat products in Italy in the period 198795. The objective of this retrospective study was to compare the genetic profiles to determine the existence of predominant clones and to evaluate their association with the sporadic cases of listeriosis reported in recent

M Gianfranceschi; M Pourshaban; A Gattuso; C Wedell-Neergaard; P Aureli

2002-01-01

160

Use of pulsed-field gel electrophoresis to link sporadic cases of invasive listeriosis with recalled chocolate milk.  

PubMed Central

Pulsed-field gel electrophoresis established the linkage between recalled chocolate milk and a multistate invasive listeriosis outbreak during a four-product recall period. Listeria monocytogenes isolates from four hospitalized patients and an environmental dairy sample displayed AscI restriction endonuclease digestion profiles identical to that of the chocolate milk isolate. PMID:7487050

Proctor, M E; Brosch, R; Mellen, J W; Garrett, L A; Kaspar, C W; Luchansky, J B

1995-01-01

161

Changes in Bacterial Species Composition in Enrichment Cultures with Various Dilutions of Inoculum as Monitored by Denaturing Gradient Gel Electrophoresis  

PubMed Central

Denaturing gradient gel electrophoresis revealed changes in the bacterial species obtained from enrichment cultures with different inoculum dilutions. This inoculum dilution enrichment approach may facilitate the detection and isolation of a greater number of bacterial species than traditional enrichment techniques. PMID:9835607

Jackson, Colin R.; Roden, Eric E.; Churchill, Perry F.

1998-01-01

162

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

Microsoft Academic Search

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

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

163

Easy washing of lysed cell plugs for bacterial typing by pulsed-field gel electrophoresis using simple equipment.  

PubMed

We designed and tested equipment to wash plugs following cell lysis in pulsed-field gel electrophoresis (PFGE). Our system can wash 30 plugs simultaneously in 1h using 15L of Tris-EDTA buffer, which makes plug washing for PFGE less labor-intensive. PMID:24739397

Murakami, Koichi; Noda, Tamie; Maeda, Eriko; Sera, Nobuyuki; Fujimoto, Shuji

2014-06-01

164

GC Fractionation Enhances Microbial Community Diversity Assessment and Detection of Minority Populations of Bacteria by Denaturing Gradient Gel Electrophoresis  

Microsoft Academic Search

Effectively and accurately assessing total microbial community diversity is one of the primary challenges in modern microbial ecology. This is particularly true with regard to the detection and characterization of unculturable populations and those present only in low abundance. We report a novel strategy, GC fraction- ation combined with denaturing gradient gel electrophoresis (GC-DGGE), which combines mechanistically different community analysis

William E. Holben; Kevin P. Feris; Anu Kettunen; Juha H. A. Apajalahti

2004-01-01

165

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

SciTech Connect

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.

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

1986-01-01

166

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

PubMed

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

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

2012-12-01

167

A combined radiolabelling and silver staining technique for improved visualisation, localisation, and identification of proteins separated by two-dimensional gel electrophoresis.  

PubMed

Two-dimensional gel electrophoresis (2-DE) remains the method of choice for the Separation of protein mixtures whilst mass spectrometry (MS) is rapidly becoming the premier tool for protein identification. When combined, 2-DE and MS form the current operating paradigm for classical proteomics. One of the key challenges of proteome research is that of detecting and identifying all of the elements (proteins) of a proteome. Silver staining and radiolabelling, e.g. with 35S-methionine ([35S]-met), represent two sensitive methods used to visualise many of the constitutive and synthesised elements of a proteome, respectively. The latter method allows a very low total protein loading on a two-dimensional (2-D) gel and challenges protein identification using current MS-based technology. Therefore, it is necessary to refer to and locate a radiolabelled spot's cognate on a preparatively loaded stained gel, or Western blot, and use that protein spot for identification. Unfortunately, the images of autoradiographs and preparative gels or blots, even of the same sample, often do not correspond making it difficult to accurately locate and select spots of interest by visual comparison. We have established a technique that permits the unambiguous localisation of radiolabelled proteins on the same silver stained 2-D gel. Protein identification of superimposed spots is described by peptide mass fingerprinting and database searching using matrix-assisted laser desorption/ionization-time of flight mass spectrometry and by peptide sequencing using tandem MS by hybrid quadrupole/orthogonal acceleration time of flight MS (Q-TOF). PMID:11680883

Westbrook, J A; Yan, J X; Wait, R; Dunn, M J

2001-03-01

168

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

PubMed

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 (2.5-15kDa). 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

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

2015-01-01

169

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

PubMed Central

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

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

2013-01-01

170

Modification of the electrokinetic properties of reversible electrophoresis gels for the separation and preparation of DNA. Addition of linear polymers.  

PubMed

The effect of adding linear polymers to a novel reversible electrophoretic was measured. Reversible gels are formed using the polyanionic carbohydrate polymer, gellan gum. Gellan gum forms strong stable gels in the presence of divalent cations or diamines. The gels are reversible (return to solution) by changing the ionic environment or pH. Gellan gum is an anionic polymer, and the electrophoresis gels have considerable electroosmotic flow (EOF) toward the negative electrode. We measured the EOF in gellan gum electrophoresis gels as a function of gel concentration, buffer composition, and linear polymer additive. The linear polymers used in this study were polyethylene oxide and hydroxyethyl cellulose. Both polymers reduced EOF in the gels, in a manner dependent on molecular weight. Polymers with high molecular weight were more effective at reducing EOF. The addition of polymers increased the resolution of low molecular weight DNA. Native gellan gum resolved DNA from approx 50,000 to 1000 bp. Addition of the polymers resolved DNA down to approx 50 bp, in some instances. The influence of the polymers on circular plasmid DNA was also investigated. Addition of high molecular weight polyethylene oxide reduced the electrophoretic mobility of the nicked circular form compared to the supercoiled form. PMID:10707807

Cole, K D; Tellez, C M; Nguyen, R B

1999-10-01

171

Disease proteomics of high-molecular-mass proteins by two-dimensional gel electrophoresis with agarose gels in the first dimension (Agarose 2-DE).  

PubMed

Agarose gel is the preferred electrophoretic medium currently used for separating high molecular mass (HMM) proteins (MW>100 kDa). Agarose gels are widely used for both SDS-agarose gel electrophoresis and agarose isoelectric focusing (IEF). A two-dimensional gel electrophoresis method employing agarose gels in the first dimension (agarose 2-DE) that is sufficiently good at separating up to 1.5mg of HMM proteins with molecular masses as large as 500 kDa has been used to separate proteins from various diseased tissues and cells. Although resolution of the agarose 2-DE pattern always depends on the tissue being analyzed, sample preparation procedures including (i) protein extraction with an SDS sample buffer; (ii) ultracentrifugation of a tissue homogenate; and (iii) 1% SDS in both stacking and separation gels of the second-dimension SDS-PAGE gel, are generally effective for HMM protein detection. In a comprehensive prostate cancer proteome study using agarose 2-DE, the HMM region of the gel was rich in proteins of particular gene/protein expression groups (39.1% of the HMM proteins but only 28.4% of the LMM ones were classified as transcription/translation-related proteins). Examples include transcription factors, DNA or RNA binding proteins, and ribosomal proteins. To understand oxidative stress-induced cellular damage at the protein level, a novel proteomic method, in which protein carbonyls were derivatized with biotin hydrazide followed by agarose 2-DE, was useful for detecting HMM protein carbonyls in tissues of both a diabetes model Ostuka Long-Evans Tokushima Fatty (OLETF) rat and a control Long-Evans Tokushima Otsuka (LETO) rat. In this paper, we review the use of agarose gels for separation of HMM proteins and disease proteomics of HMM proteins in general, with particular attention paid to our proteome analyzes based on the use of agarose 2-DE for protein separation followed by the use of mass spectrometry for protein identification. PMID:17141588

Oh-Ishi, Masamichi; Maeda, Tadakazu

2007-04-15

172

Proteomic analysis of a decellularized human vocal fold mucosa scaffold using 2D electrophoresis and high-resolution mass spectrometry  

PubMed Central

Natural biologic scaffolds for tissue engineering are commonly generated by decellularization of tissues and organs. Despite some preclinical and clinical success, in vivo scaffold remodeling and functional outcomes remain variable, presumably due to the influence of unidentified bioactive molecules on the scaffold-host interaction. Here, we used 2D electrophoresis and high-resolution mass spectrometry-based proteomic analyses to evaluate decellularization effectiveness and identify potentially bioactive protein remnants in a human vocal fold mucosa model. We noted proteome, phosphoproteome and O-glycoproteome depletion post-decellularization, and identified >200 unique protein species within the decellularized scaffold. Gene ontology-based enrichment analysis revealed a dominant set of functionally-related ontology terms associated with extracellular matrix assembly, organization, morphology and patterning, consistent with preservation of a tissue-specific niche for later cell seeding and infiltration. We further identified a subset of ontology terms associated with bioactive (some of which are antigenic) cellular proteins, despite histological and immunohistochemical data indicating complete decellularization. These findings demonstrate the value of mass spectrometry-based proteomics in identifying agents potentially responsible for variation in host response to engineered tissues derived from decellularized scaffolds. This work has implications for the manufacturing of biologic scaffolds from any tissue or organ, as well as for prediction and monitoring of the scaffold-host interaction in vivo. PMID:23102991

Welham, Nathan V.; Chang, Zhen; Smith, Lloyd M.; Frey, Brian L.

2012-01-01

173

Immunoreactive Coxiella burnetii Nine Mile proteins separated by 2D electrophoresis and identified by tandem mass spectrometry.  

PubMed

Coxiella burnetii is a Gram-negative obligate intracellular pathogen and the causative agent of Q fever in humans. Q fever causes acute flu-like symptoms and may develop into a chronic disease leading to endocarditis. Its potential as a bioweapon has led to its classification as a category B select agent. An effective inactivated whole-cell vaccine (WCV) currently exists but causes severe granulomatous/necrotizing reactions in individuals with prior exposure, and is not licensed for use in most countries. Current efforts to reduce or eliminate the deleterious reactions associated with WCVs have focused on identifying potential subunit vaccine candidates. Both humoral and T cell-mediated responses are required for protection in animal models. In this study, nine novel immunogenic C. burnetii proteins were identified in extracted whole-cell lysates using 2D electrophoresis, immunoblotting with immune guinea pig sera, and tandem MS. The immunogenic C. burnetii proteins elicited antigen-specific IgG in guinea pigs vaccinated with whole-cell killed Nine Mile phase I vaccine, suggesting a T cell-dependent response. Eleven additional proteins previously shown to react with immune human sera were also antigenic in guinea pigs, showing the relevance of the guinea pig immunization model for antigen discovery. The antigens described here warrant further investigation to validate their potential use as subunit vaccine candidates. PMID:21030434

Deringer, James R; Chen, Chen; Samuel, James E; Brown, Wendy C

2011-02-01

174

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

PubMed

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 approximately 10-150 kDa; sample recovery rates were 50% 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 microL/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 microg per channel and reduced resolution. PMID:20119951

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

2010-01-01

175

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

SciTech Connect

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.

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

2009-10-02

176

Neutron-scattering probe of complexes of sodium dodecyl sulfate and serum albumin during polyacrylamide gel electrophoresis.  

PubMed

Small-angle neutron scattering (SANS) is used to probe the conformation of SDS-BSA protein surfactant complexes during electrophoresis in cross-linked polyacrylamide gels. Contrast variation permits independent probing of the structure of protein-surfactant complexes with negligible scattering contributions from the polyacrylamide matrix. The conformation of the protein complexes in the gel is found to be independent of the electric fields that are applied in this work (10 V/cm). Furthermore, there are no signs of large-scale macromolecular orientation (anisotropy) in the scattering patterns. However, the scattering shows that there are significant interparticle correlations between the protein-surfactant complexes that are electrophoretically inserted into the gel. These interactions develop when the total concentration of protein in the gels reaches values that are larger than approximately 1 mg/mL. The correlations are due to molecular crowding in the small fraction of pores that are available for protein migration. PMID:19125631

Pozzo, Danilo C

2009-02-01

177

Characterization of Erwinia amylovora strains from Bulgaria by pulsed-field gel electrophoresis.  

PubMed

The aim of this study was to characterize genetically Bulgarian Erwinia amylovora strains using pulsed-field gel electrophoresis (PFGE) analysis. Fifty E. amylovora strains isolated from different hosts, locations, as well as in different years were analysed by PFGE after XbaI, SpeI, and XhoI digestion of the genomic DNA. The strains were distributed into four groups according to their XbaI-generated profile. About 82% of the strains displayed a PFGE profile identical to that of type Pt2. Three strains belonged to the Central Europe Pt1 type. Two new PFGE profiles, not reported so far, were established--one for a strain isolated from Malus domestica and another for all Fragaria spp. strains. The same grouping of the strains was obtained after analysis of the SpeI digestion patterns. On the basis of PFGE profiles, after XbaI and SpeI digestion, a genetic differentiation between the strains associated with subfamily Maloideae and subfamily Rosoideae was revealed. The presence of more than one PFGE profile in the population of E. amylovora in Bulgaria suggests a multiple source of inoculum. PMID:22624335

Atanasova, Iliana; Urshev, Zoltan; Hristova, Petya; Bogatzevska, Nevena; Moncheva, Penka

2012-01-01

178

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

PubMed

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

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

179

Profenofos induced DNA damage in freshwater fish, Channa punctatus (Bloch) using alkaline single cell gel electrophoresis.  

PubMed

The aim of the present study was to evaluate the induced genotoxicity (DNA damage) due to organophosphate pesticide profenofos (PFF) in gill cells of freshwater fish Channa punctatus using single cell gel electrophoresis (SCGE)/Comet assay. The 96h LC(50) value of PFF (50% EC) was estimated for the fish species in a semistatic system and then three sub-lethal of LC(50) concentrations viz the sub-lethal 1, sub-lethal 2 and sub-lethal 3 concentrations were determined as 0.58ppb, 1.16ppb and 1.74ppb, respectively. The fish specimens were exposed to these concentrations of the pesticide and the gill tissue samplings were done on 24h, 48h, 72h and 96h post exposure for assessment of DNA damage in terms of percentage of DNA in comet tails. In general, a concentration dependent response was observed in the gill cells with induction of maximum DNA damage at the highest concentration of PFF. The results of the present investigation indicated that PFF could potentially induce genotoxic effect in fish, even in sub-lethal concentrations and SCGE as a sensitive and reliable tool for in vivo assessment of DNA damage caused by the genotoxic agents. PMID:21971292

Pandey, Atindra Kumar; Nagpure, N S; Trivedi, Sunil P; Kumar, Ravindra; Kushwaha, Basdeo

2011-12-24

180

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

PubMed

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

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

2001-09-01

181

Pulsed field gel electrophoresis typing of human and retail foodstuff Campylobacters: an Irish perspective.  

PubMed

Campylobacter enteritis is a zoonosis, an infectious disease transmissible under normal conditions from vertebrate animals to man, presenting a major global public health burden. In this study, Pulsed Field Gel Electrophoresis (PFGE) was employed to identify common genotypes in a collection of 600 Campylobacter isolates in order to investigate if profiles obtained from retail samples of foodstuffs matched genotypes causing illness in the community in Ireland. The Campylobacters were isolated from retail foodstuffs, and cases of gastroenteritis, over the same 20-month period in three population centres in Ireland. The major observation made was of a high level of PFGE-genotype heterogeneity; 236 SmaI discrete genotypes were found in 507 strains successfully analysed. Analysis of the PFGE profiles revealed 22 common profiles amongst food isolates and those causing enteritis in humans. These cojoint PFGE genotypes indicate that 56 (38%) of the human clinical isolates are genetically related to 129 (36%) of the food isolates. The identification of these recurrent PFGE types, in the sampled Campylobacter coli and Campylobacter jejuni populations, indicates that a high proportion of Campylobacter isolates found in foods of animal origin also occur in patients with symptoms of enteritis. This data adds weight to the epidemiological hypothesis that a high proportion of human Campylobacter cases are contracted via the handling and consumption of contaminated foodstuffs, in particular poultry. PMID:21356447

O'Leary, Aoife M; Whyte, Paul; Madden, Robert H; Cormican, Martin; Moore, John E; Mc Namara, Eleanor; Mc Gill, Kevina; Kelly, Lorraine; Cowley, Damien; Moran, Lynn; Scates, Pam; Collins, J Dan; Carroll, Cyril V

2011-05-01

182

Comparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and restricted fragment length polymorphism among fenugreek accessions.  

PubMed

Protein and DNA polymorphismswere surveyed among seven accessions of wild fenugreek (Trigonellafoenum-graecum L.) to estimate their genetic diversity and relationships. Samples were obtained from diverse ecogeographical areas in Saudi Arabia and Yemen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of seed storage protein showed genetic variations among fenugreek germplasms, both quantitatively and qualitatively, generating a total of 168 polypeptide bands with different molecular weights ranging from 4.5 to 300 kDa. Twenty-six of these bands were polymorphic, with a considerable polymorphism value (80.00%). Furthermore, restriction fragment length polymorphism (RFLP) analysis was also employed, which was based on the ability of four restriction enzymes (EagI, EcoRI, FspI, and HindIII) to cleave genomic DNA of the plant materials at specific target nucleotide sequences into different numbers of DNA fragments. RFLP analysis revealed 166 fragments with known sequences and variable lengths ranging from 80 to 4000 bp with a highly degree of polymorphism (88.71%). Data derived from SDS-PAGE or RFLP analyses were used to produce dendrograms, which clustered the studied fenugreek accessions into different groups based on the unweighted pair group method with arithmetic mean (UPGMA). The resulting relationships indicated that these two marker techniques were nearly equivalent, but not identical, with respect to phylogenetic information. In conclusion, SDS-PAGE analysis of seed proteins should be augmented with RFLP analysis of DNA for reliable estimates of genetic diversity among fenugreek germplasms. PMID:24338424

Haliem, E A; Al-Huqail, A A

2013-01-01

183

Ammonia oxidizing bacteria community in activated sludge monitored by denaturing gradient gel electrophoresis (DGGE).  

PubMed

Biological treatment based on activated sludge is efficient in municipal wastewater treatment. The removal of nitrogen compounds is crucial for sewage purification. Due to the fact that ammonia is toxic for a water environment and causes eutrophication, ammonia oxidation is of the utmost importance in wastewater treatment. Observing the changeability of ammonia oxidizing bacteria (AOB) and identifying their most abundant species can be helpful in the optimization of wastewater treatment. In this study we used denaturing gradient gel electrophoresis (DGGE), combined with cloning and sequencing of 16S rRNA and AmoA gene fragments in order to estimate AOB biodiversity and temporal community changes. Activated sludge samples were collected from the municipal WWTP in Gliwice (Poland) at 2-week intervals. Ammonia concentration in the influent during the experiment was 30.2-57.6 mg N-NH4+/L. The research revealed a high diversity of uncultured bacteria. It is suspected that these bacteria could be involved in the nitrification, which points to the fact that these bacteria might be efficient in the process. However such a situation is not confirmed and it requires further research. The appearance of Ferribacterium-like bacteria together with Nitrosomonas sp. as the most abundant bacteria was found. PMID:19940383

Ziembi?ska, Aleksandra; Ciesielski, S?awomir; Miksch, Korneliusz

2009-10-01

184

Proteomic Profiling of Aging in Glomeruli of Mice by using Two-Dimensional Differential Gel Electrophoresis.  

PubMed

Background Glomerular proteins were analyzed by proteomics to screen proteins participating in maturation of glomeruli before senescence and to find key proteins involved in the aging process. Material and Methods Glomeruli of C57BL/6 mice at 8 and 20 weeks were separated by kidney perfusion. Proteomic profiles of glomeruli were investigated by using two-dimensional differential gel electrophoresis and MALDI-TOF mass spectrometry. Results We identified 22 differentially expressed proteins. Among them, 3 proteins were significantly up-regulated and 19 proteins were significantly down-regulated in mature mice. Out of these 22 proteins, 18% take part in protein transport, protein targeting, and proteolysis; 5% in glycolysis; 14% in transcription; 9% in electron transport; 9% were chaperones; and 9% were hydrolases. Conclusions Our results provide insights into the glomerular differentially expressed proteins correlated with renal aging. In this study we found that aging altered the expression of ATP synthase subunit beta. Further studies on this protein might help to understand the mechanism of renal aging. PMID:25659849

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

2015-01-01

185

Analysis of secondary modifications of mouse mammary tumor virus proteins by two-dimensional gel electrophoresis.  

PubMed Central

The structural proteins of mouse mammary tumor virus (MMTV) were analyzed by two-dimensional electrophoresis on isoelectric focusing and sodium dodecyl sulfate gels. Many of the viral proteins displayed heterogeneity in charge due to variable contents of carbohydrates (in particular, sialic acid) and phosphate residues. Neuraminidase treatment of the virions influenced the isoelectric pattern of the envelope glycoproteins. The glycoproteins of an MMTV variant which was attenuated by replication in feline kidney cells had different isoelectric points. This suggested that the acquisition of an altered carbohydrate configuration had changed the host range of the virus. The major MMTV structural core protein, p27, consisted of two species, which had identical iodinated tryptic peptide compositions but differed in phosphate contents. Another MMTV phosphoprotein, p21, was separated into four different phosphorylated species. Phosphorylation of p21 could be performed in vitro by the MMTV virion-associated protein kinase. This enzyme also has a high affinity for MMTV p30 as a substrate. Possible functions of this enzyme are discussed. Images PMID:6255175

Nusse, R; Janssen, H; de Vries, L; Michalides, R

1980-01-01

186

Monitoring the lactic acid bacterial diversity during shochu fermentation by PCR-denaturing gradient gel electrophoresis.  

PubMed

The presence of lactic acid bacteria (LAB) during shochu fermentation was monitored by PCR-denaturing gradient gel electrophoresis (DGGE) and by bacteriological culturing. No LAB were detected from fermented mashes by PCR-DGGE using a universal bacterial PCR primer set. However, PCR-DGGE using a new primer specific for the 16S rDNA of Lactococcus, Streptococcus, Tetragenococcus, Enterococcus, and Vagococcus and two primers specific for the 16S rDNA of Lactobacillus, Pediococcus, Leuconostoc, and Weissella revealed that Enterococcus faecium, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus nagelii, Lactobacillus plantarum, Lactococcus lactis, Leuconostoc citreum, Leuconostoc mesenteroides, and Weissella cibaria inhabited in shochu mashes. It was also found that the LAB community composition during shochu fermentation changed after the main ingredient and water were added during the fermentation process. Therefore, we confirmed that PCR-DGGE using all three primers specific for groups of LAB together was well suited to the study of the LAB diversity in shochu mashes. The results of DGGE profiles were similar to the results of bacteriological culturing. In conclusion, LAB are present during shochu fermentation but not dominant. PMID:16233780

Endo, Akihito; Okada, Sanae

2005-03-01

187

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

PubMed

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

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

2006-02-01

188

Myoglobin intron variation in the Gouldian Finch Erythrura gouldiae assessed by temperature gradient gel electrophoresis.  

PubMed

In this study we show how the use of exon-primed, intron-crossing (EPIC) polymerase chain reaction (PCR) of a diploid intronic region, in conjunction with temperature gradient gel electrophoresis (TGGE), can be used to detect and rapidly assess allelic variation at the nucleotide level. We developed passerine-specific primers to amplify and sequence a 762 bp region including the second intron of the myoglobin gene in the Gouldian Finch, Erythrura gouldiae. A POLAND plot based on this sequence indicated that TGGE in combination with heteroduplex analysis (TGGE/HA) should reveal nucleotide variation in the 160 bp low-melting domain. Sequencing of the entire fragment from 19 Er. gouldiae revealed five nucleotide substitution differences within the low-melt domain, all of which could be detected and differentiated by TGGE/HA, and an additional substitution in a section of the high-melt domain which characterised another allele. A total of 181 individuals from four populations were screened for these six alleles. PMID:9548272

Heslewood, M M; Elphinstone, M S; Tidemann, S C; Baverstock, P R

1998-02-01

189

Molecular epidemiologic analysis of Vibrio cholerae O1 isolates by pulsed-field gel electrophoresis.  

PubMed Central

Isolates of Vibrio cholerae O1 El Tor from two well-defined cholera outbreaks in Malaysia were analyzed by using pulsed-field gel electrophoresis (PFGE). Isolates from sporadic cases occurring during the same time period were also studied. Digestion of chromosomal DNA from these isolates of V. cholerae O1 with restriction endonucleases NotI (5'-GCGGCCGC-3') and SfiI (5'-GGCCNNNN-3'), followed by PFGE, produced restriction endonuclease analysis (REA) patterns consisting of 13 to 24 bands (ranging in size from 46 to 398 kbp). Analysis of the REA patterns generated by PFGE after digestion with NotI and SfiI suggested the clonal nature and close genetic identity of the isolates obtained during each of the two outbreaks (Dice coefficient, 0.93 to 1.0). Although they had very similar REA patterns, the two outbreak clones were not identical. Isolates of V. cholerae O1 from sporadic cases, on the other hand, appeared to be much more heterogeneous (five different REA patterns detected in the five isolates tested; Dice coefficient, 0.31 to 0.81) than those obtained during the two outbreaks. We conclude that PFGE of V. cholerae O1 chromosomal DNA digested with infrequently cutting restriction endonucleases is a useful method for molecular typing of V. cholerae isolates for epidemiological purposes. Images PMID:7883885

Mahalingam, S; Cheong, Y M; Kan, S; Yassin, R M; Vadivelu, J; Pang, T

1994-01-01

190

Difference gel electrophoresis (DiGE) identifies differentially expressed proteins in endoscopically-collected pancreatic fluid  

PubMed Central

Alterations in the pancreatic fluid proteome of individuals with chronic pancreatitis may offer insights into the development and progression of the disease. The endoscopic pancreas function test (ePFT) can safely collect large volumes of pancreatic fluid that are potentially amenable to proteomic analyses using difference gel electrophoresis (DiGE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Pancreatic fluid was collected endoscopically using the ePFT method following secretin stimulation from three individuals with severe chronic pancreatitis and three chronic abdominal pain controls. The fluid was processed to minimize protein degradation and the protein profiles of each cohort, as determined by DiGE and LC-MS/MS, were compared. This DiGE-LC-MS/MS analysis reveals proteins that are differentially expressed in chronic pancreatitis compared to chronic abdominal pain controls. Proteins with higher abundance in pancreatic fluid from chronic pancreatitis individuals include: actin, desmoplankin, alpha-1-antitrypsin, SNC73, and serotransferrin. Those of relatively lower abundance include carboxypeptidase B, lipase, alpha-1-antichymotrypsin, alpha-2-macroglobulin, Arp2/3 subunit 4, glyceraldehyde-3-phosphate dehydrogenase, and protein disulfide isomerase. Endoscopic collection (ePFT) in tandem with DiGE-LC-MS/MS is a suitable approach for pancreatic fluid proteome analysis, however, further optimization of our protocol, as outlined herein, may improve proteome coverage in future analyses. PMID:21792986

Paulo, Joao A.; Lee, Linda S.; Banks, Peter A.; Steen, Hanno; Conwell, Darwin L.

2012-01-01

191

Happy bicentennial, electrophoresis!  

PubMed

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

Righetti, Pier Giorgio

2009-12-01

192

Thermal denaturation of double-stranded nucleic acids: prediction of temperatures critical for gradient gel electrophoresis and polymerase chain reaction.  

PubMed Central

A program is described which calculates the thermal stability and the denaturation behaviour of double-stranded DNAs and RNAs up to a length of 1000 base pairs. The algorithm is based on recursive generation of conditional and a priori probabilities for base stacking. Output of the program may be compared directly to experimental results; thus the program may be used to optimize the nucleic acid fragments, the primers and the experimental conditions prior to experiments like polymerase chain reactions, temperature-gradient gel electrophoresis, denaturing-gradient gel electrophoresis and hybridizations. The program is available in three versions; the first version runs interactively on VAXstations producing graphics output directly, the second is implemented as part of the HUSAR package at GENIUSnet, the third runs on any computer producing text output which serves as input to available graphics programs. Images PMID:8052531

Steger, G

1994-01-01

193

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

NASA Astrophysics Data System (ADS)

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

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

2009-11-01

194

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

PubMed

Pulsed-field gel electrophoresis (PFGE) separates large DNA molecules by the use of an alternating electrical field, such that greater size resolution can be obtained when compared to normal agarose gel electrophoresis. PFGE is often employed to track pathogens and is a valuable typing scheme to detect and differentiate strains. Particularly, the contour-clamped homogeneous electric field (CHEF) PFGE system is considered to be the gold standard for use in epidemiological studies of many bacterial pathogens. Here we describe a PFGE protocol that was applicable to the study of bovine streptococci, namely, Streptococcus agalactiae (group B Streptococcus, GBS), Streptococcus dysgalactiae subsp. dysgalactiae (group C Streptococcus, GCS), and Streptococcus uberis-which are relevant pathogens causing mastitis, a highly prevalent and costly disease in dairy industry due to antibiotherapy and loss in milk production. PMID:25399106

Santos-Sanches, Ilda; Chambel, Llia; Tenreiro, Rogrio

2015-01-01

195

Use of an acid-labile surfactant as an SDS substitute for gel electrophoresis and proteomic analysis.  

PubMed

Modern protein identification and analysis relies largely on proteolytic in-gel digestion of proteins separated during polyacrylamide gel electrophoresis (PAGE) followed by mass spectrometric (MS) measurement of the extracted peptides. Sodium dodecyl sulfate (SDS) is routinely used in nonnative PAGE. However, SDS can interfere with MS. We report the use of an acid-labile surfactant (ALS-I) in place of SDS. ALSI is a long chain derivative of 1,3-dioxolane sodium propyloxy sulfate and has similar denaturing and electrophoretic properties as SDS, but it decomposes at low pH and enhances MS detection of proteins. PMID:19498958

Zeller, Martin; Brown, Elizabeth K; Bouvier, Edouard S P; Knig, Simone

2002-03-01

196

Identification of Listeria monocytogenes contamination sources in two fresh sauce production plants by pulsed-field gel electrophoresis  

Microsoft Academic Search

Twenty-three Listeria monocytogenes strains isolated from two food-processing plants, which produce fresh sauces, were serologically characterized and tested by the mouse biological assay and molecular typing by pulsed-field gel electrophoresis (PFGE). The use of PFGE for the characterization of these L. monocytogenes strains provided, in plant A, valuable information about potential sites of cross-contamination and in plant B, a valuable

M Pourshaban; M Gianfranceschi; A Gattuso; F Menconi; P Aureli

2000-01-01

197

Comparison of Ribotyping, Arbitrarily Primed PCR, and Pulsed-Field Gel Electrophoresis for Molecular Typing ofListeria monocytogenes  

Microsoft Academic Search

Fifty-one clinical isolates ofListeria monocytogenes(15 isolates from two outbreaks and 36 epidemiologically unrelated isolates) were typed by conventional serotyping, ribotyping (RT), pulsed-field gel electrophoresis (PFGE), and arbitrarily primed PCR (AP-PCR). Serotyping was unable to distinguish between related and unrelated strains of L. monocytogenes. Each of the three molecular methods showed excellent typeability and reproducibility. Restriction with EcoRI and PvuII gave

M. LOUIE; P. JAYARATNE; I. LUCHSINGER; J. DEVENISH; J. YAO; W. SCHLECH

198

Phylogenetic diversity of nitrogen-fixing bacteria in mangrove sediments assessed by PCRdenaturing gradient gel electrophoresis  

Microsoft Academic Search

Culture-independent PCRdenaturing gradient gel electrophoresis (DGGE) was employed to assess the composition of diazotroph\\u000a species from the sediments of three mangrove ecosystem sites in Sanya, Hainan Island, China. A strategy of removing humic\\u000a acids prior to DNA extraction was conducted, then total community DNA was extracted using the soil DNA kit successfully for\\u000a nifH PCR amplification, which simplified the current

Yanying Zhang; Junde Dong; Zhihao Yang; Si Zhang; Youshao Wang

2008-01-01

199

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

Microsoft Academic Search

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

Christoph Helma; Maria Uhl

2000-01-01

200

Direct calculation of the sizes of DNA fragments separated by gel electrophoresis using programmes written for a pocket calculator.  

PubMed Central

In order to facilitate the direct computation of the sizes of DNA fragments separated by gel electrophoresis, we have written and evaluated programmes for the Hewlett-Packard 41C programmable calculator. The sizes estimated for DNA fragments of known length using some of these programmes were found to be more accurate than the estimates obtained by conventional graphical procedures. These programmes should be adaptable to other programmable calculators. Images PMID:6320110

Gough, E J; Gough, N M

1984-01-01

201

Denaturing Gradient Gel Electrophoresis Used To Monitor the Enrichment Culture of Aerobic Chemoorganotrophic Bacteria from a Hot Spring Cyanobacterial Mat  

Microsoft Academic Search

Previous studies investigating microbial diversity in the Octopus Spring cyanobacterial mat community (Yellowstone National Park) have shown a discrepancy between bacterial populations observed by molecular retrieval and cultivation techniques. To investigate how selective enrichment culture techniques affect species composition, we used denaturing gradient gel electrophoresis (DGGE) separation of PCR-amplified 16S rRNA gene fragments to monitor the populations contained within enrichment

CECILIA M. SANTEGOEDS; STEPHEN C. NOLD; ANDDAVID M. WARD

1996-01-01

202

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

Microsoft Academic Search

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

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

1999-01-01

203

Classification of Lactobacillus plantarum by Restriction Endonuclease Analysis of Total Chromosomal DNA Using Conventional Agarose Gel Electrophoresis  

Microsoft Academic Search

A total of 17 Lactobacillus plantarum strains that originated from different environments and 24 reference strains were classified by performing a restriction endonuclease analysis of total chromosomal DNAs digested with EcolRI, HindIII, and ClaI, and the resulting patterns were visualized after the fragments were separated according to size by agarose gel electrophoresis. The patterns were analyzed by using the Pearson

M.-L. JOHANSSON; M. QUEDNAU; G. MOLIN

204

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

NASA Astrophysics Data System (ADS)

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

Kim, Joong Hyun; Chaudhary, Sumit; Ozkan, Mihrimah

2007-05-01

205

Evaluation of pulsed-field gel electrophoresis and rep-PCR for the epidemiological analysis of Ochrobactrum anthropi strains  

Microsoft Academic Search

Pulsed-field gel electrophoresis and polymerase chain reaction genome fingerprinting based on repetitive chromosomal sequences (rep-PCR) were used for typing 14 strains ofOchrobactrum anthropi. Six strains isolated during an outbreak of bacteraemia in patients who had received a contaminated rabbit anti-thymocyte globulin gave identical patterns by both techniques. Different patterns were found in sporadic and reference strains, except for one clinical

P. van Dijck; M. Delme; H. Ezzedine; A. Deplano; M. J. Struelens

1995-01-01

206

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

Three serological assays, protein gel electrophoresis hics. and the polymerase chain reaction assays were evaluated in psittacine birds. Birds suspected of Chlamydia psittaci infections were identified and tested with the following assays...

Hofle, Michael David

1999-01-01

207

Combination of static-field gel electrophoresis and densitometric scanning for the determination of radiation-induced DNA double-strand breaks in CHO cells  

Microsoft Academic Search

An experimental setup using static-field gel electrophoresis (SFGE) was developed to determine radiation-induced DNA double-strand breaks (DSBs) in CHO-K1 cells after exposure to X-rays or heavy charged particles. The fraction of DNA eluted into the gel matrix depends on the quantity of DSBs introduced. In agreement with a recent report, SFGE and pulsed-field electrophoresis were found to be equally sensitive

M. Schneider; G. Taucher-Scholz; J. Heilmann; G. Kraft

1994-01-01

208

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

PubMed

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

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

2015-01-01

209

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

PubMed Central

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

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

210

Efficient subtyping of pathogenic Yersinia enterocolitica strains by pulsed-field gel electrophoresis.  

PubMed Central

Yersinia enterocolitica is an enteropathogen that has recently and rapidly expanded over the world. There is a close correlation between the biotypes, serotypes, and phage types of the strains, making it virtually impossible to distinguish isolates of the same serotype with the classical phenotypic markers. In the present study, pulsed-field gel electrophoresis (PFGE) was used to compare the NotI genomic profile (i.e., pulsotype) of 20 strains each of serotypes O:3, O:9, and O:5. Eleven, 12, and 18 different pulsotypes were obtained, respectively, indicating that this technique is very efficient for subtyping pathogenic isolates of Y. enterocolitica. Within strains of serotype O:5, PFGE differentiated two subgroups that corresponded to two biotypes (biotypes 1A and 3). Comparison of the pulsotypes of three strains of biotype 3 and serotype O:3 (referred to as 3/O:3) with those of strains 4/O:3 and 3/O:5 suggested that the pulsotype is closer to the biotype than to the serotype. The pulsotypes of five pairs of strains isolated from the same patient or siblings were also analyzed. In four pairs, the two strains displayed identical pulsotypes, indicating that PFGE might be a powerful epidemiological tool. In the fifth pair, one restriction fragment differed, suggesting that genomic polymorphism may occur in vivo in Y. enterocolitica. Finally, the in vitro genomic stabilities of one strain each of Y. enterocolitica O:3, O:9, and O:5 were investigated. The pulsotypes of 10 isolated colonies were identical within each strain, indicating that in vitro, the genome of Y. enterocolitica is much more stable than that of Y. pestis. Images PMID:7883877

Najdenski, H; Iteman, I; Carniel, E

1994-01-01

211

Population dynamics of chesapeake bay virioplankton: total-community analysis by pulsed-field gel electrophoresis  

PubMed

Recognition of viruses as the most abundant component of aquatic microbial communities has stimulated investigations of the impact of viruses on bacterio- and phytoplankton host communities. From results of field studies to date, it is concluded that in most aquatic environments, a reduction in the number of bacteria on a daily basis is caused by viral infection. However, the modest amount of in situ virus-mediated mortality may be less significant than viral infection serving to maintain clonal diversity in the host communities directly, through gene transmission (i.e., transduction), and indirectly, by elimination of numerically dominant host species. If the latter mechanism for controlling community diversity prevails, then the overall structure of aquatic viral communities would be expected to change as well over short seasonal and spatial scales. To determine whether this occurs, pulsed-field gel electrophoresis (PFGE) was used to monitor the population dynamics of Chesapeake Bay virioplankton for an annual cycle (1 year). Virioplankton in water samples collected at six stations along a transect running the length of the bay were concentrated 100-fold by ultrafiltration. Viruses were further concentrated by ultracentrifugation, and the concentrated samples were embedded in agarose. PFGE analysis of virus DNA in the agarose plugs yielded several distinct bands, ranging from 50 to 300 kb. Principal-component and cluster analyses of the virus PFGE fingerprints indicated that changes in virioplankton community structure were correlated with time, geographical location, and extent of water column stratification. From the results of this study, it is concluded that, based on the dynamic nature of the Chesapeake Bay virioplankton community structure, the clonal diversity of bacterio- and phytoplankton host communities is an important component of the virus community. PMID:9872784

Wommack; Ravel; Hill; Chun; Colwell

1999-01-01

212

Typing of Staphylococcus aureus by pulsed-field gel electrophoresis, zymotyping, capsular typing, and phage typing: resolution of clonal relationships.  

PubMed

Sixty-nine Staphylococcus aureus isolates from two epidemiologically unrelated sources were typed by pulsed-field gel electrophoresis after SmaI digestion of chromosomal DNA (genome typing), and the results were compared with those obtained by other typing methods: phage typing with the international set of phages, capsular serotyping with monoclonal antibodies against capsular polysaccharides type 5 and 8, and zymotyping by polyacrylamide agarose electrophoresis for esterase polymorphism. A good correlation of S. aureus types was found by these four typing methods. Differentiation increased in the order capsular typing < zymotyping < phage typing < genome typing, yielding 2, 10, 20, and 26 different S. aureus types, respectively. Five of the 26 genome types were further divided into several subtypes revealing clonal relationships. When 36 French S. aureus isolates were compared with 33 German S. aureus isolates, 3 strains representing clonal populations were identical in both groups. S. aureus isolates from patients with cystic fibrosis were also typed at the beginning and the end of a 4-week summer camp for these patients. The results suggested a possible strain transmission during the summer camp. We conclude that genome typing by pulsed-field gel electrophoresis is a powerful tool not only for strain identification but also for the resolution of the clonal relationships of S. aureus strains. PMID:8432807

Schlichting, C; Branger, C; Fournier, J M; Witte, W; Boutonnier, A; Wolz, C; Goullet, P; Dring, G

1993-02-01

213

Typing of Staphylococcus aureus by pulsed-field gel electrophoresis, zymotyping, capsular typing, and phage typing: resolution of clonal relationships.  

PubMed Central

Sixty-nine Staphylococcus aureus isolates from two epidemiologically unrelated sources were typed by pulsed-field gel electrophoresis after SmaI digestion of chromosomal DNA (genome typing), and the results were compared with those obtained by other typing methods: phage typing with the international set of phages, capsular serotyping with monoclonal antibodies against capsular polysaccharides type 5 and 8, and zymotyping by polyacrylamide agarose electrophoresis for esterase polymorphism. A good correlation of S. aureus types was found by these four typing methods. Differentiation increased in the order capsular typing < zymotyping < phage typing < genome typing, yielding 2, 10, 20, and 26 different S. aureus types, respectively. Five of the 26 genome types were further divided into several subtypes revealing clonal relationships. When 36 French S. aureus isolates were compared with 33 German S. aureus isolates, 3 strains representing clonal populations were identical in both groups. S. aureus isolates from patients with cystic fibrosis were also typed at the beginning and the end of a 4-week summer camp for these patients. The results suggested a possible strain transmission during the summer camp. We conclude that genome typing by pulsed-field gel electrophoresis is a powerful tool not only for strain identification but also for the resolution of the clonal relationships of S. aureus strains. Images PMID:8432807

Schlichting, C; Branger, C; Fournier, J M; Witte, W; Boutonnier, A; Wolz, C; Goullet, P; Dring, G

1993-01-01

214

Plasma protein electrophoresis in birds: comparison of a semiautomated agarose gel system with an automated capillary system.  

PubMed

Plasma agarose gel electrophoresis (AGE) is recognized as a very reliable diagnostic tool in avian medicine. Within the last 10 years, new electrophoresis techniques such as capillary zone electrophoresis (CZE) have emerged in human laboratory medicine but have never been investigated in birds. To investigate the use of CZE in birds and to compare it with AGE, plasma samples from 30 roosters (Gallus gallus), 20 black kites (Milvus migrans), and 10 racing pigeons (Columba livia) were analyzed by both AGE and CZE. For the 3 species studied, values determined by AGE and CZE were well correlated for albumin and beta and gamma fractions whereas other values differed significantly. Values for alpha-3 fraction in the rooster, alpha-1 fraction in the black kite, and alpha fractions in the pigeon obtained by AGE were very well correlated with the prealbumin fraction values obtained by CZE. Repeatability and reproducibility appeared higher with CZE than with AGE. Although the interpretation of CZE electrophoresis patterns seems to produce results similar to those obtained with AGE, some proteins present in the alpha fraction measured with AGE migrated to the prealbumin fraction found with CZE. Although CZE requires the use of specific reference intervals and a much higher sample volume, this method has many advantages when compared with AGE, including better repeatability and reproducibility and higher analysis output. PMID:23971218

Roman, Yannick; Bomsel-Demontoy, Marie-Claude; Levrier, Julie; Chaste-Duvernoy, Daniel; Saint Jalme, Michel

2013-06-01

215

[Investigation of Salmonella serotype Enteritidis isolates by plasmid profile analysis and pulsed field gel electrophoresis].  

PubMed

In this study a total of 122 Salmonella serotype Enteritidis stock strains selected from the culture collection of Enterobacteriaceae Laboratory of Ankara University Faculty of Medicine, Department of Medical Microbiology, were investigated by plasmid profile analysis with the method defined by Kado and Liu and pulsed field gel electrophoresis (PFGE) according to World Health Organization protocols using SpeI and XbaI macrorestriction enzymes, for better understanding of the molecular epidemiology of S. Enteritidis. The study strains were selected from a collection of previously isolated epidemic (n= 13) and sporadic (n= 109) strains (103 stool, 16 blood and one each bile, urine and cerebrospinal fluid) obtained from 10 different cities after the year 2000. PFGE patterns were analyzed with Gene Directory software (Syngene, UK) and a similarity index was determined by using Dice coefficient and the unweighted pair group method with mathematical averaging (UPGMA). Plasmid-carrying 110 (90%) strains that harbored 1-4 plasmids with sizes ranging from 2.0 to 100 kb were separated into patterns more than 14 (p1-p14). A total of 85 (69.7%) isolates harbored the 57 kb plasmid solely or in combination with other plasmids. By PFGE, 11 distinct patterns were shown with each enzyme SpeI and XbaI. S. Enteritidis strains after digestion with macrorestriction enzyme SpeI generated 11 different PFGE patterns (A to K), whereas XbaI generated also 11 different PFGE patterns (a to k). PFGE pattern A consisted of 93 strains (76.2%) after digestion with macrorestriction enzyme SpeI, while PFGE pattern a consisted 53 (43.4%) and PFGE pattern b 42 strains (34.4%) after digestion with macrorestriction enzyme XbaI. Using two macrorestriction enzymes two PFGE cluster profiles Aa (50 strains, 40.9%) and Ab (42 strains, 34.4%) were found to be predominating among 17 different PFGE clusters. Our results confirmed the clonal nature of S. Enteritidis strains in Turkey. The use of two enzymes in PFGE analysis appeared to increase the discriminatory power of PFGE, leading to greater diversity among strains. PFGE analysis performed by SpeI and XbaI enzymes combined with plasmid profiling could be established as a useful tool for detection of genetic relationship between isolates. PMID:21644064

Us, Ebru; Erdem, Birsel; Tekeli, Alper; Gereker, Devran; Saran, Begm; Bayramova, Mehseti; Sahin, Fikret

2011-04-01

216

Typing and subtyping of haptoglobin from native serum using disc gel electrophoresis in alkaline buffer: application to routine screening.  

PubMed

A method with which the six common phenotypes of human haptoglobin can be identified using unseparated serum is described. In contrast to other reported methods, both typing and subtyping of haptoglobin can be performed by polyacrylamide gel electrophoresis in alkaline buffer using 0.1-4.0 microliter of native serum with hemoglobin added. Haptoglobin-hemoglobin complexes are visualized by their peroxidase activity using benzidine and barium peroxide. This relatively inexpensive and fast method seems particularly well suited for the typing and subtyping of haptoglobin from minute amounts in large series of sera and other body fluids and thus may be useful in medical genetics and forensic medicine. PMID:6496936

Linke, R P

1984-08-15

217

Effect of antisense peptide binding on the dimerization of human cystatin C--gel electrophoresis and molecular modeling studies.  

PubMed

Human cystatin C (HCC) shows a tendency to dimerize. This process is particularly easy in the case of the L68Q HCC mutant and might lead to formation of amyloid deposits in brain arteries of young adults. Our purpose was to find ligands of monomeric HCC that can prevent its dimerization. Eleven antisense peptide ligands of monomeric HCC were designed and synthesized. The influence of these ligands on HCC dimerization was studied using gel electrophoresis and molecular modeling methods. The results suggest that all the designed peptides interact with monomeric HCC facilitating its dimerization rather than preventing it. PMID:15094836

Stachowiak, Krystyna; Rodziewicz-Motowid?o, Sylwia; Sosnowska, Renata; Kasprzykowski, Franciszek; ?ankiewicz, Leszek; Grubb, Anders; Grzonka, Zbigniew

2004-01-01

218

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

NASA Technical Reports Server (NTRS)

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.

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

2003-01-01

219

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

PubMed

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

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

2015-06-01

220

Modification of the electrokinetic properties of reversible electrophoresis gels for the separation and preparation of DNA  

Microsoft Academic Search

The effect of adding linear polymers to a novel reversible electrophoretic media was measured. Reversible gels are formed\\u000a using the polyanionic carbohydrate polymer, gellan gum. Gellan gum forms strong stable gels in the presence of divalent cations\\u000a or diamines. The gels are reversible (return to solution) by changing the ionic environment or pH. Gellan gum is an anionic\\u000a polymer, and

Kenneth D. Cole; Carlos M. Tellez; Richard B. Nguyen

1999-01-01

221

A New Standard-Based Polynomial Interpolation (SBPIn) Method to Address Gel-to-Gel Variability for the Comparison of Multiple Denaturing Gradient Gel Electrophoresis Profile Matrices  

PubMed Central

The Standard-Based Polynomial Interpolation (SBPIn) method is a new simple three-step protocol proposed to address common gel-to-gel variations for the comparison of sample profiles across multiple DGGE gels. The advantages of this method include no requirement for additional software or modification of the standard DGGE protocol. PMID:23234884

Valentn-Vargas, Alexis; Chorover, Jon; Maier, Raina M.

2013-01-01

222

Effects of coffee bean aroma on the rat brain stressed by sleep deprivation: a selected transcript- and 2D gel-based proteome analysis.  

PubMed

The aim of this study was 2-fold: (i) to demonstrate influences of roasted coffee bean aroma on rat brain functions by using the transcriptomics and proteomics approaches and (ii) to evaluate the impact of roasted coffee bean aroma on stress induced by sleep deprivation. The aroma of the roasted coffee beans was administered to four groups of adult male Wistar rats: 1, control group; 2, 24 h sleep deprivation-induced stress group (the stress group); 3, coffee aroma-exposed group without stress (the coffee group); and 4, the stress with coffee aroma group (the stress with coffee group). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of some known genes responsive to aroma or stress was performed using total RNA from these four groups. A total of 17 selected genes of the coffee were differently expressed over the control. Additionally, the expression levels of 13 genes were different between the stress group and the stress with coffee group: Up-regulation was found for 11 genes, and down-regulation was seen for two genes in the stress with coffee group. We also looked to changes in protein profiles in these four samples using two-dimensional (2D) gel electrophoresis; 25 differently expressed gel spots were detected on 2D gels stained by silver nitrate. Out of these, a total of nine proteins were identified by mass spectrometry. Identified proteins belonged to five functional categories: antioxidant; protein fate; cell rescue, defense, and virulence; cellular communication/signal transduction mechanism; and energy metabolism. Among the differentially expressed genes and proteins between the stress and the stress with coffee group, NGFR, trkC, GIR, thiol-specific antioxidant protein, and heat shock 70 kDa protein 5 are known to have antioxidant or antistress functions. In conclusion, the roasted coffee bean aroma changes the mRNA and protein expression levels of the rat brain, providing for the first time clues to the potential antioxidant or stress relaxation activities of the coffee bean aroma. PMID:18517217

Seo, Han-Seok; Hirano, Misato; Shibato, Junko; Rakwal, Randeep; Hwang, In Kyeong; Masuo, Yoshinori

2008-06-25

223

Proteomic Analysis of Human Saliva From Lung Cancer Patients Using Two-Dimensional Difference Gel Electrophoresis and Mass Spectrometry*  

PubMed Central

Lung cancer is often asymptomatic or causes only nonspecific symptoms in its early stages. Early detection represents one of the most promising approaches to reduce the growing lung cancer burden. Human saliva is an attractive diagnostic fluid because its collection is less invasive than that of tissue or blood. Profiling of proteins in saliva over the course of disease progression could reveal potential biomarkers indicative of oral or systematic diseases, which may be used extensively in future medical diagnostics. There were 72 subjects enrolled in this study for saliva sample collection according to the approved protocol. Two-dimensional difference gel electrophoresis combined with MS was the platform for salivary proteome separation, quantification, and identification from two pooled samples. Candidate proteomic biomarkers were verified and prevalidated by using immunoassay methods. There were 16 candidate protein biomarkers discovered by two-dimensional difference gel electrophoresis and MS. Three proteins were further verified in the discovery sample set, prevalidation sample set, and lung cancer cell lines. The discriminatory power of these candidate biomarkers in lung cancer patients and healthy control subjects can reach 88.5% sensitivity and 92.3% specificity with AUC = 0.90. This preliminary data report demonstrates that proteomic biomarkers are present in human saliva when people develop lung cancer. The discriminatory power of these candidate biomarkers indicate that a simple saliva test might be established for lung cancer clinical screening and detection. PMID:22096114

Xiao, Hua; Zhang, Lei; Zhou, Hui; Lee, Jay M.; Garon, Edward B.; Wong, David T. W.

2012-01-01

224

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

PubMed Central

Purpose: The increasing use of herbal drugs and their easy availability have necessitated the use of mutagenicity test to analyze their toxicity and safety. The aim of this study was to evaluate the genotoxicity of Supermint herbal medicine in DNA breakage of rat hepatocytes in comparison with sodium dichromate by single cell gel electrophoresis technique or comet assay. Methods: Hepatocytes were prepared from male wistar rats and were counted and kept in a bioreactor for 30 minutes. Then cells were exposed to the Supermint herbal medicine at doses of 125, 250 and 500 l/ml. Buffer 4 (incubation buffer) and sodium dichromate were used as negative and positive control for one hour respectively. Then cell suspension with low melting point agarose were put on precoated slides and covered with agarose gel. Then lysing, electrophoresis, neutralization and staining were carried out. Finally the slides were analyzed with fluorescence microscope. The parameter under this analysis was the type of migration which was determined according to Kobayashi pattern. Results: With increased dose of Supermint herbal medicine the DNA damage was slightly increased (P<0001). Conlusion: In overall compared to the positive control significant differences is observed which convinced that the crude extract of Supermint in vitro did not have mutagenic effect. Conlusion: In overall compared to the positive control significant differences is observed which convinced that the crude extract of Supermint in vitro did not have mutagenic effect. PMID:24312800

Kalantari, Heibatullah; Rezaei, Mohsen; Mahdavinia, Masoud; Kalantar, Mojtaba; Amanpour, Zivar; varnaseri, golnaz

2012-01-01

225

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

PubMed

Small-angle X-ray (SAXS) and neutron (SANS) scattering is used to probe the structure of protein-surfactant complexes in solution and to correlate this information with their performance in gel electrophoresis. Proteins with sizes between 6.5 to 116 kDa are denatured with sodium alkyl sulfates (SC(x)S) of variable tail lengths. Several combinations of proteins and surfactants are analyzed to measure micelle radii, the distance between micelles, the extension of the complex, the radius of gyration, and the electrophoretic mobility. The structural characterization shows that most protein-surfactant complexes can be accurately described as pearl-necklace structures with spherical micelles. However, protein complexes with short surfactants (SC(8)S) bind with micelles that deviate significantly from spherical shape. Sodium decyl (SC(10)S) and dodecyl (SC(12)S, more commonly abbreviated as SDS) sulfates result in the best protein separations in standard gel electrophoresis. Particularly, SC(10)S shows higher resolutions for complexes of low molecular weight. The systematic characterization of alkyl sulfate surfactants demonstrates that changes in the chain architecture can significantly affect electrophoretic migration so that protein-surfactant structures could be optimized for high resolution protein separations. PMID:21182321

Ospinal-Jimnez, Mnica; Pozzo, Danilo C

2011-02-01

226

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

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

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

2012-01-01

227

Comparison of the Serum Protein Fractions of the Newly Hatched Chick with those of Adult Birds using Starch-gel Electrophoresis  

Microsoft Academic Search

IT has been established by several investigators that serum protein components of chick embryo undergo qualitative as well as quantitative changes throughout the developmental period until the time of hatching1-4. The serum protein composition of the newly hatched chick, however, resembles that of the adult bird, as revealed by moving-boundary and paper electro-phoresis. The technique of starch-gel electrophoresis suggested by

A. Amin

1961-01-01

228

Typing of nosocomial strains of Serratia marcescens: comparison of pulsed-field gel electrophoresis of macrorestriction fragments with biotyping, esterase typing and ribotyping  

Microsoft Academic Search

Fifty nosocomial isolates of Serratia marcescens, collected in six Belgian hospitals between 1986 and 1990, were characterized by using pulsed-field gel electrophoresis (PFGE) with Xbal. The results were compared with those previously obtained by three other methods: biotyping, esterase electrophoresis typing and ribotyping with EcoRl and Hindlll. Macrorestriction analysis (42 PFGE groups) and esterase typing (42 zymo-types) proved to be

H. Chetoui; E. Delhalle; P. Melin; M. J. Struelens; R. De Ryck; P. Osterrieth; P. De Mol

1998-01-01

229

Distribution of the Serum Proteins of Syrian Hamster as revealed by Starch-gel Electrophoresis  

Microsoft Academic Search

ALTHOUGH considerable work has been published on the distribution of the serum proteins of various laboratory animals, little has been reported on the electrophoretic pattern of the serum proteins of hamster. Moore1 investigated the serum proteins of several species of animals, including that of the hamster, by moving boundary electrophoresis. He identified six components in the serum of hamster corresponding

Abolghassem Amin; K. D. Shamloo

1963-01-01

230

Zinc-imidazole positive: a new method for DNA detection after electrophoresis on agarose gels not interfering with DNA biological integrity.  

PubMed

Nucleic acids separated by gel electrophoresis are commonly detected within the gel matrix with ethidium bromide staining, followed by gel irradiation with ultraviolet (UV) light. When the separated nucleic acids are to be recovered for further characterization or use, this methodology is unsuitable (i) because a significant number of chemical lesions to the nucleic acid molecules are caused, heavily compromising their biological activity, and (ii) because of health hazards due to accumulative direct contact with ethidium bromide and exposure to UV-light. As an alternative, for preparative purposes, a new nontoxic detection method employing zinc and imidazole salts is described. After electrophoresis, the gel is first washed in distilled water to substantially remove remaining electrophoresis reagents, then incubated in 40 mM zinc sulfate for 10 min to allow binding of Zn2+ to the DNA, and subsequently washed with distilled water to remove unbound Zn2+ from gel regions devoid of DNA. On soaking in 0.2 M imidazole for a few minutes, zinc-DNA complexes are visualized as deep-white (positive) stained bands against a slightly opaque background. The sensitivity is similar to that of ethidium bromide. Gels can be kept in distilled water for months without loss of staining. After zinc chelation, e.g. with EDTA, it is feasible to quantitatively recover chemically intact and biologically active DNA from the gels, as shown by reelectrophoresis and transformation experiments. PMID:8907513

Hardy, E; Sosa, A E; Pupo, E; Casalvilla, R; Fernandez-Patron, C

1996-01-01

231

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

PubMed

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

Davalieva, Katarina; Kiprijanovska, Sanja; Polenakovic, Momir

2014-04-01

232

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...

233

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

PubMed Central

The pmetH probe, tightly linked to the locus for cystic fibrosis (CF), detected a NotI polymorphism with allele sizes of 1,000 and 550 kb that could be separated by pulsed-field gel electrophoresis. Both alleles were found on chromosomes bearing either the normal or the CF allele. Preliminary data showed that this polymorphism was not in strong linkage disequilibrium with the CF locus. Preliminary large-scale restriction mapping of the fragment showed that the NotI polymorphic site is 200-370 kb distant from the MET locus; thus it defines a new polymorphic locus in the CF region. This locus is of potential interest for studying CF families that show recombinants between CF and its tightly linked probes, in order to increase the precision of the genetic map of the region. Images Figure 1 Figure 2 PMID:2892401

Julier, C; White, R

1988-01-01

234

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

PubMed Central

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

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

2014-01-01

235

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

PubMed

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

Gornatti Churria, Carlos D; Vigo, Germn B; Origlia, Javier; Campos, Josefina; Caffer, Mara; Pscopo, Miguel; Loyola, Miguel Herrero; Petruccelli, Miguel; Pichel, Mariana

2014-01-01

236

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

PubMed Central

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

Furukawa, Takashi; Suzuki, Yoshihiro

2013-01-01

237

Determination of vegetal proteins in milk powder by sodium dodecyl sulfate-capillary gel electrophoresis: interlaboratory study.  

PubMed

An interlaboratory study, with the participation of 8 laboratories, was conducted to evaluate a sodium dodecyl sulfate-capillary gel electrophoresis method for determination of adulteration of milk powder with soy and pea proteins. Calibration standards (0-8%, w/w, soy and pea protein in total protein) and adulterated skim milk powders (0-5%, w/w, soy and pea proteins in total protein) were produced. Vegetal proteins were determined after removal of milk proteins by pretreatment of the samples with tetraborate-EDTA buffer, pH 8.3. Repeatability standard deviations ranged from 9 to 15% and reproducibility standard deviations ranged from 25 to 30% in the samples containing 5% vegetal protein in total protein. PMID:12374408

Manso, Mara A; Cattaneo, Tiziana Maria; Barzaghi, Stefania; Olieman, Cornelis; Lpez-Fandio, Rosina

2002-01-01

238

Expanding the proteome two-dimensional gel electrophoresis reference map of human renal cortex by peptide mass fingerprinting.  

PubMed

Proteomics methodologies hold great promise in basic renal research and clinical nephrology. The classical approach for proteomic analysis couples two-dimensional gel electrophoresis (2-DE) with protein identification by mass spectrometry, to produce more global information regarding normal protein expression and alterations in different physiological and pathological states. In this report we have expanded the identification of proteins in the renal cortex, improving the previously published map to facilitate the study of different diseases affecting the human kidney. About 250 spots were analyzed by peptide mass fingerprinting, 89 proteins and 74 isoforms for some of them were identified and implemented in the normal human renal cortex 2-DE reference map. This more comprehensive view of the proteome of the human renal cortex could be of invaluable help to the differential proteomic display of urological diseases. PMID:15668995

Magni, Fulvio; Sarto, Cecilia; Valsecchi, Cristina; Casellato, Stefano; Bogetto, Stefano Ferrero; Bosari, Silvano; Di Fonzo, Andrea; Perego, Roberto A; Corizzato, Matteo; Doro, Giancarlo; Galbusera, Carmen; Rocco, Francesco; Mocarelli, Paolo; Galli Kienle, Marzia

2005-02-01

239

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

PubMed

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

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

2007-11-01

240

A proposal for source tracking of fecal pollution in recreational waters by pulsed-field gel electrophoresis.  

PubMed

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

Furukawa, Takashi; Suzuki, Yoshihiro

2013-01-01

241

Use of polyacrylamide gel moving boundary electrophoresis to enable low-power protein analysis in a compact microdevice.  

PubMed

In designing a protein electrophoresis platform composed of a single-inlet, single-outlet microchannel powered solely by voltage control (no pumps, values, injectors), we adapted the original protein electrophoresis format-moving boundary electrophoresis (MBE)-to a high-performance, compact microfluidic format. Key to the microfluidic adaptation is minimization of injection dispersion during sample injection. To reduce injection dispersion, we utilize a photopatterned free-solution-polyacrylamide gel (PAG) stacking interface at the head of the MBE microchannel. The nanoporous PAG molecular sieve physically induces a mobility shift that acts to enrich and sharpen protein fronts as proteins enter the microchannel. Various PAG configurations are characterized, with injection dispersion reduced by up to 85%. When employed for analysis of a model protein sample, microfluidic PAG MBE baseline-resolved species in 5 s and in a separation distance of less than 1 mm. PAG MBE thus demonstrates electrophoretic assays with minimal interfacing and sample handling, while maintaining separation performance. Owing to the short separation lengths needed in PAG MBE, we reduced the separation channel length to demonstrate an electrophoretic immunoassay powered with an off-the-shelf 9 V battery. The electrophoretic immunoassay consumed less than 3 ?W of power and was completed in 30 s. To our knowledge, this is the lowest voltage and lowest power electrophoretic protein separation reported. Looking forward, we see the low-power PAG MBE as a basis for highly multiplexed protein separations (mobility shift screening assays) as well as for portable low-power diagnostic assays. PMID:22971048

Duncombe, Todd A; Herr, Amy E

2012-10-16

242

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

PubMed

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

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

2014-08-01

243

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

PubMed Central

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

Mao, Xiaoying; Hua, Yufei; Chen, Guogang

2014-01-01

244

Comparative analysis of excretory-secretory antigens of Trichinella spiralis and Trichinella britovi muscle larvae by two-dimensional difference gel electrophoresis and immunoblotting  

PubMed Central

Background Trichinellosis is a zoonotic disease in humans caused by Trichinella spp. The present study was undertaken to discover excretory-secretory (E-S) proteins from T. spiralis and T. britovi muscle larvae (ML) that hold promise for species-specific diagnostics. To that end, the purified E-S proteins were analyzed by fluorescent two-dimensional difference gel electrophoresis (2-D DIGE) coupled with protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). To search for immunoreactive proteins that are specifically recognized by host antibodies the E-S proteins were subjected to two-dimensional (2-DE) immunoblotting with antisera derived from pigs experimentally infected with T. spiralis or T. britovi. Results According to 2-D DIGE analysis, a total of twenty-two proteins including potentially immunogenic proteins and proteins produced only by one of the two Trichinella species were subjected to LC-MS/MS for protein identification. From these proteins seventeen could be identified, of which many were identified in multiple spots, suggesting that they have undergone post-translational modification, possibly involving glycosylation and/or proteolysis. These proteins included 5'-nucleotidase, serine-type protease/proteinase, and p43 glycoprotein (gp43) as well as 49 kDa E-S protein (p49). Our findings also suggest that some of the commonly identified proteins were post-translationally modified to different extents, which in certain cases seemed to result in species-specific modification. Both commonly and specifically recognized immunoreactive proteins were identified by 2-DE immunoblotting; shared antigens were identified as gp43 and different protease variants, whereas those specific to T. britovi included multiple isoforms of the 5'-nucleotidase. Conclusions Both 2-D DIGE and 2-DE immunoblotting approaches indicate that T. spiralis and T. britovi produce somewhat distinctive antigen profiles, which contain E-S antigens with potential as species-specific diagnostic markers for Trichinella. Our results also demonstrate the value of 2-D DIGE as a versatile tool to compare secretomes of different Trichinella species for pinpointing factors contributing to the interaction with the host. PMID:22325190

2012-01-01

245

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

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

MICHAEL G. SONNENBERG; JOHN T. BELISLE

1997-01-01

246

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...

247

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...

248

Use of alkaline Comet assay (single cell gel electrophoresis technique) to detect DNA damages in lymphocytes of operating room personnel occupationally exposed to anaesthetic gases  

Microsoft Academic Search

Here, we report the possible in vivo induction DNA damage by exposure to various waste anaesthetic gases such as halothane, nitrous oxide and isoflurane. The alkaline comet assay (single cell gel electrophoresis technique) was carried out on 66 operating room personnel (anaesthetists [doctors]; anaesthesia nurses and anaesthesia unit technicians) currently employed at the Ankara Hospital in Turkey. A significant increase

S ?arda?; N Aygn; M Gamli; Y nal; N nal; N Berk; A. E Karakaya

1998-01-01

249

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

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

Tweedie, John W.; Stowell, Kathryn M.

2005-01-01

250

Molecular Identification of Bacteria from a Coculture by Denaturing Gradient Gel Electrophoresis of 16S Ribosomal DNA Fragments as a Tool for Isolation in Pure Cultures  

Microsoft Academic Search

Molecular information about the bacterial composition of a coculture capable of sulfate reduction after exposure to oxic and microoxic conditions was used to identify and subsequently to isolate the components of the mixture in pure culture. PCR amplification of 16S ribosomal DNA fragments from the coculture, analyzed by denaturing gradient gel electrophoresis, resulted in two distinct 16S ribosomal DNA bands,

ANDREAS TESKE; PAVEL SIGALEVICH; YEHUDA COHEN; ANDGERARD MUYZER; Moshe Shilo; Alexander Silberman

1996-01-01

251

Identification and typing of Malassezia yeasts using amplified fragment length polymorphism (AFLP Tm), random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE)  

Microsoft Academic Search

Three molecular tools, amplified fragment length polymorphism (AFLPTm), denaturing gradient gel electrophoresis (DGGE) and random amplified polymorphic DNA (RAPD) analysis, were explored for their usefulness to identify isolates of Malassezia yeasts. All seven species could be separated by AFLP and DGGE. Using AFLP, four genotypes could be distinguished within M. furfur. AFLP genotype 4 contained only isolates from deep human

Bart Theelen; Massimiliano Silvestri; Eveline Guho; Alex van Belkum; Teun Boekhout

2001-01-01

252

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

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.

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

253

Genotyping Campylobacter jejuni by comparative genome indexing: an evaluation with pulsed-field gel electrophoresis and flaA SVR sequencing  

Technology Transfer Automated Retrieval System (TEKTRAN)

Aims: Comparative genome indexing (CGI) using whole-genome DNA microarrays was evaluated as a means of genotying Campylobacter jejuni relative to two standard methods, pulsed-field gel electrophoresis (PFGE) and flaA short variable region sequencing (flaA SVR typing). Methods and Results: Thirty-six...

254

Pulsed field gel electrophoresis and physical mapping of large DNA fragments in the Tm2a region of chromosome 9 in tomato  

Microsoft Academic Search

A method has been developed which allows the isolation of very high molecular weight DNA (>2 million bp) from leaf protoplasts of tomato (Lycopersicon esculentum). The DNA isolated in this manner was digested in agarose with rare-cutting restriction enzymes and separated by pulsed field gel electrophoresis. The size range of the reslting fragments was determined by hybridization to a number

Martin W. Ganal; Nevin D. Young; Steven D. Tanksley

1989-01-01

255

Validation of Pulsed-Field Gel Electrophoresis and spa Typing for Long-Term, Nationwide Epidemiological Surveillance Studies of Staphylococcus aureus Infections  

Microsoft Academic Search

Pulsed-field gel electrophoresis (PFGE) of genomic macrorestriction fragments has been used by the Belgian Reference Laboratory for Staphylococci for national hospital surveys of methicillin-resistant Staphylococcus aureus since 1992. The sequencing of the polymorphic X region of the protein A gene (spa typing) offers significant advantages over PFGE in terms of speed, ease of interpretation, and exportability. To validate its potential

M. Hallin; A. Deplano; O. Denis; R. De Mendonca; R. De Ryck; M. J. Struelens

2007-01-01

256

Comparison of Arbitrarily Primed PCR and Macrorestriction (Pulsed-Field Gel Electrophoresis) Typing ofPseudomonas aeruginosaStrains from Cystic Fibrosis Patients  

Microsoft Academic Search

Arbitrarily primed PCR fingerprinting was carried out on 43 Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients. Seventeen major groups of strains that coincided with groups also distinguished by macrorestriction (pulsed-field gel electrophoresis) typing were identified. Our results illustrated that a CF patient can carry more than one strain and can carry a given strain for long periods of time

DANGERUTA KERSULYTE; MARC J. STRUELENS; ARIANE DEPLANO; ANDDOUGLAS E. BERG

1995-01-01

257

Detection of specific sequences among DNA fragments separated by gel electrophoresis  

Microsoft Academic Search

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.

E. M. Southern

1975-01-01

258

Turning a PAGE: the overnight sensation of SDS-polycrylamide gel electrophoresis  

Microsoft Academic Search

The zonal separation of proteins on the basis of net charge was initially conducted on paper, then in columns of sucrose and later in gels of starch and polyacrylamide, with appropriate electric fields. Then, in 1964, a graduate student at MIT discovered the power of sodium dodecyl sulfate (SDS) to dissoci- ate the envelope proteins of Escherichia coli and to

Thoru Pederson

2007-01-01

259

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

PubMed Central

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

Brown, Hilda H.; Borchelt, David R.

2014-01-01

260

Comparison of MIDI Sherlock system and pulsed-field gel electrophoresis in characterizing strains of methicillin-resistant Staphylococcus aureus from a recent hospital outbreak.  

PubMed Central

An outbreak of methicillin-resistant Staphylococcus aureus infections at the University of Utah Health Sciences Center occurred over a 7-month period. While the isolates phenotypically appeared to be similar in gross morphology and have similar Vitek antibiotic susceptibility patterns, two additional methods of strain characterization were evaluated to enhance the epidemiological investigation: pulsed-field gel electrophoresis and gas chromatography with the MIDI Sherlock system. Sherlock uses gas chromatography to qualitatively and quantitatively analyze the cellular fatty acid composition of organisms and creates two-dimensional plots based on principal-component analysis to define groups of closely related organisms. All isolates were also evaluated by digesting their chromosomal DNAs with the low-frequency-cutting enzyme SmaI and separating the restriction fragments by contour-clamped homogeneous electric field gel electrophoresis. Sample preparation for this pulsed-field gel electrophoresis included a novel cell lysis procedure involving achromopeptidase, greatly reducing the turnaround time. Isolates tested were recovered from the following: 45 suspected outbreak patients, 6 hospitalized patients believed to be unrelated to the outbreak, 6 patients from outside the hospital, and one health care practitioner implicated in the outbreak. Of 45 phenotypically similar suspect strains, 43 clustered tightly on the Sherlock two-dimensional plot. All outbreak patient isolates were also identical by pulsed-field gel electrophoresis with the exception of the same two outliers identified by Sherlock. In this epidemiologic investigation, we found an excellent correlation between the Sherlock and pulsed-field gel electrophoresis results for strain characterization of methicillin-resistant S. aureus. PMID:8567913

Leonard, R B; Mayer, J; Sasser, M; Woods, M L; Mooney, B R; Brinton, B G; Newcomb-Gayman, P L; Carroll, K C

1995-01-01

261

Efficient method of protein extraction from Theobroma cacao L. roots for two-dimensional gel electrophoresis and mass spectrometry analyses.  

PubMed

Theobroma cacao is a woody and recalcitrant plant with a very high level of interfering compounds. Standard protocols for protein extraction were proposed for various types of samples, but the presence of interfering compounds in many samples prevented the isolation of proteins suitable for two-dimensional gel electrophoresis (2-DE). An efficient method to extract root proteins for 2-DE was established to overcome these problems. The main features of this protocol are: i) precipitation with trichloroacetic acid/acetone overnight to prepare the acetone dry powder (ADP), ii) several additional steps of sonication in the ADP preparation and extractions with dense sodium dodecyl sulfate and phenol, and iii) adding two stages of phenol extractions. Proteins were extracted from roots using this new protocol (Method B) and a protocol described in the literature for T. cacao leaves and meristems (Method A). Using these methods, we obtained a protein yield of about 0.7 and 2.5 mg per 1.0 g lyophilized root, and a total of 60 and 400 spots could be separated, respectively. Through Method B, it was possible to isolate high-quality protein and a high yield of roots from T. cacao for high-quality 2-DE gels. To demonstrate the quality of the extracted proteins from roots of T. cacao using Method B, several protein spots were cut from the 2-DE gels, analyzed by tandem mass spectrometry, and identified. Method B was further tested on Citrus roots, with a protein yield of about 2.7 mg per 1.0 g lyophilized root and 800 detected spots. PMID:25062492

Bertolde, F Z; Almeida, A-A F; Silva, F A C; Oliveira, T M; Pirovani, C P

2014-01-01

262

Identification and population dynamics of yeasts in sourdough fermentation processes by PCR-denaturing gradient gel electrophoresis.  

PubMed

Four sourdoughs (A to D) were produced under practical conditions, using a starter obtained from a mixture of three commercially available sourdough starters and baker's yeast. The doughs were continuously propagated until the composition of the microbiota remained stable. A fungi-specific PCR-denaturing gradient gel electrophoresis (DGGE) system was established to monitor the development of the yeast biota. The analysis of the starter mixture revealed the presence of Candida humilis, Debaryomyces hansenii, Saccharomyces cerevisiae, and Saccharomyces uvarum. In sourdough A (traditional process with rye flour), C. humilis dominated under the prevailing fermentation conditions. In rye flour sourdoughs B and C, fermented at 30 and 40 degrees C, respectively, S. cerevisiae became predominant in sourdough B, whereas in sourdough C the yeast counts decreased within a few propagation steps below the detection limit. In sourdough D, which corresponded to sourdough C in temperature but was produced with rye bran, Candida krusei became dominant. Isolates identified as C. humilis and S. cerevisiae were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively. The yeast species isolated from the sourdoughs were also detected by PCR-DGGE. However, in the gel, additional bands were visible. Because sequencing of these PCR fragments from the gel failed, cloning experiments with 28S rRNA amplicons obtained from rye flour were performed, which revealed Cladosporium sp., Saccharomyces servazii, S. uvarum, an unculturable ascomycete, Dekkera bruxellensis, Epicoccum nigrum, and S. cerevisiae. The last four species were also detected in sourdoughs A, B, and C. PMID:14660398

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

2003-12-01

263

Quantification of Isotope Encoded Proteins in 2-D Gels Using Surface Enhanced Resonance Raman  

PubMed Central

A strategy for quantification of multiple protein isoforms from a complex sample background is demonstrated, combining isotopomeric rhodamine 6G (R6G) labels and surface enhanced Raman in polyacrylamide matrix. The procedure involves isotope-encoding by lysine-labeling with (R6G) active ester reagents, isoform separation by 2-DGE, fluorescence quantification using internal standardization to water, and silver nanoparticle deposition followed by surface enhanced Raman detection. R6G sample encoding and standardization enabled the determination of total protein concentration and the distribution of specific isoforms using the combined detection approach of water-referenced fluorescence spectral imaging and ratiometric quantification. A detection limit of approximately 13.5 picomolar R6G-labeled protein was determined for the surface-enhanced Raman in a gel matrix (15-fold lower than fluorescence). High quantification accuracies for small differences in protein populations at low nanogram abundance were demonstrated for human GMP synthetase (hGMPS) either as purified protein samples in a single-point determination mode (3% relative standard deviation, RSD%) or HCT116 human cancer cellular lysate in an imaging application (with 16% RSD%). These results represent a prototype for future applications of isotopic surface enhanced resonance Raman scatter to quantification of protein distributions. PMID:18925772

Knudsen, Giselle M.; Davis, Brandon M.; Deb, Shirshendu K.; Loethen, Yvette; Gudihal, Ravindra; Perera, Pradeep; Ben-Amotz, Dor; Davisson, V. Jo

2009-01-01

264

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

SciTech Connect

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.

Carrasco, L.; Bravo, R.

1986-05-01

265

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...

266

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

E-print Network

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

Shoubridge, Eric

267

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

SciTech Connect

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.

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

1994-09-01

268

Application of Pulsed Field Gel Electrophoresis for Study of Genetic Diversity in Mycobacterium tuberculosis Strains Isolated From Tuberculosis Patients  

PubMed Central

Background: Mycobacterium tuberculosis genotyping can effectively improve tuberculosis (TB) control programs by controlling disease transmission. Pulsed field gel electrophoresis (PFGE) is a particularly powerful tool for determination of clonal identity of bacteria providing information for understanding and controlling the spread of disease. Objectives: The aim of present study was to investigate the genetic diversity of M. tuberculosis strains in Khuzestan province by the PFGE technique. Patients and Methods: In total, 80 M. tuberculosis positive cultures were obtained from tuberculosis patients. PFGE was performed on 60 PCR-confirmed isolates by using DraI and XbaI restriction enzymes according to standard protocols. Plugs containing digested DNA were then loaded on agarose gels and run using contour-clamped homogenous electric fields. Results: Fifty distinct DNA banding patterns were obtained by digestion of DNA with DraI and 38 DNA banding patterns by digestion with XbaI restriction enzymes. The patterns comprised of 17 different clusters in which cluster I was the major one, containing six strains. Three clusters contained three strains each and the 13 remaining clusters comprised of two strains each. Digestion with DraI yielded 15-20 DNA fragments with 50-485 kb size, while digestion by XbaI produced DNA fragments with a size smaller than 50-242 kb. Conclusions: Despite the ability of PFGE for study of genetic diversity of many mycobacterial species and it being considered as a robust and useful tool, in this study we only found a 15% epidemiological relationship amongst the isolates. Thus, for higher discrimination of genotypic clusters among M. tuberculosis clinical isolates, the application of more sophisticated complementary techniques is required. PMID:25147723

Khosravi, Azar Dokht; Vatani, Shideh; Feizabadi, Mohammad Mehdi; Abasi Montazeri, Effat; Jolodar, Abbas

2014-01-01

269

Effects of renovascular hypertension on myocardial protein patterns: analysis by computer-assisted two-dimensional gel electrophoresis.  

PubMed

Hypertensive heart disease caused by renovascular hypertension reflects the response of the heart to an increased afterload and neurohormonal stimulation. We hypothesized that in this condition the composition of the myocardial proteins of rats was altered. To identify yet unknown quantitative and qualitative differences in myocardial proteins in renovascular hypertensive heart disease, we analyzed protein patterns by computer-assisted two-dimensional polyacrylamide large gel electrophoresis. Renovascular hypertension was induced by placing a silver clip on the left renal artery in 9-week-old rat siblings. Sham-operated animals served as controls. Systolic blood pressure (197 +/- 19 mm Hg) and heart/body weight ratios (0.36 +/- 0.04%) were significantly increased in the hypertensive animals. Twenty protein patterns from the left ventricle of five hypertensive and five control rats were compared. The molecular mass and isoelectric point (pI) of proteins spots ranging from 13 to 100 kDa and from 4.5 to 8.5, respectively, were determined using marker proteins. In total, 761 +/- 88 protein spots were resolved in all twenty gels. For the quantitative data analysis a univariate (Mann-Whitney test) as well as a multivariate statistical approach (correspondence analysis) were applied. Only one myocardial protein spot (molecular mass = 41.3 kDa; pI = 6.3) was decreased by more than twofold (p < 0.05) in renovascular hypertension. The vast majority of spots did not indicate a significant alteration of intensity. Left ventricular hypertrophy in early renovascular hypertension induces a form of myocardial hypertrophy that conserves the naturally occurring protein expression pattern. PMID:9740066

Pleissner, K P; Regitz-Zagrosek, V; Krdewagen, B; Trenkner, J; Hocher, B; Fleck, E

1998-08-01

270

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

PubMed Central

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

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

1999-01-01

271

An enhanced discriminatory pulsed-field gel electrophoresis scheme for subtyping Salmonella serotypes Heidelberg, Kentucky, SaintPaul, and Hadar.  

PubMed

Conventional pulsed-field gel electrophoresis (PFGE) protocols, used extensively as a successful approach for subtyping many salmonellae, may be inadequate for discriminating strains sharing levels of homogeneity within the same serotype. Four additional restriction enzymes (SpeI, PacI, SfiI, and NotI), in addition to XbaI and BlnI, were used in PFGE typing of 33 Salmonella Heidelberg, 27 Salmonella Kentucky, 27 Salmonella SaintPaul, and 27 Salmonella Hadar isolates that were recovered from poultry and porcine retail meats from different states of the United States. A dendrogram derived from the combined analysis of six enzymes was highly discriminatory with a Simpson index of diversity value of over 0.950. The ratio of nodes to isolates was more than 0.75 with an average of fewer than three isolates in each polytomy for all four serotypes. Two three-enzyme combinations, SpeI/NotI/SfiI for Salmonella Heidelberg and Salmonella Hadar, and SpeI/BlnI/SfiI for Salmonella Kentucky and Salmonella SaintPaul, were found to have comparable discriminatory abilities of differentiating isolates of these Salmonella serotypes with the six-enzyme combination. The enhanced discriminatory PFGE-based subtyping scheme can be used effectively for the differentiation of strains of the four Salmonella serotypes. The findings also highlight PFGE analysis as a continued essential and informative subtyping method for source tracking and outbreak investigations of these and other Salmonella pathogens. PMID:18939754

Xi, Meili; Zheng, Jie; Zhao, Shaohua; Brown, Eric W; Meng, Jianghong

2008-10-01

272

Genotyping of Yersinia enterocolitica biotype 1A strains from clinical and nonclinical origins by pulsed-field gel electrophoresis.  

PubMed

Yersinia enterocolitica biotype 1A (B1A) strains are considered mainly nonpathogenic. However, some studies considered strains of this biotype to be the causal agents of infections in humans and animals. In South America, there are no studies that have compared clinical and nonclinical strains of B1A typed by pulsed-field gel electrophoresis (PFGE) and none that have compared the capability of different enzymes on typing these strains. This study typed 51 Y. enterocolitica B1A strains isolated in Brazil and Chile by PFGE, testing the enzymes XbaI, NotI, and XhoI. The resulting dendrograms discriminated the strains in 47, 40, and 49 pulsotypes generated by the cleavage with the enzymes XbaI, NotI, and XhoI, respectively. The majority of the strains were grouped independently of their clinical or nonclinical origins. The high discriminatory power of PFGE confirmed the heterogeneity of B1A strains but could not divide the strains studied into clusters that differed in the frequency of some virulence genes as observed in studies using other methodologies. PMID:24869470

Campioni, Fbio; Falco, Juliana P

2014-06-01

273

Performance Assessment of DNA Fragment Sizing by High-Sensitivity Flow Cytometry and Pulsed-Field Gel Electrophoresis  

PubMed Central

The sizing of restriction fragments is the chief analytical technique utilized in the production of DNA fingerprints. Few techniques have been able to compete with pulsed-field gel electrophoresis (PFGE), which is capable of discriminating among bacteria at species and strain levels by resolving restriction fragments. However, an ultrasensitive flow cytometer (FCM) developed in our lab has also demonstrated the ability to discriminate bacteria at species and strain levels. The abilities of FCM warrant a quantitative parallel comparison with PFGE to assess and evaluate the accuracy and precision of DNA fragment sizing by both techniques. Replicate samples of Staphylococcus aureus Mu50 were analyzed along with two clinical S. aureus isolates. The absolute fragment sizing accuracy was determined for PFGE (5% 2%) and FCM (4% 4%), with sequence-predicted Mu50 SmaI fragment sizes used as a reference. Precision was determined by simple arithmetic methods (relative standard deviation for PFGE [RSDPFGE ] = 3% 2% and RSDFCM = 1.2% 0.8%) as well as by the use of dendrograms derived from Dice coefficient-unweighted pair group method with arithmetic averages (UPGMA) and Pearson-UPGMA analyses. All quantitative measures of PFGE and FCM precision were equivalent, within error. The precision of both methods was not limited by any single sample preparation or analysis step that was tracked in this study. Additionally, we determined that the curve-based clustering of fingerprint data provided a more informative and useful assessment than did traditional band-based methods. PMID:15131156

Ferris, Matthew M.; Yan, Xiaomei; Habbersett, Robbert C.; Shou, Yulin; Lemanski, Cheryl L.; Jett, James H.; Yoshida, Thomas M.; Marrone, Babetta L.

2004-01-01

274

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

PubMed

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

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

2015-01-01

275

Actively Growing Bacteria in the Inland Sea of Japan, Identified by Combined Bromodeoxyuridine Immunocapture and Denaturing Gradient Gel Electrophoresis?  

PubMed Central

A fundamental question in microbial oceanography concerns the relationship between prokaryote diversity and biogeochemical function in an ecosystem context. We combined bromodeoxyuridine (BrdU) magnetic bead immunocapture and PCR-denaturing gradient gel electrophoresis (BUMP-DGGE) to examine phylotype-specific growth in natural marine assemblages. We also examined a broad range of marine bacterial isolates to determine their abilities to incorporate BrdU in order to test the validity of the method for application to diverse marine assemblages. We found that 27 of 29 isolates belonging to different taxa could incorporate BrdU. BUMP-DGGE analysis revealed phylogenetic affiliations of DNA-synthesizing, presumably actively growing bacteria across a eutrophic to mesotrophic transect in the Inland Sea of Japan. We found that the BrdU-incorporating (growing) communities were substantially different from the total communities. The majority (34/56) of phylotypes incorporated BrdU and were presumably growing, and these phylotypes comprised 10 alphaproteobacteria, 1 betaproteobacterium, 11 gammaproteobacteria, 11 Cytophaga-Flavobacterium-Bacteroides group bacteria, and 1 unclassified bacterium. All BrdU-responsive alphaproteobacteria were members of the Rhodobacterales, suggesting that such bacteria were dominant in the growing alphaproteobacterial populations in our samples. The BrdU-responsive gammaproteobacteria belonged to the Oceanospirillales, the SAR86 cluster, the Pseudomonadales, the Alteromonadales, and the Vibrionales. Thus, contemporaneous cooccurrence of diverse actively growing bacterial taxa was a consistent pattern in our biogeochemically varied study area. PMID:17337555

Hamasaki, Koji; Taniguchi, Akito; Tada, Yuya; Long, Richard A.; Azam, Farooq

2007-01-01

276

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

PubMed

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

Zhang, Sheng; Jiang, Cheng; Jia, Li

2011-01-15

277

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

PubMed Central

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

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

2015-01-01

278

Non-denaturating isoelectric focusing gel electrophoresis for uranium-protein complexes quantitative analysis with LA-ICP MS.  

PubMed

A non-denaturating isoelectric focusing (ND-IEF) gel electrophoresis protocol has been developed to study and identify uranium (U)-protein complexes with laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS) and electrospray ionization mass spectrometry (ESI-MS). The ND-IEF-LA-ICP MS methodology set-up was initiated using in vitro U-protein complex standards (i.e., U-bovine serum albumin and U-transferrin) allowing the assessment of U recovery to 64.4??0.4 %. This methodology enabled the quantification of U-protein complexes at 9.03??0.23, 15.27??0.36, and 177.31??25.51 nmol U L(-1) in digestive gland cytosols of the crayfish, Procambarus clarkii, exposed respectively to 0, 0.12, and 2.5 ?mol of waterborne depleted U L(-1) during 10 days. ND-IEF-LA-ICP MS limit of detection was 19.3 pmol U L(-1). Elemental ICP MS signals obtained both in ND-IEF electropherograms and in size exclusion chromatograms of in vivo U-protein complexes revealed interactions between U- and Fe- and Cu-proteins. Moreover, three proteins (hemocyanin, pseudohemocyanin-2, and arginine kinase) out of 42 were identified as potential uranium targets in waterborne-exposed crayfish cytosols by microbore reversed phase chromatography coupled to molecular mass spectrometry (RPC-ESI-MS/MS) after ND-IEF separation. PMID:23665639

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

2014-02-01

279

Analysis of HLA-DR from alveolar macrophages and blood monocytes by two-dimensional gel electrophoresis  

SciTech Connect

Human blood monocytes (BM) are more effective than alveolar macrophages (AM) in promoting lymphocyte proliferation to antigen. To further understand these differences, the HLA-DR molecules synthesized by these two cell types were compared. AM were prepared by adherence of cells obtained by bronchoscopic lavage; BM were prepared by adherence of blood mononuclear cells from the same normal volunteer. Cells were cultured for 7 hours with /sup 3/H-leucine and HLA-DR was immunoprecipitated with the murine monoclonal antibody L243. Immunoprecipitates were analyzed by two-dimensional gel electrophoresis. In three experiments, protein synthetic rate was greater and more HLA-DR was immunoprecipitated per cell in BM than in AM. Isoelectric focusing showed identical charge variation for BM and AM. However, molecular weight analysis of AM HLA-DR revealed multiple bands of slightly different molecular weight for each beta-chain peptide, whereas only a single band occurred with BM HLA-DR. Neuraminidase treatment reduced the charge heterogeneity but did not affect the molecular weight differences. These findings may relate to the differential ability of AM and BM to promote lymphocyte proliferation to antigen.

Ferro, T.J.; Monos, D.S.; Spear, B.T.; Rossman, M.D.; Zmijewski, C.M.; Kamoun, M.; Daniele, R.P.

1986-03-01

280

SURFEIT-1 gene analysis and two-dimensional blue native gel electrophoresis in cytochrome c oxidase deficiency.  

PubMed

Leigh syndrome, a progressive, often fatal, neurodegenerative disorder, is frequently associated with a deficiency in the activity of cytochrome c oxidase (COX), the last enzyme of the mitochondrial respiratory chain. In contrast to NADH:ubiquinone oxidoreductase and succinate dehydrogenase deficiencies, no mutations in nuclear genes encoding COX subunits have been identified thus far. Very recently, however, a Leigh syndrome complementation group has been identified which showed mutations in the SURFEIT-1 (SURF-1) gene. The results of a mutational detection study in 16 new randomly selected COX-deficient patients revealed a new mutation (C688T) in 2 patients and the earlier reported 845delCT mutation in 2 additional patients. In addition, we evaluated the diagnostic value of two-dimensional blue native gel electrophoresis. We show that this technique reveals distinct patterns of both fully and partially assembled COX complexes and is thereby capable of discrimination between COX-deficient SURF-1 and non-SURF-1-mutated patients. PMID:10558868

Coenen, M J; van den Heuvel, L P; Nijtmans, L G; Morava, E; Marquardt, I; Girschick, H J; Trijbels, F J; Grivell, L A; Smeitink, J A

1999-11-19

281

Correlation of Particular Bacterial PCR-Denaturing Gradient Gel Electrophoresis Patterns with Bovine Ruminal Fermentation Parameters and Feed Efficiency Traits ?  

E-print Network

The influence of rumen microbial structure and functions on host physiology remains poorly understood. This study aimed to investigate the interaction between the ruminal microflora and the host by correlating bacterial diversity with fermentation measurements and feed efficiency traits, including dry matter intake, feed conversion ratio, average daily gain, and residual feed intake, using culture-independent methods. Universal bacterial partial 16S rRNA gene products were amplified from ruminal fluid collected from 58 steers raised under a low-energy diet and were subjected to PCR-denaturing gradient gel electrophoresis (DGGE) analysis. Multivariate statistical analysis was used to relate specific PCR-DGGE bands to various feed efficiency traits and metabolites. Analysis of volatile fatty acid profiles showed that butyrate was positively correlated with daily dry matter intake (P < 0.05) and tended to have higher concentration in inefficient animals (P ? 0.10), while isovalerate was associated with residual feed intake (P < 0.05). Our results suggest that particular bacteria and their metabolism in the rumen may contribute to differences in host feed efficiency under a low-energy diet. This is the first study correlating PCR-DGGE bands representing specific bacteria to metabolites in the bovine rumen and to host feed efficiency traits. A fundamental understanding of microbial ecology and relationships to ruminant physiology is essential for successful

Emma Hern; Le Luo Guan; Laksiri A. Goonewardene; Meiju Li; Denis F. Mujibi; Paul Stothard; Stephen S. Moore; Monica C. Leon-quintero

2010-01-01

282

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

PubMed Central

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

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

2003-01-01

283

Intraspecific characterization of Vibrio tapetis strains by use of pulsed-field gel electrophoresis, ribotyping, and plasmid profiling.  

PubMed Central

A total of twenty-two strains of Vibrio tapetis, the causative agent of brown ring disease affecting cultured clams, were compared and evaluated in an investigation of strain heterogeneity using pulsed-field gel electrophoresis (PFGE), ribotyping, and plasmid profile analysis. A total of 90.9% of V. tapetis strains tested by using NotI showed the same PFGE pattern, consisting of 15 bands. In contrast, the V. tapetis strains showed a low degree of similarity with six reference Vibrio species tested. All V. tapetis strains harbored a large plasmid of 74.5 kb. This plasmid was not detected in any of the other Vibrio species. In addition, endonuclease restriction analysis of the plasmid content of the strains using EcoRI and HindIII clearly showed that all the strains of V. tapetis possessed the same cleavage pattern. The three enzymes used for ribotyping, PvuII, SmaI, and SalI, yielded patterns with 8 to 12 bands ranging in size from 2 to 23 kb. The application of the SalI and SmaI endonuclease rendered the separation of the strains tested in two ribotypes, while all the V. tapetis strains belonged to the same ribotype when the enzyme PvuII was used. PMID:9097443

Castro, D; Romalde, J L; Vila, J; Magarios, B; Luque, A; Borrego, J J

1997-01-01

284

Pulsed-field gel electrophoresis typing, antibiotic resistance, and plasmid profiles of Escherichia coli strains isolated from foods.  

PubMed

Bacterial contamination in foods and antimicrobial resistance levels of common pathogenic strains causing food-borne disease are important in human health. Thus, typing technologies are important tools to determine primary sources of bacterial contamination. In this study, 40 Escherichia coli strains isolated from 85 food samples were evaluated in terms of genetic diversity, susceptibility to certain antibiotics, and plasmid profiles. Pulsed-field gel electrophoresis was used to identify the genetic relations of E. coli isolates. It was determined that the 40 E. coli strains revealed 32 different pulsotypes represented by 6 subtypes. Antibiotic susceptibility tests conducted by using a disc diffusion method against 15 antibiotics showed that although the isolates revealed 14 different types of resistance profiles, the strains showed the greatest resistance to ampicillin (77.5%), followed by ticarcillin-clavulanic acid (30%), tetracycline (22.5%), and cephalothin (14.5%). Plasmid isolations studies of the strains conducted by the method of alkaline lysis revealed that 18 (45%) of 40 E. coli strains contain 31 different plasmid bands ranging between 64.4 and 1 kb. The results showed that PFGE was a powerful method in tracking sources of food contamination and that the antibiotic resistance levels of food isolates were high and should be monitored. PMID:23145825

Uysal, Ahmet; Durak, Yusuf

2012-11-01

285

Frequencies of virulence genes and pulse field gel electrophoresis fingerprints in Escherichia coli isolates from canine pyometra.  

PubMed

Escherichia coli is the most common bacterial agent isolated from canine pyometra. The frequencies of 24 virulence genes and pulsed field gel electrophoresis (PFGE) profiles were determined for 23 E. coli isolates from cases of canine pyometra in Brazil. The frequencies of virulence genes were 91.3% fimH, 91.3% irp-2, 82.6% fyuA, 56.5% iroN, 47.8% traT, 39.1% usp, 34.8% sfaD/E, 34.8% tsh, 30.4% papC, 30.4% hlyA, 26.1% papGIII, 26.1% cnf-1, 21.7% papE/F, 21.7% iss, 17.4% iutA, 17.4% ompT, 17.4% cvaC, 17.4% hlyF, 17.4% iucD, 13.0% iucC, 13.0% astA, 4.3% papGII, 0% afaB/C and 0% papGI. The high frequency of yersiniabactin (fyuA and irp2) and salmochelin (iroN) genes suggests that iron uptake systems might be important in the pathogenesis of canine pyometra. PFGE profiles of 19 isolates were heterogeneous, confirming that E. coli isolates from canine pyometra are unlikely to be epidemic clones. PMID:25201253

Maluta, Renato P; Borges, Clarissa A; Beraldo, Lvia G; Cardozo, Marita V; Voorwald, Fabiana A; Santana, Andr M; Rigobelo, Everlon C; Toniollo, Gilson H; Avila, Fernando A

2014-11-01

286

High background radiation areas of Ramsar in Iran: evaluation of DNA damage by alkaline single cell gel electrophoresis (SCGE).  

PubMed

The hot springs in special areas in Ramsar, a northern coastal town in Iran, contain (226)Ra and (222)Rn. The natural radiation effects, radiosensitivity or adaptive responses, on the inhabitants of high natural radiation in Ramsar were studied. The single cell gel electrophoresis was used to monitor DNA damages. Three groups of volunteers were selected, one from high natural background radiation areas as the case group and two from normal background radiation areas as controls (control 1 and control 2). The latter one had the similar living situation to case group while the other (control 2) had different living situation from the other groups. Peripheral blood mononuclear cells (PMNCs) were separated and irradiated by (60)Co source at five different gamma doses. It was found that the spontaneous level of DNA damage and the induced DNA damage in all challenging doses in case group was considerably higher than control groups (p < 0.05). On the other hand, the repair rate in those volunteers, who received less than 10.2 mSv/y was significantly more than the control groups. In the contrary, individuals who live in homes with more than 10.2 mSv/y had incomplete repair. Additionally the plasma and urinary levels of vitamin C were measured spectrophotometrically. Although the concentration of vitamin C of plasma was equal in case and control 1 groups, the urinary level of vitamin C was found to be lower in the case group. PMID:16376699

Masoomi, J R; Mohammadi, Sh; Amini, M; Ghiassi-Nejad, M

2006-01-01

287

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

PubMed

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

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

2014-01-01

288

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

PubMed

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

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

2014-04-01

289

A gel electrophoresis study of the competitive effects of monovalent counterion on the extent of divalent counterions binding to DNA.  

PubMed Central

The behavior of alkaline earth metal cations (Mg2+ and Ca2+) and transition metal cations (Zn2+ and Cu2+) interacting with lambda-DNA-HindIII fragments ranging from 2,027 to 23,130 bp in Tris-borate-EDTA buffer solutions was investigated. The divalent counterions competed with Tris+ and Na+ for binding to polyion DNA, and the competition binding situations were investigated by measuring the reduction of the DNA mobility, by pulsed- or constant-field gel electrophoresis. The interaction of Mg2+ with DNA was intensively studied over a wide range of Mg2+ concentrations. In addition, we examined the competition binding as a function of ionic strength and DNA size. To compare valence effects, we studied Co(NH3)6(3+) interaction with DNA fragments under conditions similar to that of Mg2+. At relatively low Mg2+ concentration, the normalized titration curves of DNA mobility were well fit by Manning's two-variable counterion condensation (CC) theory. The agreement between the predicted value (total charge neutralization fraction theta) from Manning's CC theory and the data based on our measured DNA electrophoretic mobility reduction was consistent under our experimental conditions. In contrast to alkaline earth metal cations (Mg2+ and Ca2+), different binding behaviors were observed for the transition metal cations (Zn2+ and Cu2+). These differences highlight the usefulness of our reduced DNA electrophoretic mobility measurement approach to describing cation interactions with polyelectrolyte DNA. PMID:9533707

Li, A Z; Huang, H; Re, X; Qi, L J; Marx, K A

1998-01-01

290

Improved recognition of MRSA case clusters by the application of molecular subtyping using pulsed-field gel electrophoresis.  

PubMed

Methicillin-resistant Staphylococcus aureus (MRSA) is increasingly common in hospital and community populations, making the recognition of true nosocomial outbreaks more difficult. We have used pulsed-field gel electrophoresis (PFGE) with Sma I digestion to analyse retrospectively two perceived outbreaks of epidemic methicillin-resistant Staphylococcus aureus 15 (EMRSA 15) colonization. The first cluster of cases in patients and staff on a general ward (ward D) revealed three different antibiograms based on differences in ciprofloxacin and rifampicin sensitivities. All isolates typed using PFGE, which was more discriminatory than phage-typing. One PFGE banding profile labelled type 5 was predominant, but 12 isolates proved to be subtypes of type 5 and two were PFGE type 11. Four staff members carried a strain not found in patients, three carried strains found in patients and transient carriage was highlighted as a problem when screening staff. PFGE enhanced the epidemiological data and proved that the cases on this ward did not comprise one large outbreak but numerous sporadic cases and smaller clusters. In contrast, isolates from a second cluster of cases which occurred on ward F were indistinguishable using antibiograms, phage-typing and PFGE, confirming this was more likely to be a true outbreak of colonization. We conclude that PFGE usefully augments epidemiological information and allows more logical infection control decisions to be made, with better utilization of scarce resources. PMID:9949962

Macfarlane, L; Walker, J; Borrow, R; Oppenheim, B A; Fox, A J

1999-01-01

291

Comparison of PCR-Ribotyping, Arbitrarily Primed PCR, and Pulsed-Field Gel Electrophoresis for Typing Clostridium difficile  

PubMed Central

Clostridium difficile is now recognized as the major agent responsible for nosocomial diarrhea in adults. Among the genotyping methods available, arbitrarily primed PCR (AP-PCR), PCR-ribotyping, and pulsed-field gel electrophoresis (PFGE) have been widely used for investigating outbreaks of C. difficile infections. However, the comparative typing ability, reproducibility, discriminatory power, and efficiency of these methods have not been fully investigated. We compared the results of three methodsAP-PCR with three different primers (AP3, AP4, and AP5), PCR-ribotyping, and PFGE (with SmaI endonuclease)to differentiate 99 strains of C. difficile that had been previously serogrouped. Typing abilities were 100% for PCR-ribotyping and AP-PCR with AP3 and 90% for PFGE, due to early DNA degradation in strains from serogroup G. Reproducibilities were 100% for PCR-ribotyping and PFGE but only 88% for AP-PCR with AP3, 67% for AP-PCR with AP4, and 33% for AP-PCR with AP5. Discriminatory power for unrelated strains was >0.95 for all the methods but was lower for PCR-ribotyping among serogroups D and C. PCR-based methods were easier and quicker to perform, but their fingerprints were more difficult to interpret than those of PFGE. We conclude that PCR-ribotyping offers the best combination of advantages as an initial typing tool for C. difficile. PMID:10878030

Bidet, Philippe; Lalande, Valrie; Salauze, Batrice; Burghoffer, Batrice; Avesani, Vronique; Delme, Michel; Rossier, Anne; Barbut, Frdric; Petit, Jean-Claude

2000-01-01

292

High-throughput glycosylation analysis of therapeutic immunoglobulin G by capillary gel electrophoresis using a DNA analyzer  

PubMed Central

The Fc glycosylation of therapeutic antibodies is crucial for their effector functions and their behavior in pharmacokinetics and pharmacodynamics. To monitor the Fc glycosylation in bioprocess development and characterization, high-throughput techniques for glycosylation analysis are needed. Here, we describe the development of a largely automated high-throughput glycosylation profiling method with multiplexing capillary-gel-electrophoresis (CGE) with laser induced fluorescence (LIF) detection using a DNA analyzer. After PNGaseF digestion, the released glycans were labeled with 9-aminopyrene-1,3,6-trisulfonic acid (APTS) in 96-well plates, which was followed by the simultaneous analysis of up to 48 samples. The peak assignment was conducted by HILIC-UPLC-MS/MS of the APTS-labeled glycans combined with peak fractionation and subsequent CGE-LIF analysis of the MS-characterized fractions. Quantitative data evaluation of the various IgG glycans was performed automatically using an in-house developed software solution. The excellent method accuracy and repeatability of the test system was verified by comparison with two UPLC-based methods for glycan analysis. Finally, the practical value of the developed method was demonstrated by analyzing the antibody glycosylation profiles from fermentation broths after small scale protein A purification. PMID:24135630

Reusch, Dietmar; Haberger, Markus; Kailich, Tobias; Heidenreich, Anna-Katharina; Kampe, Michael; Bulau, Patrick; Wuhrer, Manfred

2014-01-01

293

Seven New Mutations in hMSH2, an HNPCC Gene, Identified by Denaturing Gradient-Gel Electrophoresis  

PubMed Central

Hereditary nonpolyposis colorectal cancer (HNPCC) is a relatively common autosomal dominant cancer-susceptibility condition. The recent isolation of the DNA mismatch repair genes (hMSH2, hMLH1, hPMS1, and hPMS2) responsible for HNPCC has allowed the search for germ-line mutations in affected individuals. In this study we used denaturing gradient-gel electrophoresis to screen for mutations in the hMSH2 gene. Analysis of all the 16 exons of hMSH2, in 34 unrelated HNPCC kindreds, has revealed seven novel pathogenic germ-line mutations resulting in stop codons either directly or through frameshifts. Additionally, nucleotide substitutions giving rise to one missense, two silent, and one useful polymorphism have been identified. The proportion of families in which hMSH2 mutations were found is 21%. Although the spectrum of mutations spread at the hMSH2 gene among HNPCC patients appears extremely heterogeneous, we were not able to establish any correlation between the site of the individual mutations and the corresponding tumor spectrum. Our results indicate that, given the genomic size and organization of the hMSH2 gene and the heterogeneity of its mutation spectrum, a rapid and efficient mutation detection procedure is necessary for routine molecular diagnosis and presymptomatic detection of the disease in a clinical setup. ImagesFigure 1 PMID:7726159

Wijnen, Juul; Vasen, Hans; Khan, P. Meera; Menko, Fred H.; van der Klift, Heleen; van Leeuwen, Claus; van den Broek, Marianne; van Leeuwen-Cornelisse, Inge; Nagengast, Fokko; Meijers-Heijboer, Anne; Lindhout, Dick; Griffioen, Gerrit; Cats, Annemieke; Kleibeuker, Jan; Varesco, Liliana; Bertario, Lucio; Bisgaard, Marie Luise; Mohr, Jan; Fodde, Riccardo

1995-01-01

294

Disease proteomics of endocrine disorders revealed by two-dimensional gel electrophoresis and mass spectrometry.  

PubMed

Endocrine disorders such as dwarfism and diabetes show abnormalities in many different organs even if a certain hormone is the primary cause of the disease. One of the aims of proteomics is to elucidate an abnormal hormone network underlying dysfunction in the disease through quantitative and qualitative proteome analyses of various organs. In a comprehensive study of the rdw rat with hereditary dwarfism, we found the accumulation of ER proteins in the rdw thyroid. Contrary to the initial notion that the dwarfism of the rat was caused by genetic mutations related to pituitary hormones, the primary cause is a missense mutation in the thyroglobulin gene. To understand at the protein level cellular damage caused by oxidative stress, we developed a proteomic method and applied to detecting protein carbonyls in various organs of a diabetes model OLETF rat. The method would provide a means toward clarifying a comprehensive view of oxidative modifications of proteins in diabetes. We review 2-DE-based disease proteomics of endocrine disorders in general, with particular attention paid to our proteome projects by a 2-DE method with an agarose IEF gel in the first dimension (agarose 2-DE) and LC-MS/MS. PMID:21136837

Oh-Ishi, Masamichi; Kodera, Yoshio; Furudate, Sen-Ichi; Maeda, Tadakazu

2008-03-01

295

Identification of S-nitrosated mitochondrial proteins by S-nitrosothiol difference in gel electrophoresis (SNO-DIGE): implications for the regulation of mitochondrial function by reversible S-nitrosation.  

PubMed

The S-nitrosation of mitochondrial proteins as a consequence of NO metabolism is of physiological and pathological significance. We previously developed a MitoSNO (mitochondria-targeted S-nitrosothiol) that selectively S-nitrosates mitochondrial proteins. To identify these S-nitrosated proteins, here we have developed a selective proteomic methodology, SNO-DIGE (S-nitrosothiol difference in gel electrophoresis). Protein thiols in control and MitoSNO-treated samples were blocked, then incubated with copper(II) and ascorbate to selectively reduce S-nitrosothiols. The samples were then treated with thiol-reactive Cy3 (indocarbocyanine) or Cy5 (indodicarbocyanine) fluorescent tags, mixed together and individual protein spots were resolved by 2D (two-dimensional) gel electrophoresis. Fluorescent scanning of these gels revealed S-nitrosated proteins by an increase in Cy5 red fluorescence, allowing for their identification by MS. Parallel analysis by Redox-DIGE enabled us to distinguish S-nitrosated thiol proteins from those which became oxidized due to NO metabolism. We identified 13 S-nitrosated mitochondrial proteins, and a further four that were oxidized, probably due to evanescent S-nitrosation relaxing to a reversible thiol modification. We investigated the consequences of S-nitrosation for three of the enzymes identified using SNO-DIGE (aconitase, mitochondrial aldehyde dehydrogenase and alpha-ketoglutarate dehydrogenase) and found that their activity was selectively and reversibly inhibited by S-nitrosation. We conclude that the reversible regulation of enzyme activity by S-nitrosation modifies enzymes central to mitochondrial metabolism, whereas identification and functional characterization of these novel targets provides mechanistic insight into the potential physiological and pathological roles played by this modification. More generally, the development of SNO-DIGE facilitates robust investigation of protein S-nitrosation across the proteome. PMID:20533907

Chouchani, Edward T; Hurd, Thomas R; Nadtochiy, Sergiy M; Brookes, Paul S; Fearnley, Ian M; Lilley, Kathryn S; Smith, Robin A J; Murphy, Michael P

2010-08-15

296

Identification of S-nitrosated mitochondrial proteins by S-nitrosothiol difference in gel electrophoresis (SNO-DIGE): implications for the regulation of mitochondrial function by reversible S-nitrosation  

PubMed Central

The S-nitrosation of mitochondrial proteins as a consequence of NO metabolism is of physiological and pathological significance. We previously developed a MitoSNO (mitochondria-targeted S-nitrosothiol) that selectively S-nitrosates mitochondrial proteins. To identify these S-nitrosated proteins, here we have developed a selective proteomic methodology, SNO-DIGE (S-nitrosothiol difference in gel electrophoresis). Protein thiols in control and MitoSNO-treated samples were blocked, then incubated with copper(II) and ascorbate to selectively reduce S-nitrosothiols. The samples were then treated with thiol-reactive Cy3 (indocarbocyanine) or Cy5 (indodicarbocyanine) fluorescent tags, mixed together and individual protein spots were resolved by 2D (two-dimensional) gel electrophoresis. Fluorescent scanning of these gels revealed S-nitrosated proteins by an increase in Cy5 red fluorescence, allowing for their identification by MS. Parallel analysis by Redox-DIGE enabled us to distinguish S-nitrosated thiol proteins from those which became oxidized due to NO metabolism. We identified 13 S-nitrosated mitochondrial proteins, and a further four that were oxidized, probably due to evanescent S-nitrosation relaxing to a reversible thiol modification. We investigated the consequences of S-nitrosation for three of the enzymes identified using SNO-DIGE (aconitase, mitochondrial aldehyde dehydrogenase and ?-ketoglutarate dehydrogenase) and found that their activity was selectively and reversibly inhibited by S-nitrosation. We conclude that the reversible regulation of enzyme activity by S-nitrosation modifies enzymes central to mitochondrial metabolism, whereas identification and functional characterization of these novel targets provides mechanistic insight into the potential physiological and pathological roles played by this modification. More generally, the development of SNO-DIGE facilitates robust investigation of protein S-nitrosation across the proteome. PMID:20533907

Chouchani, EdwardT.; Hurd, ThomasR.; Nadtochiy, SergiyM.; Brookes, PaulS.; Fearnley, IanM.; Lilley, KathrynS.; Smith, RobinA.J.; Murphy, MichaelP.

2010-01-01

297

Analysis of Leishmania chagasi by 2-D difference gel electrophoresis (2-D DIGE) and immunoproteomic: identification of novel candidate antigens for diagnostic tests and vaccine.  

PubMed

Identification of novel antigens is essential for developing new diagnostic tests and vaccines. We used DIGE to compare protein expression in amastigote and promastigote forms of Leishmania chagasi. Nine hundred amastigote and promastigote spots were visualized. Five amastigote-specific, 25 promastigote-specific, and 10 proteins shared by the two parasite stages were identified. Furthermore, 41 proteins were identified in the Western blot employing 2-DE and sera from infected dogs. From these proteins, 3 and 38 were reactive with IgM and total IgG, respectively. The proteins recognized by total IgG presented different patterns in terms of their recognition by IgG1 and/or IgG2 isotypes. All the proteins selected by Western blot were mapped for B-cell epitopes. One hundred and eighty peptides were submitted to SPOT synthesis and immunoassay. A total of 25 peptides were shown of interest for serodiagnosis to visceral leishmaniasis. In addition, all proteins identified in this study were mapped for T cell epitopes by using the NetCTL software, and candidates for vaccine development were selected. Therefore, a large-scale screening of L. chagasi proteome was performed to identify new B and T cell epitopes with potential use for developing diagnostic tests and vaccines. PMID:21355625

Costa, Mriam M; Andrade, Hlida M; Bartholomeu, Daniella C; Freitas, Leandro M; Pires, Simone F; Chapeaurouge, Alexander D; Perales, Jonas; Ferreira, Andr T; Giusta, Mrio S; Melo, Maria N; Gazzinelli, Ricardo T

2011-05-01

298

Analysis of -subgroup proteobacterial ammonia oxidizer populations in soil by denaturing gradient gel electrophoresis analysis and hierarchical phylogenetic probing  

Microsoft Academic Search

A combination of denaturing gradient gel electrophoresis (DGGE) and oligonucleotide probing was used to investigate the influence of soil pH on the compositions of natural populations of autotrophic beta-subgromp proteobacterial ammonia oxidizers. PCR primers specific to this group were used to amplify 16S ribosomal DIVA (rDNA) from soils maintained for 36 years at a range of pH values, and PCR

JOHN R. STEPHEN; GEORGE A. KOWALCHUK; MARY-ANN V. BRUNS; ALLISON E. MCCAIG; CAROL J. PHILLIPS; T. MARTIN EMBLEY; JAMES I. PROSSER

1998-01-01

299

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

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

Gerard Muyzer; Andreas Teske; Carl O. Wirsen; Holger W. Jannasch

1995-01-01

300

Pulsed-Field Gel Electrophoresis Supports the Presence of Host-Adapted Salmonella enterica subsp. enterica Serovar Typhimurium Strains in the British Garden Bird Population?  

PubMed Central

Salmonellosis is a frequently diagnosed infectious disease of passerine birds in garden habitats within Great Britain with potential implications for human and domestic animal health. Postmortem examinations were performed on 1,477 garden bird carcasses of circa 50 species from England and Wales, 1999 to 2007 inclusive. Salmonellosis was confirmed in 263 adult birds of 10 passerine species in this 11-year longitudinal study. A subset of 124 fully biotyped Salmonella enterica subsp. enterica serovar Typhimurium isolates was examined using pulsed-field gel electrophoresis to investigate the hypothesis that these strains are host adapted and to determine whether this molecular technique offers greater resolution in understanding the epidemiology of Salmonella Typhimurium infection than phage typing alone. For the two most common phage types, definitive type (DT) 40 and DT56v, which together accounted for 97% (120/124) of isolates, pulsed-field gel electrophoresis groupings closely correlated with phage type with remarkably few exceptions. A high degree of genetic similarity (>90%) was observed within and between the two most common pulsed-field gel electrophoresis groups. No clustering or variation was found in the pulsed-field gel electrophoresis groupings by bird species, year, or geographical region beyond that revealed by phage typing. These findings support the hypothesis that there are currently two host-adapted Salmonella phage types, S. Typhimurium DT40 and DT56v, circulating widely in British garden birds and that the reservoir of infection is maintained within wild bird populations. Large-scale multilocus sequence typing studies are required to further investigate the epidemiology of this infection. PMID:21948838

Lawson, Becki; Hughes, Laura A.; Peters, Tansy; de Pinna, Elizabeth; John, Shinto K.; Macgregor, Shaheed K.; Cunningham, Andrew A.

2011-01-01

301

Temperature gradient gel electrophoresis analysis of 16S rRNA from human fecal samples reveals stable and host-specific communities of active bacteria  

Microsoft Academic Search

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.

ERWIN G. ZOETENDAL; ANTOON D. L. AKKERMANS; Vos de W. M

1998-01-01

302

Development and Validation of PCR Primers To Assess the Diversity of Clostridium spp. in Cheese by Temporal Temperature Gradient Gel Electrophoresis  

Microsoft Academic Search

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

Anne-Gaelle Le Bourhis; Katiana Saunier; Joel Dore; Jean-Philippe Carlier; Jean-Francois Chamba; Michel-Robert Popoff; Jean-Luc Tholozan

2005-01-01

303

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

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

EMILIO O. CASAMAYOR; HENDRIK SCHAFER; LLUIS BANERAS; CARLOS PEDROS-ALIO; G. Muyzer

2000-01-01

304

Seasonal Distributions of Dominant 16S rRNA-Defined Populations in a Hot Spring Microbial Mat Examined by Denaturing Gradient Gel Electrophoresis  

Microsoft Academic Search

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

M. J. FERRIS; ANDD. M. WARD

1997-01-01

305

Evaluation of denaturing gradient gel electrophoresis in the detection of 16S rDNA sequence variation in rhizobia and methanotrophs  

Microsoft Academic Search

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

Tatiana Vallaeys; Edward Topp; Gerard Muyzer; Valrie Macheret; Gisle Laguerre; Annabel Rigaud; Guy Soulas

1997-01-01

306

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  

Microsoft Academic Search

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,

Laura Wonderling; Rachel Pearce; F. Morgan Wallace; Jeffrey E. Call; Ingrid Feder; Mark Tamplin; John B. Luchansky

2003-01-01

307

Genetic Heterogeneity of Shiga Toxin-Producing Escherichia coli Strains Isolated in Sao Paulo, Brazil, from 1976 through 2003, as Revealed by Pulsed-Field Gel Electrophoresis  

Microsoft Academic Search

The pulsed-field gel electrophoresis (PFGE) patterns of 46 Shiga toxin-producing Escherichia coli (STEC) strains isolated in Sao Paulo, Brazil, during the period from 1976 to 2003 were compared with those found among 30 non-STEC strains that carried eae and that belonged to the same serogroups as the STEC strains. All except two of the STEC and non-STEC strains of human

Tnia Mar; I. Va; Kinue Irino; Lucilia S. Nishimura; Maria Cec li

2006-01-01

308

Comparison of arbitrarily-primed polymerase chain reaction, restriction enzyme analysis and pulsed-field gel electrophoresis for typing Clostridium difficile  

Microsoft Academic Search

Three methods of molecular typing of Clostridium difficile [arbitrarily-primed polymerase chain reaction (AP-PCR), restriction enzyme analysis (REA) and pulsed-field gel electrophoresis (PFGE)] were compared using 33 isolates collected during a prospective study of Clostridium difficile transmission. Sixteen isolates (from 13 patients and 3 environmental sites) represented a cluster of C. difficile diarrhea on 2 wards, whereas the other 17 isolates

M. H. Samore; M. Kristjansson; L. Venkataraman; P. C. DeGirolami; R. D. Arbeit

1996-01-01

309

Comparison of Pulsed-Field Gel Electrophoresis and Randomly Amplified DNA Polymorphism Analysis for Typing Extended Spectrum-b-Lactamase-ProducingKlebsiella pneumoniae  

Microsoft Academic Search

The incidence and transmission patterns of extended-spectrum-b-lactamase (ESBL)-producing Klebsiella pneumoniaeinpatientsadmittedtotheintensivecareunit(ICU)ofauniversityhospitalwereinvestigatedover a 3-year period. K. pneumoniae isolates were characterized by antibiotic susceptibility, capsular serotyping, plasmid profiles, and pulsed-field gel electrophoresis (PFGE) of genome macrorestriction patterns withXbaI, and the results were compared with those obtained by typing with the randomly amplified polymorphic DNA (RAPD) patterns. The discriminatory power of RAPD typing was

ANDREA GORI; FLORENCE ESPINASSE; ARIANE DEPLANO; CLAIRE NONHOFF; MARIE HELENE NICOLAS; ANDMARC J. STRUELENS

1996-01-01

310

Comparison of Pulsed-Field Gel Electrophoresis and Coagulase Gene Restriction Profile Analysis Techniques in the Molecular Typing of Staphylococcus aureus  

Microsoft Academic Search

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

CHIEN-SHUN CHIOU; HSIAO-LUN WEI; LI-CHU YANG

2000-01-01

311

Comparison of Ribotyping, Randomly Amplified Polymorphic DNA Analysis, and Pulsed-Field Gel Electrophoresis in Typing of Lactobacillus rhamnosus and L. casei Strains  

Microsoft Academic Search

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 discrimi- natory power of the methods. Species-specific primers for L. rhamnosus and L. casei

SOILE TYNKKYNEN; REETTA SATOKARI; MARIA SAARELA; TIINA MATTILA-SANDHOLM; MAIJA SAXELIN

1999-01-01

312

Coupling sodium dodecyl sulfate-capillary polyacrylamide gel electrophoresis with matrix-assisted laser desorption ionization time-of-flight mass spectrometry via a poly(tetrafluoroethylene) membrane.  

PubMed

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

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

2011-03-01

313

2D gel-based multiplexed proteomic analysis during larval development and metamorphosis of the biofouling polychaete tubeworm Hydroides elegans.  

PubMed

Larval settlement and metamorphosis of a common biofouling polychaete worm, Hydroides elegans, involve remarkable structural and physiological changes during this pelagic to sessile habitat shift. The endogenous protein molecules and post-translational modifications that drive this larval transition process are not only of interest to ecologists but also to the antifouling paint industry, which aims to control the settlement of this biofouling species on man-made structures (e.g., ship hulls). On the basis of our recent proteomic studies, we hypothesize that rapid larval settlement of H. elegans could be mediated through changes in phosphorylation status of proteins rather than extensive de novo synthesis of proteins. To test this hypothesis, 2D gel-based multiplexed proteomics technology was used to monitor the changes in protein expression and phosphorylation status during larval development and metamorphosis of H. elegans. The protein expression profiles of larvae before and after they reached competency to attach and metamorphose were similar in terms of major proteins, but the percentage of phosphorylated proteins increased from 41% to 49% after competency. Notably, both the protein and phosphoprotein profiles of the metamorphosed individuals (adult) were distinctly different from that of the larvae, with only 40% of the proteins phosphorylated in the adult stage. The intensity ratio of all phosphoprotein spots to all total protein spots was also the highest in the competent larval stage. Overall, our results indicated that the level of protein phosphorylation might play a crucial role in the initiation of larval settlement and metamorphosis. PMID:20666481

Zhang, Yu; Sun, Jin; Xiao, Kang; Arellano, Shawn M; Thiyagarajan, Vengatesen; Qian, Pei-Yuan

2010-09-01

314

Two-step denaturing gradient gel electrophoresis (2S-DGGE), a gel-based strategy to capture full-length 16S rRNA gene sequences.  

PubMed

Obtaining full-length 16S rRNA gene sequences is important for generating accurate taxonomy assignments of bacteria, which normally is realized via clone library construction. However, the application of clone library has been hindered due to its limitations in sample throughput and in capturing minor populations (<1% of total microorganisms). To overcome these limitations, a new strategy, two-step denaturing gradient gel electrophoresis (2S-DGGE), is developed to obtain full-length 16S rRNA gene sequences. 2S-DGGE can compare microbial communities based on its first-round DGGE profiles and generate partial 16S rRNA gene sequences (8-534bp, Escherichia coli numbering). Then, strain-specific primers can be designed based on sequence information of bacteria of interest to PCR amplify their remaining 16S rRNA gene sequences (515-1541bps, E. coli numbering). The second-round DGGE can confirm DNA sequence purity of these PCR products. Finally, the full-length 16S rRNA gene sequences can be obtained through combining the two partial DNA sequences. By employing 2S-DGGE, taxonomies of a group of dehalogenating bacteria have been assigned based on their full-length 16S rRNA gene sequences, several of which existed in dehalogenating microcosms as minor populations. In all, 2S-DGGE can be utilized as a medium throughput method for taxonomic identification of interested/minor populations from single or multiple microbial consortia. PMID:22772864

Wang, Shanquan; He, Jianzhong

2012-09-01

315

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

PubMed Central

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 to 18 selected isolates. Of the 41 isolates examined, 7 were ?-lactamase producing and 8 were vancomycin resistant. By PFGE, 17 isolates had distinct patterns; the other 24 were classified into eight different clonal groups. By PCR using the BOXA2R primer, 16 isolates generated distinct patterns; the other 25 were classified into nine different clonal groups. There were only minor differences in the PCR results obtained by using the BOXA2R primer and the REP1R-Dt and REP2-Dt primers. Two isolates among vancomycin-resistant enterococci from the greater Houston, Tex., area were related by PFGE, distinct by PCR with the BOXA2R primer, and related by PCR with the REP1R-Dt and REP2-Dt primers. Clonal relationships among the remaining 39 isolates were similar by both PFGE and PCR. PCR reliably discriminated all epidemiologically unrelated isolates. Although PCR is less time consuming than PFGE, PCR results were more difficult to interpret than PFGE results, perhaps because fewer bands were generated by PCR than by PFGE and some PCR products were inconsistently seen. PMID:9431949

Malathum, Kumthorn; Singh, Kavindra V.; Weinstock, George M.; Murray, Barbara E.

1998-01-01

316

Detection of DNA damage in response to cooling injury in equine spermatozoa using single-cell gel electrophoresis.  

PubMed

Single-cell gel electrophoresis (SCGE), or comet assay, has the ability to detect damage at the single cell level and has not been reported for equine sperm. The ability to detect nuclear damage at the single cell level could aid in the advancement of protocols for optimal semen preservation. The goals of these experiments were to adapt this assay for use with equine sperm and to utilize the assay for determining the integrity of equine sperm DNA following treatments with storage at various decreased temperatures (-20 degrees C and 5 degrees C). Results from experiments in which sperm were frozen (-20 degrees C) in the absence of cryoprotectants revealed that significantly more cells with fragmented tails of DNA, or comets, occurred among those exposed to 1, 3, and 5 freeze-thaw cycles (65% +/- 6%, 76% +/- 11%, 92% +/- 6%, respectively) compared with fresh, untreated sperm (19% +/- 16%, P < .05). In addition DNA damage was different (P < .05) between the three freeze-thaw treatments. Sensitivity of SCGE on equine sperm was further tested with known ratios of frozen-thawed and fresh cells. The amount of detectable DNA damage was positively correlated with the percentage of cryo-damaged cells in each treatment (r2 = 0.92, P < .05). Potential damage as a result of cooled storage was also investigated and results revealed that sperm stored for 48 hours (at 5 degrees C) had a higher percentage of comets than that of fresh sperm (63% +/- 13.9% and 28% +/- 15.6%, respectively, P < .05). The percentage of viable sperm also decreased linearly over time and was inversely correlated with percent of comets (r2 = 0.805, P < .001). Detection of sublethal and/or uncompensable fertility factors in semen, such as DNA fragmentation, could be useful for detecting male differences in semen for cooling or cryopreservation potential and could provide a tool for monitoring and preserving fertility for individual stallions. PMID:11780916

Linfor, Jennifer J; Meyers, Stuart A

2002-01-01

317

Plasmid Profile and PulsedField Gel Electrophoresis Analysis of Salmonella enterica Isolates from Humans in Turkey  

PubMed Central

This study was conducted for typing Salmonella enterica subspecies enterica strains in Turkey using pulsedfield gel electrophoresis (PFGE) and plasmid DNA profile analysis. Fourty-two strains were isolated from clinical samples obtained from unrelated patients with acute diarrhea. The samples were collected from state hospitals and public health laboratories located at seven provinces in different regions of Turkey at different times between 2004 and 2010. The strains were determined to belong to 4 different serovars. The Salmonella enterica strains belonged to the serovars Salmonella Enteritidis (n?=?23), Salmonella Infantis (n?=?14), Salmonella Munchen (n?=?2), and Salmonella Typhi (n?=?3). Forty-two Salmonella enterica strains were typed with PFGE methods using XbaI restriction enzyme and plasmid analysis. At the end of typing, 11 different PFGE band profiles were obtained. Four different PFGE profiles (type 1, 4, 9, and 10) were found among serotype S. Enteritidis species, 3 different PFGE profiles (type 3, 5, 6) were found among S. Infantis species, 2 different PFGE profiles were found among S. Typhi species (type 2 and 11), and 2 different PFGE profiles were found among S. Munchen species (type 7, 8). The UPGMA dendrogram was built on the PFGE profiles. In this study, it was determined that 4 strains of 42 Salmonella enterica strains possess no plasmid, while the isolates have 13 plasmids ranging from 5.0 to 150 kb and making 12 different plasmid profiles (P1P12). In this study, we have applied the analysis of the PFGE patterns and used bioinformatics methods to identify both inter and intra serotype relationships of 4 frequently encountered serotypes for the first time in Turkey. PMID:24852084

Ozdemir, Kerem; Acar, Sumeyra

2014-01-01

318

Typing of Campylobacter jejuni Isolates from Dogs by Use of Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis ?  

PubMed Central

Campylobacter is a major cause of human gastroenteritis worldwide. Risk of Campylobacter infection in humans has been associated with many sources, including dogs. This study aimed to investigate whether C. jejuni carried by dogs could potentially be a zoonotic risk for humans and if there were common sources of C. jejuni infection for both humans and dogs. Multilocus sequence typing (MLST) together with macrorestriction analysis of genomic DNA using SmaI and pulsed-field gel electrophoresis (PFGE) were both used to analyze 33 C. jejuni isolates obtained from various dog populations, including those visiting veterinary practices and from different types of kennels. MLST data suggested that there was a large amount of genetic diversity between dog isolates and that the majority of sequence types found in isolates from these dogs were the same as those found in isolates from humans. The main exception was ST-2772, which was isolated from four samples and could not be assigned to a clonal complex. The most commonly identified clonal complex was ST-45 (11 isolates), followed by ST-21 (4 isolates), ST-508 (4 isolates), and ST-403 (3 isolates). The profiles obtained by macrorestriction PFGE were largely in concordance with the MLST results, with a similar amount of genetic diversity found. The diversity of sequence types found within dogs suggests they are exposed to various sources of C. jejuni infection. The similarity of these sequence types to C. jejuni isolates from humans suggests there may be common sources of infection for both dogs and humans. Although only a small number of household dogs may carry C. jejuni, infected dogs should still be considered a potential zoonotic risk to humans, particularly if the dogs originate from kennelled or hunt kennel dog populations, where the prevalence may be higher. PMID:19794053

Parsons, B. N.; Cody, A. J.; Porter, C. J.; Stavisky, J. H.; Smith, J. L.; Williams, N. J.; Leatherbarrow, A. J. H.; Hart, C. A.; Gaskell, R. M.; Dingle, K. E.; Dawson, S.

2009-01-01

319

Comparison of multilocus sequence typing and pulsed-field gel electrophoresis for Salmonella spp. identification in surface water  

NASA Astrophysics Data System (ADS)

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

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

320

Diversity of pulsed-field gel electrophoresis profiles of Campylobacter jejuni and Campylobacter coli from broiler chickens in France.  

PubMed

In 2003 to 2004, 26 free-range broilers flocks excreting Campylobacter were studied for identification of Campylobacter species and genotype diversity. Seventeen flocks were sampled at the end of the indoor rearing period and 9 before departure to the slaughterhouse after access to an open area. Out of 513 isolates, 315 were identified as Campylobacter jejuni and 198 as Campylobacter coli. Pulsed-field gel electrophoresis analysis revealed 35 genotypes for C. jejuni and 43 genotypes for C. coli; 38.4% of the isolates were clustered into 16 genetic groups. This kind of poultry production system is characterized by a large number of Campylobacter coli isolates. Flocks sampled during the indoor phase were predominantly contaminated by C. jejuni, whereas those sampled during warm months were predominantly contaminated by C. coli. The Campylobacter population was genetically highly diverse: multiple genotypes were detected in a single flock. Six flocks were each found to harbor a mixture of genotypes; these isolates were genetically closely related and were grouped into clusters of at least 80% genetic similarity. Isolates with genotypes found in different flocks and strains, but sharing the same genetic clusters, were detected in different farms and at different times in the year. Flocks sampled during the indoor rearing period and when farmers use fresh farm-made litter were associated with a small number of clusters. In conclusion, Campylobacter species were genetically highly diverse. Our findings suggest the presence of genomic rearrangements in Campylobacter colonizing the chick intestine and that some Campylobacter strains are adapted to poultry. In addition, the species diversity in the flocks was affected by season and cloistering measures. Litter and exposure to an open area were likely sources of flock Campylobacter contamination. PMID:18648064

Denis, M; Rose, V; Huneau-Salan, A; Balaine, L; Salvat, G

2008-08-01

321

Protein composition of wheat gluten polymer fractions determined by quantitative two-dimensional gel electrophoresis and tandem mass spectrometry  

PubMed Central

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

2014-01-01

322

Proteome signatures for stress and starvation in Bacillus subtilis as revealed by a 2-D gel image color coding approach.  

PubMed

In this paper we have defined proteome signatures of Bacillus subtilis in response to heat, salt, peroxide, and superoxide stress as well as after starvation for ammonium, tryptophan, glucose, and phosphate using the 2-D gel-based approach. In total, 79 stress-induced and 155 starvation-induced marker proteins were identified including 50% that are not expressed in the vegetative proteome. Fused proteome maps and a color coding approach have been used to define stress-specific regulons that are involved in specific adaptative functions (HrcA for heat, PerR and Fur for oxidative stress, RecA for peroxide, CymR and S-box for superoxide stress). In addition, starvation-specific regulons are defined that are involved in the uptake or utilization of alternative nutrient sources (TnrA, sigmaL/BkdR for ammonium; tryptophan-activated RNA-binding attenuation protein for tryptophan; CcpA, CcpN, sigmaL/AcoR for glucose; PhoPR for phosphate starvation). The general stress or starvation proteome signatures include the CtsR, Spx, sigmaL/RocR, sigmaB, sigmaH, CodY, sigmaF, and sigmaE regulons. Among these, the Spx-dependent oxidase NfrA was induced by all stress conditions indicating stress-induced protein damages. Finally, a subset of sigmaH-dependent proteins (sporulation response regulator, YvyD, YtxH, YisK, YuxI, YpiB) and the CodY-dependent aspartyl phosphatase RapA were defined as general starvation proteins that indicate the transition to stationary phase caused by starvation. PMID:16847875

Tam, Le Thi; Antelmann, Haike; Eymann, Christine; Albrecht, Dirk; Bernhardt, Jrg; Hecker, Michael

2006-08-01

323

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

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

Richardson, David

324

Two Electrophoresis Experiments for Freshmen in the Health Professions.  

ERIC Educational Resources Information Center

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)

Brabson, G. Dana; Waugh, David S.

1986-01-01

325

Metabolic activation of herbicide products by Vicia faba detected in human peripheral lymphocytes using alkaline single cell gel electrophoresis.  

PubMed

Ametryn and metribuzin S-triazines derivatives and EPTC thiocarbamate are herbicides used extensively in Mexican agriculture, for example in crops such as corn, sugar cane, tomato, wheat, and beans. The present study evaluated the DNA damage and cytotoxic effects of three herbicides after metabolism by Vicia faba roots in human peripheral lymphocytes using akaline single cell gel electrophoresis. Three parameters were scored as indicators of DNA damage: tail length, percentage of cells with DNA damage (with comet), and level DNA damage. The lymphocytes were treated for 2 h with 0.5-5.0 microg/ml ametryn or metribuzin and 1.5-10 microg/ml EPTC. Lymphocytes also were coincubated for 2 h with 20 microl V. faba roots extracts that had been treated for 4 h with 50-500 mg/l of the two triazines or with the thiocarbamate herbicide or with ethanol (3600 mg/l), as positive control. The lymphocytes treated with three pesticides without in vivo metabolic activation by V. faba root did not show significant differences in the mean values between genotoxic parameters compared with negative control. But when human cells were exposed to three herbicides after they had been metabolized the frequency of cell comet, tail length and level DNA damage all increased. At highest concentrations of the three herbicides produced severe DNA damage compared with S10 fraction and negative control. The linear regression analysis of the tail length values of three herbicides indicated that there was genotoxic effect concentration-response relationship with ametryn and ametribuzin but no EPTC. The ethanol induced major increase DNA damage compared with S10 fraction and the three pesticides. There were not effects in cell viability with treatment EPTC and metribuzin whether or not it had been metabolized. High concentrations of ametryn alone and after it had been metabolized decreased cell viability compared with the negative control. The results demonstrated that the three herbicides needed to be activated by the V. faba root metabolism to produce DNA damage in human peripheral lymphocyte. The alkaline comet technique is a rapid and sensitive assay, to quickly evaluate DNA damage the metabolic activation of herbicide products by V. faba root in human cells in vitro. PMID:17482794

Caldern-Segura, Mara Elena; Gmez-Arroyo, Sandra; Molina-Alvarez, Bertha; Villalobos-Pietrini, Rafael; Caldern-Ezquerro, Carmen; Corts-Eslava, Josefina; Valencia-Quintana, Pedro Rafael; Lpez-Gonzlez, Lucina; Ziga-Reyes, Rubn; Snchez-Rincn, Jos

2007-09-01

326

Resolution of clonal subgroups among Neisseria gonorrhoeae IB-2 and IB-6 serovars by pulsed-field gel electrophoresis.  

PubMed Central

OBJECTIVE--Analysis of macrorestriction patterns by PFGE to resolve the relatedness of clonal subgroups amongst N gonorrhoeae IB-2 and IB-6 serovar strains. MATERIALS AND METHODS--Nineteen IB-2 and eight IB-6 serovar strains that differed in either auxotype or penicillin sensitivity were isolated over a two and a half-year period from patients attending several STD clinics in Sydney. During this period, a major clone, Wt/IB-2 (FS), established on epidemiological grounds, was circulating amongst homosexual males. The genetic relation of this major clone to the other strains present in the community was determined by pulsed-field gel electrophoretic (PFGE) analysis of DNA restriction fragments. Genomic DNA from the 27 isolates were prepared, digested with SpeI and BglII and the restriction patterns were analysed by contour-clamped homogeneous electric field electrophoresis (CHEF) in a CHEF DRIII equipment. RESULTS--Phenotypic characterisation of the 27 isolates by the combined use of auxotype, serological characterisation and penicillin sensitivity indicated the presence of subgroups within each of the two serovars. In the present study, PFGE analysis of SPeI and BglII-generated genomic DNA restriction patterns from six of the ten Wt/IB-2 (FS) correlated well with phenotypic characterisation of this major clone. Four of the ten Wt/IB-2 (FS) were found to be clonally-derived variants of this major clone as minor genome variations (less than 3 DNA fragments) were observed. Distinct clones were represented by three Wt/IB-2 (LS) isolates as the DNA fingerprints generated from these were unrelated to the major clone. Analysis of PFGE patterns of 6 Pro/IB-2 isolates showed that one was genotypically identical to the major clone, two were clonal variants and three had significantly different patterns to indicate that they were genotypically unrelated. Wt/IB-6 isolates had heterogenous PFGE patterns that were clearly unrelated to the Wt/IB-2 serovar strains. Within the IB-6 serovar, there were three isolates with the Wt/IB-6 (FS) phenotype that could be considered as clonal variants whilst the rest were genotypically distinct. CONCLUSIONS--PFGE analysis of macrorestriction patterns generated from SpeI- and BglII-cleavage of genomic DNA has enabled the establishment of clonal origins of strains present in the Sydney community during the period of study. The delineation of strains belonging to major A/S groups by PFGE analysis presents a clearer epidemiological picture than phenotypic characterisation alone. Images PMID:7635488

Poh, C L; Loh, G K; Tapsall, J W

1995-01-01

327

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

SciTech Connect

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

Kinashi, Yuko; Nagasawa, Hatsumi; Little, J.B. [Harvard School of Public Health, Boston, MA (United States); Okayasu, Ryuichi; Iliakis, G.E. [Thomas Jefferson Univ., Philadelphia, PA (United States)

1995-02-01

328

Evaluations of Different Hypervariable Regions of Archaeal 16S rRNA Genes in Profiling of Methanogens by Archaea-Specific PCR and Denaturing Gradient Gel Electrophoresis?  

PubMed Central

Different hypervariable (V) regions of the archaeal 16S rRNA gene (rrs) were compared systematically to establish a preferred V region(s) for use in Archaea-specific PCR-denaturing gradient gel electrophoresis (DGGE). The PCR products of the V3 region produced the most informative DGGE profiles and permitted identification of common methanogens from rumen samples from sheep. This study also showed that different methanogens might be detected when different V regions are targeted by PCR-DGGE. Dietary fat appeared to transiently stimulate Methanosphaera stadtmanae but inhibit Methanobrevibacter sp. strain AbM4 in rumen samples. PMID:18083874

Yu, Zhongtang; Garca-Gonzlez, Rubn; Schanbacher, Floyd L.; Morrison, Mark

2008-01-01

329

Rapid pulsed-field gel electrophoresis protocol for typing of Escherichia coli O157:H7 and other gram-negative organisms in 1 day.  

PubMed Central

Genomic DNA patterns generated by pulsed-field gel electrophoresis are highly specific for different strains of an organism and have significant value in epidemiologic investigations of infectious-disease outbreaks. Unfortunately, time-consuming and tedious specimen processing is an inherent problem which limits the use of this powerful technology as a real-time epidemic investigational tool. Here, I describe a rapid method to improve the response time and provide specific bacterial strain identification for the typing of Escherichia coli O157:H7 and other gram-negative organisms in a single day. PMID:9350772

Gautom, R K

1997-01-01

330

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

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

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

331

Simplification and improvement of protein detection in two-dimensional electrophoresis gels with SERVA HPE lightning red.  

PubMed

A new fluorescent amino-reactive dye has been tested for both labelling proteins prior to electrophoretic separations and between the two steps of two-dimensional electrophoresis. A series of experiments showed, that the labelling of lysines with this dye is compatible with all standard additives used for sample preparation, including reducing substances and carrier ampholytes. Using this dye for pre-labelling considerably simplifies the electrophoresis and detection workflow and provides highly sensitive and quantitative visualisation of proteins. PMID:23786184

Griebel, Anja; Obermaier, Christian; Westermeier, Reiner; Moche, Martin; Bttner, Knut

2013-07-01

332

Bacterial population dynamics in a reverse-osmosis water purification system determined by fluorescent staining and PCR-denaturing gradient gel electrophoresis.  

PubMed

The bacterial population dynamics in an industrial scale reverse-osmosis (RO) water purification system were analyzed by fluorescent staining methods and denaturing gradient gel electrophoresis (DGGE). Bacterial numbers increased with storage in a tank, and bacterial diversity changed during the water purification process. A DNA sequence-based analysis of the major bands on the DGGE gel revealed that Simonsiella sp. (Betaproteobacteria) was abundant in the source water (activated sludge-treated waste effluent), while Bosea sp. and Rhizobium sp. (Alphaproteobacteria), which usually exist in an oligotrophic environment, became abundant during the water purification process. These results suggest the importance of microbiological monitoring by culture-independent methods for quality control in RO water purification systems. These methods could provide an early warning of impending problems and clarify critical steps in controlling specific bacteria contributing to the contamination of RO water systems. PMID:21566369

Baba, Takashi; Matsumoto, Rie; Yamaguchi, Nobuyasu; Nasu, Masao

2009-01-01

333

The structure and thermodynamics of proteinSDS complexes in solution and the mechanism of their transports in gel electrophoresis process  

NASA Astrophysics Data System (ADS)

A unified exposition of the structure, thermodynamics and transport of complexes formed by association of a surfactant sodium dodecyl sulfate (SDS) with water-soluble globular proteins in solution is presented. From small angle neutron scattering experiments we have determined the structure of the complex to be a polymer-like object consisting of a string of SDS micelles decorating the hydrophobic patches of the unfolded polypeptide chain in solution. Based on this structural model we have predicted and observed a polymer-like phase separation of the denatured protein solution at suitable pH. This model can also successfully predict the mobility of the complexes in polyacrylamide gel during an electrophoresis process, if we adapt the well known reptation concept of diffusion of a single chain of polymer in gels.

Guo, Xuan-Hui; Chen, Sow-Hsin

1990-12-01

334

Proteins pattern alteration in AZT-treated K562 cells detected by two-dimensional gel electrophoresis and peptide mass fingerprinting  

PubMed Central

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

D'Andrea, Gabriele; Lizzi, Anna R; Venditti, Sara; Di Francesco, Laura; Giorgi, Alessandra; Mignogna, Giuseppina; Oratore, Arduino; Bozzi, Argante

2006-01-01

335

Influence of nearest neighbor sequence on the stability of base pair mismatches in long DNA; determination by temperature-gradient gel electrophoresis.  

PubMed Central

Temperature-gradient gel electrophoresis (TGGE) was employed to determine the thermal stabilities of 48 DNA fragments that differ by single base pair mismatches. The approach provides a rapid way for studying how specific base mismatches effect the stability of a long DNA fragment. Homologous 373 bp DNA fragments differing by single base pair substitutions in their first melting domain were employed. Heteroduplexes were formed by melting and reannealing pairs of DNAs, one of which was 32P-labeled on its 5'-end. Product DNAs were separated based on their thermal stability by parallel and perpendicular temperature-gradient gel electrophoresis. The order of stability was determined for all common base pairs and mismatched bases in four different nearest neighbor environments; d(GXT).d(AYC), d(GXG).d(CYC), d(CXA).d(TYG), and d(TXT).d(AYA) with X,Y = A, T, C, or G. DNA fragments containing a single mismatch were destabilized by 1 to 5 degrees C with respect to homologous DNAs with complete Watson-Crick base pairing. Both the bases at the mismatch site and neighboring stacking interactions influence the destabilization caused by a mismatch. G.T, G.G and G.A mismatches were always among the most stable mismatches for all nearest neighbor environments examined. Purine.purine mismatches were generally more stable than pyrimidine.pyrimidine mispairs. Our results are in very good agreement with data where available from solution studies of short DNA oligomers. Images PMID:8255768

Ke, S H; Wartell, R M

1993-01-01

336

Ultrafast haplotyping of putative microRNA-binding sites in the WFS1 gene by multiplex polymerase chain reaction and capillary gel electrophoresis.  

PubMed

The transmembrane protein wolframin (WSF1) plays a crucial role in cell integrity in pancreatic beta cells and maintaining ER homeostasis. Genetic variations in the WFS1 gene have been described to be associated with Wolfram syndrome or type 2 diabetes mellitus. In this paper we report on an efficient double-tube allele-specific amplification method in conjunction with ultrafast capillary gel electrophoresis for direct haplotyping analysis of the SNPs in two important miRNA-binding sites (rs1046322 and rs9457) in the WFS1 gene. An automated single-channel capillary gel electrophoresis system was utilized in the method that provided dsDNA fragment analysis in less than 240 s. The light-emitting diode induced fluorescence (LEDIF) detection system enabled excellent sensitivity for automated haplotyping of a large number of clinical samples. The detection limit was 0.002 ng/?L using field amplified injection from water diluted samples. The dynamic quantitation range was 0.08-10.00 ng/?L (R(2)=0.9997) in buffer diluted samples. PMID:23499253

Kerkgyrt, Mrta; Nmeth, Nra; Kerekes, Tams; Rnai, Zsolt; Guttman, Andrs

2013-04-19

337

ProteinAnalysis Electrophoresis  

E-print Network

ProteinDetectionKit(Colorimetric)--GLYCO-PRO GoldSolution,Colloidal--50755 OilRedO--O9755 PonceauSSolution--P7170 ProteoSilverTM--PROT-SIL1 ProteoSilver (G 1041). #12;ProteinAnalysis Electrophoresis sigma-aldrich.com 104 ELECTROPHORESIS Silver Stain Markers Designed for molecular weight determinations on silver stained gels, Silver Stain SDS

Lebendiker, Mario

338

Spot quantification in two dimensional gel electrophoresis image analysis: comparison of different approaches and presentation of a novel compound fitting algorithm  

PubMed Central

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

2014-01-01

339

Modification of the melting properties of duplex DNA by attachment of a GC-rich DNA sequence as determined by denaturing gradient gel electrophoresis.  

PubMed Central

The melting behavior of a DNA fragment carrying the mouse beta maj-globin promoter was investigated as a means of establishing procedures for separating DNA fragments differing by any single base substitution using the denaturing gradient gel electrophoresis procedure of Fischer and Lerman (1,2). We find that attachment of a 300 base pair GC-rich DNA sequence, termed a GC-clamp, to a 135 bp DNA fragment carrying the mouse beta-globin promoter significantly alters the pattern of DNA melting within the promoter. When the promoter is attached to the clamp, the promoter sequences melt without undergoing strand dissociation. The calculated distribution of melting domains within the promoter differs markedly according to the relative orientation of the clamp and promoter sequences. We find that the behavior of DNA fragments containing the promoter and clamp sequences on denaturing gradient polyacrylamide gels is in close agreement with the theoretical melting calculations. These studies provide the basis for critical evaluation of the parameters for DNA melting calculations, and they establish conditions for determining whether all single base substitutions within the promoter can be separated on denaturing gradient gels. Images PMID:2987873

Myers, R M; Fischer, S G; Maniatis, T; Lerman, L S

1985-01-01

340

Strategies and techniques for testing the precision, reliability and reproducibility of computerized two-dimensional gel electrophoresis analysis systems.  

PubMed

A set of test procedures is described for evaluating the reliability, precision and reproducibility of computer systems designed to analyze two-dimensional gel electrophoretograms. The Elsie 4 gel analysis system (Analyt. Biochem., 169, 49-70 (1988) is used to demonstrate the use of these tests. Three major groups of tests are described: analysis of the scanner; analysis of mathematically constructed model gels; and analysis of real gel images. Scanner tests involve evaluating the stability and reproducibility of the scanner. The tests consist primarily of measuring the output of the scanner over a time period to determine its stability, and evaluating the consistency of the scanner at different points in the scanning field. Commonly encountered real gel situations are simulated and analyzed by arranging computer-generated 'ideal spots' in different ways. With such gels we can determine such things as the ability of the computer system to separate closely resolved and/or shoulder spots; whether or not streaks and/or smears are handled correctly; how random noise affects measurements; and, in idealized situation, the accuracy of the quantitation. The idealized spots are generated using the two-dimensional Gaussian as a model of density distribution; other spot models can be used. The Elsie 4 system is capable of finding virtually any spot (if detection parameters are set low enough), and of resolving different-sized spots whose peaks are separated by one and one-half their mean half width). The most significant tests are done on a set of actual gels.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2488595

Miller, M J; Merril, C

1989-01-01

341

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

PubMed

Sm14 was the first fatty acid-binding protein homologue identified in helminths. Thereafter, members of the same family were identified in several helminth species, with high aminoacid sequence homology between them. In addition, immune crossprotection was also reported against Fasciola hepatica infection, in animals previously immunized with the Schistosoma mansoni vaccine candidate, r-Sm14. In the present study, data on preliminary sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting analysis of nine different helminth extracts focusing the identification of Sm14 related proteins, is reported. Out of these, three extracts - Ascaris suum (males and females), Echinostoma paraensei, and Taenia saginata - presented components that comigrated with Sm14 in SDS-PAGE, and that were recognized by anti-rSm14 policlonal serum, in Western blotting tests. PMID:12426606

Thaumaturgo, Nilton; Vilar, Mnica Magno; Edelenyi, Ricardo; Tendler, Miriam

2002-01-01

342

Comparison of Campylobacter Isolates from Poultry and Humans: Association between In Vitro Virulence Properties, Biotypes, and Pulsed-Field Gel Electrophoresis Clusters  

PubMed Central

The in vitro virulence properties of 197 temporally and geographically related Campylobacter isolates from chicken broilers and humans were compared. Comparisons of the virulence properties associated with genotypes and biotypes were made. All isolates adhered to, and 63% invaded, INT-407 cells, whereas 13% were cytotoxic for CHO cells. CHO cell-cytotoxic extracts were also cytotoxic for INT-407 cells, but the sensitivity for Vero cells was variable. The proportion of isolates demonstrating a high invasiveness potential (>1,000 CFU ml?1) or Vero cell cytotoxicity was significantly higher for human than for poultry isolates. Invasiveness was associated with Campylobacter jejuni isolates of biotypes 1 and 2, whereas CHO and INT-407 cell cytotoxicity was associated with C. jejuni isolates of biotypes 3 and 4. Cytotoxic isolates were also clustered according to pulsed-field gel electrophoresis profiles. PMID:14532099

Nadeau, ric; Messier, Serge; Quessy, Sylvain

2003-01-01

343

Comparison of arbitrarily primed PCR and macrorestriction (pulsed-field gel electrophoresis) typing of Pseudomonas aeruginosa strains from cystic fibrosis patients.  

PubMed Central

Arbitrarily primed PCR fingerprinting was carried out on 43 Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients. Seventeen major groups of strains that coincided with groups also distinguished by macrorestriction (pulsed-field gel electrophoresis) typing were identified. Our results illustrated that a CF patient can carry more than one strain and can carry a given strain for long periods of time and that strains can evolve by changes in drug resistance or other phenotypic traits during long-term colonization. The arbitrarily primed PCR method is recommended for first-pass screening of P. aeruginosa isolates from CF patients, especially when many strains are to be typed, because of its sensitivity and efficiency. PMID:7559985

Kersulyte, D; Struelens, M J; Deplano, A; Berg, D E

1995-01-01

344

Dosimetric verification for intensity-modulated arc therapy plans by use of 2D diode array, radiochromic film and radiosensitive polymer gel  

PubMed Central

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

Hayashi, Naoki; Malmin, Ryan L.; Watanabe, Yoichi

2014-01-01

345

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

PubMed Central

Pulsed field gel electrophoresis (PFGE) techniques have been developed to overcome the limitations of conventional electrophoresis and to increase the separation 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. However, a deep understanding of the molecular mechanisms of motion responsible for DNA separation becomes necessary for the rational optimization of these techniques. This paper shows the first clear observations of individual molecules of DNA during the reorientation process in 90 degrees PFGE and 120 degrees PFGE. Real-time visualization of the DNA dynamics during PFGE was possible with the use of an epi-illumination fluorescence microscope specifically equipped to run these experiments and by staining the DNA with YOYO-1 (1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-meth yl -2,3-dihydro-(benzo-1,3-oxazole)-2-methyl-idene]-quinolinium tetraiodide). This dye forms a very stable, highly fluorescent complex with double-stranded DNA and dramatically improves the quality of the DNA images. The results of computer simulations used to reproduce the molecular mechanisms of motion as well as the DNA separation features are also discussed. PMID:8972863

Gurrieri, S; Smith, S B; Wells, K S; Johnson, I D; Bustamante, C

1996-01-01

346

Improved protocol for isolation of Campylobacter spp. from retail broiler meat and use of pulsed field gel electrophoresis for the typing of isolates.  

PubMed

To improve the detection of Campylobacter spp. in retail broiler meat, a reference method (R subsamples) based on the enrichment of 25 g of meat in Bolton broth at 42C under microaerobiosis was compared with an alternative method (A subsamples) consisting in the rinsing of meat samples for 30s in buffered peptone water with antimicrobials with incubation at 42C under aerobiosis. One piece of meat (breasts, tenderloins and thighs) was rinse in experiment 1 (A1) and two pieces in experiment 2 (A2). Campylobacter spp. were isolated on agar plates and identified by PCR. Retail samples in Alabama had less prevalence (P ? 0.05) than samples in the state of Washington. The percentage of positive was higher (P ? 0.05) in A than in R subsamples and rinsing two pieces of meat yielded the highest percentage of positive subsamples. R subsamples showed variations in the prevalence by product. However, A subsamples had similar prevalence of positives among products compare to the result from reference method. More Campylobacter coli isolates were collected in A2 subsamples. Pulse field gel electrophoresis (PFGE) was used as subtyping method to study the genome similarity among the isolates from all methods. A larger diversity of isolates were detected by PFGE in A2 subsamples. Denaturing gradient gel electrophoresis analysis suggested that the initial bacterial populations of the meat samples impact the final bacterial profile after enrichment. Rinsing broiler meats was less time consuming, required less sample preparation and was more sensitive than the reference method for the isolation of naturally occurring Campylobacter spp. This new method could help with epidemiological and intervention studies to control Campylobacter spp. PMID:23545445

Oyarzabal, Omar A; Williams, Aretha; Zhou, Ping; Samadpour, Mansour

2013-10-01

347

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

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

Freddy Van Goethem; Dominique Lison; Micheline Kirsch-Volders

1997-01-01

348

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

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

LUCA COCOLIN; MARISA MANZANO; CARLO CANTONI; GIUSEPPE COMI

2001-01-01

349

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...

350

Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis.  

PubMed Central

Duplex DNA fragments differing by single base substitutions can be separated by electrophoresis in denaturing gradient polyacrylamide gels, but only substitutions in a restricted part of the molecule lead to a separation (1). In an effort to circumvent this problem, we demonstrated that the melting properties and electrophoretic behavior of a 135 base pair DNA fragment containing a beta-globin promoter are changed by attaching a GC-rich sequence, called a 'GC-clamp' (2). We predicted that these changes should make it possible to resolve most, if not all, single base substitutions within fragments attached to the clamp. To test this possibility we examined the effect of several different single base substitutions on the electrophoretic behavior of the beta-globin promoter fragment in denaturing gradient gels. We find that the GC-clamp allows the separation of fragments containing substitutions throughout the promoter fragment. Many of these substitutions do not lead to a separation when the fragment is not attached to the clamp. Theoretical calculations and analysis of a large number of different mutations indicate that approximately 95% of all possible single base substitutions should be separable when attached to a GC-clamp. Images PMID:4000972

Myers, R M; Fischer, S G; Lerman, L S; Maniatis, T

1985-01-01

351

Bacterial community structure in Apis florea larvae analyzed by denaturing gradient gel electrophoresis and 16S rRNA gene sequencing.  

PubMed

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

Saraithong, Prakaimuk; Li, Yihong; Saenphet, Kanokporn; Chen, Zhou; Chantawannakul, Panuwan

2014-11-13

352

Large-gel two-dimensional electrophoresis-matrix assisted laser desorption/ionization-time of flight-mass spectrometry: an analytical challenge for studying complex protein mixtures.  

PubMed

The large-gel two-dimensional electrophoresis (2-DE) technique, developed by Klose and co-workers over the past 25 years, provides the resolving power necessary to separate crude proteome extracts of higher eukaryotes. Matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) provides the sample throughput necessary to identify thousands of different protein species in an adequate time period. Spot excision, in situ proteolysis, and extraction of the cleavage products from the gel matrix, peptide purification and concentration as well as the mass spectrometric sample preparation are the crucial steps that interface the two analytical techniques. Today, these routines and not the mass spectrometric instrumentation determine how many protein digests can be analyzed per day per instrument. The present paper focuses on this analytical interface and reports on an integrated protocol and technology developed in our laboratory. Automated identification of proteins in sequence databases by mass spectrometric peptide mapping requires a powerful search engine that makes full use of the information contained in the experimental data, and scores the search results accordingly. This challenge is heading a second part of the paper. PMID:11565779

Nordhoff, E; Egelhofer, V; Giavalisco, P; Eickhoff, H; Horn, M; Przewieslik, T; Theiss, D; Schneider, U; Lehrach, H; Gobom, J

2001-08-01

353

Direct detection of an antimicrobial peptide of Pediococcus acidilactici in sodium dodecyl sulfate-polyacrylamide gel electrophoresis  

Microsoft Academic Search

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

Arun K. Bhunia; M. C. Johnson; Bibek Ray

1987-01-01

354

Double-stranded cucumovirus associated RNA 5: experimental analysis of necrogenic and non-necrogenic variants by temperature-gradient gel electrophoresis.  

PubMed Central

Cucumber mosaic virus (CMV) and peanut stunt virus (PSV) each contain a fifth major RNA in the size range of 334 to 393 nucleotides. This fifth RNA is a satellite capable of modulating the expression of viral disease symptoms. It is present in infected tissue in single-stranded and double-stranded form. Nucleotide sequence variants of the double-stranded CMV-associated RNA 5 (dsCARNA 5) and PSV-associated RNA 5 (dsPARNA 5) were analysed by temperature-gradient gel electrophoresis. Gels were 5% polyacrylamide, containing 8 M urea in 8.9 mM Tris-borate buffer, with temperature differences of 25-40 degrees C establishing gradients either perpendicular or parallel to the direction of the electric field. For dsCARNA 5 two characteristic transitions were detected with increasing temperature: at temperatures between 40 degrees C and 46 degrees C a drastic retardation in electrophoretic mobility induced by partial dissociation of the duplex structure from the ends and at temperatures above 52 degrees C an abrupt increase in mobility due to complete strand dissociation. dsPARNA 5 exhibited both transitions at up to 10 degrees C higher temperatures and an additional retardation between the transitions mentioned. Seven different variants of dsCARNA 5, 4 necrogenic and 3 non-necrogenic, were analysed. Some showed only one single band, others gave rise to up to six well separated bands corresponding to six molecular species. From all experimental results a correlation between the temperature of the retardation transition and the necrogenicity of CARNA 5 was derived. The diagnostic application of the temperature-gradient gel analysis in agriculture, particularly for the use of non-necrogenic variants as biological control agents to impede CMV-infections, is discussed. Images PMID:3601667

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

1987-01-01

355

ProteomeGRID: towards a high-throughput proteomics pipeline through opportunistic cluster image computing for two-dimensional gel electrophoresis.  

PubMed

The quest for high-throughput proteomics has revealed a number of critical issues. Whilst improved two-dimensional gel electrophoresis (2-DE) sample preparation, staining and imaging issues are being actively pursued by industry, reliable high-throughput spot matching and quantification remains a significant bottleneck in the bioinformatics pipeline, thus restricting the flow of data to mass spectrometry through robotic spot excision and protein digestion. To this end, it is important to establish a full multi-site Grid infrastructure for the processing, archival, standardisation and retrieval of proteomic data and metadata. Particular emphasis needs to be placed on large-scale image mining and statistical cross-validation for reliable, fully automated differential expression analysis, and the development of a statistical 2-DE object model and ontology that underpins the emerging HUPO PSI GPS (Human Proteome Organization Proteomics Standards Initiative General Proteomics Standards). The first step towards this goal is to overcome the computational and communications burden entailed by the image analysis of 2-DE gels with Grid enabled cluster computing. This paper presents the proTurbo framework as part of the ProteomeGRID, which utilises Condor cluster management combined with CORBA communications and JPEG-LS lossless image compression for task farming. A novel probabilistic eager scheduler has been developed to minimise make-span, where tasks are duplicated in response to the likelihood of the Condor machines' owners evicting them. A 60 gel experiment was pair-wise image registered (3540 tasks) on a 40 machine Linux cluster. Real-world performance and network overhead was gauged, and Poisson distributed worker evictions were simulated. Our results show a 4:1 lossless and 9:1 near lossless image compression ratio and so network overhead did not affect other users. With 40 workers a 32x speed-up was seen (80% resource efficiency), and the eager scheduler reduced the impact of evictions by 58%. PMID:15478217

Dowsey, Andrew W; Dunn, Michael J; Yang, Guang-Zhong

2004-12-01

356

Proteomic profiling of dog urine after European adder (Vipera berus berus) envenomation by two-dimensional difference gel electrophoresis.  

PubMed

Between April and September every year, many dogs in Finland are bitten by Vipera berus berus, also known as the European adder, the only venomous snake in the area. Exposure to snake bite venom causes local and systemic symptoms and in severe cases can lead to death. Urine samples were collected from four dogs bitten by V. berus berus and treated in the intensive care unit of the Veterinary Teaching Hospital at the University of Helsinki. The inclusion criteria were a strong suspicion of an adder bite no more than two days before admission and clinical signs of an adder bite. Exclusion criteria were defined as ongoing treatment with glucocorticoids or a known history of liver or kidney diseases. Six privately owned, healthy dogs were obtained as controls. Samples were subjected to 2D-DIGE analysis. Image analysis was performed with DeCyder 7.0 2D software, and protein spots demonstrating a minimum 1.5-fold difference in average spot volume ratios between envenomed and control dogs with a Student's t-test p-value of less than 0.05 were picked and identified using LC-MS/MS. In 2D-DIGE analysis, seven proteins were significantly (p<0.05) over-expressed in the urine of dogs bitten by V. berus berus compared to the control group. From these, five proteins were identified: beta-2-microglobulin (b2MG), alpha-1-antitrypsin (AAT), albumin, fetuin-B and superoxide dismutase (SOD1). Results indicate that envenomation by V. berus berus alter the urinary protein profile in dogs. PMID:22975087

Palviainen, Mari; Raekallio, Marja; Vainionp, Mari; Kosonen, Suvi; Vainio, Outi

2012-12-01

357

The XbaI-BlnI-CeuI genomic cleavage map of Salmonella typhimurium LT2 determined by double digestion, end labelling, and pulsed-field gel electrophoresis.  

PubMed Central

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

Liu, S L; Hessel, A; Sanderson, K E

1993-01-01

358

Use of the single cell gel electrophoresis (comet assay) for comparing apoptotic effect of conventional antibodies versus nanobodies  

PubMed Central

The large molecular size of antibodies is considered one major factor preventing them from becoming more efficient therapeutically. It is well established that all camelids have unique antibodies circulating in their blood called heavy-chain antibodies (HcAbs). Unlike antibodies from other species, these HcAbs contain a single variable domain and two constant domains (CH2 and CH3). HcAbs are a novel type of immunoglobulin-like, antigen binding protein with beneficial pharmacokinetic properties that are ideally suited to targeting cellular antigens for molecular imaging or therapeutic purposes. Since the antigen-binding site of dromedary HcAb is comprised in one single domain, it was referred to as nanobody. In the present work, the different IgG subclasses from immunized camel (Camelus dromedairus) were purified employing their different affinity for protein A column (PA) and protein G column (PG). Characterization of IgG subclasses was done by using 12% SDSPAGE under reducing conditions. Protein bands were visualized after staining with Coomassie Brilliant Blue, showing two bands at 50kDa and 30kDa in case of IgG1 while IgG2 and IgG3 produce only one band at 46kDa and 43kDa respectively. The induction of apoptosis by either conventional or nanobodies was evaluated on two different cell lines, Colon and Hepatic cancer cell (HCT116 and HepG2), using the comet assay. Induced apoptosis were confirmed by visualizing DNA fragmentation bands on 2% agarose gel, and the gel was photographed under UV light. This study demonstrates the successful targeting of human cancer colon cell lines by nanobodies in vitro. It may open perspectives for their future use as tumor target vehicle, due to their small size, soluble behavior and they interact with epitopes that are less antigenic for conventional antibodies. PMID:23960797

Shaker, Ghada H.; Melake, Nahla A.

2011-01-01

359

Vibrational spectroscopy and electrophoresis as a "golden means" in monitoring of polysaccharides in medical plant and gels  

NASA Astrophysics Data System (ADS)

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.

Pielesz, A.

360

Vibrational spectroscopy and electrophoresis as a "golden means" in monitoring of polysaccharides in medical plant and gels.  

PubMed

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. PMID:22465769

Pielesz, A

2012-07-01

361

A simple, rapid and low-cost staining method for gel-electrophoresis separated phosphoproteins via the fluorescent purpurin dye.  

PubMed

A novel fluorescence detection method for phosphoproteins in 1-D and 2-D SDS-PAGE by using purpurin is developed in this study. Phosphoproteins as low as 4-8 ng could be specifically detected by purpurin within 60 min, and the detection limit is similar to or better than that of Pro-Q Diamond staining. Only 2 steps (staining and destaining) are needed for purpurin staining without requiring excessive fixing and washing steps, and for single use, $0.8 is enough for purpurin staining. By comprehensively comparing with Pro-Q Diamond staining, it is concluded that purpurin staining is a simple, rapid and low-cost staining method for a broad application to the research of phosphoproteins. PMID:25325196

Cong, Weitao; Shen, Jiayi; Xuan, Yuanhu; Zhu, Xinliang; Ni, Maowei; Zhu, Zhongxin; Hong, Guoying; Lu, Xianghong; Jin, Litai

2014-12-01

362

Variation and Genomic Localization of Genes Encoding DROSOPHILA MELANOGASTER Male Accessory Gland Proteins Separated by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis  

PubMed Central

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

Whalen, Michael; Wilson, Thomas G.

1986-01-01

363

Prevalence of Clostridium botulinum in Finnish trout farms: pulsed-field gel electrophoresis typing reveals extensive genetic diversity among type E isolates.  

PubMed

The distribution of Clostridium botulinum serotypes A, B, E, and F in Finnish trout farms was examined. A total of 333 samples were tested with a neurotoxin-specific PCR assay. C. botulinum type E was found in 68% of the farm sediment samples, in 15% of the fish intestinal samples, and in 5% of the fish skin samples. No other serotypes were found. The spore counts determined by the most-probable-number method were considerably higher for the sediments than for the fish intestines and skin; the average values were 2,020, 166, and 310 C. botulinum type E spores kg-1, respectively. The contamination rates in traditional freshwater ponds and marine net cages were high, but in concrete ponds equipped with sediment suction devices the contamination rates were significantly lower. Pulsed-field gel electrophoresis (PFGE) typing of 42 isolates obtained in this survey and 12 North American reference strains generated 28 pulsotypes upon visual inspection, suggesting that there was extensive genetic diversity and that the discriminatory power of PFGE typing in C. botulinum type E was high. A numerical analysis of SmaI-XmaI macrorestriction profiles confirmed these findings, as it divided the 54 isolates into 15 clusters at a similarity level of 76%. For this material, this level of similarity corresponded to a three-band difference in the macrorestriction profiles, which indicated that there is no genotypic proof of a close epidemiological relationship among the clusters. PMID:9797260

Hielm, S; Bjrkroth, J; Hyyti, E; Korkeala, H

1998-11-01

364

Phylogenetic analyses and detection of viridans streptococci based on sequences and denaturing gradient gel electrophoresis of the rod shape-determining protein gene  

PubMed Central

Background Population analysis of viridans streptococci is important because these species are associated with dental caries, bacteremia, and subacute endocarditis, in addition to being important members of the human oral commensal microbiota. Design In this study, we phylogenetically analyzed the rod shape-determining protein gene (rodA), which is associated with cellular morphology, cell division, and sensitivity for antibiotics, and demonstrated that the diversity of the rodA gene is sufficient to identify viridans streptococci at the species level. Moreover, we developed a more convenient denaturing gradient gel electrophoresis (DGGE) method based on the diversity of the rodA gene (rodA-DGGE) for detecting nine dominant streptococcal species in human saliva, namely, Streptococcus sanguinis, Streptococcus oralis, Streptococcus mitis, Streptococcus parasanguinis, Streptococcus gordonii, Streptococcus vestibularis, Streptococcus salivarius, Streptococcus mutans, and Streptococcus sobrinus. Results This rodA-DGGE method proved useful in detecting viridans streptococci without cultivation, isolation, and phenotypic characterization. Conclusion Analysis of the oral microbiota by rodA-DGGE offers a higher resolution than the conventional DGGE using 16S rDNA and may be an alternative in the microbial diagnosis of streptococcal infection. PMID:21523207

Konishi, Ikuri; Hoshino, Tomonori; Kondo, Yoshio; Saito, Kan; Nishiguchi, Miyuki; Sato, Kyoko; Fujiwara, Taku

2009-01-01

365

The pathogenic potential of different pulsed-field gel electrophoresis types of Listeria monocytogenes strains isolated from food in Northeast Bosnia and Herzegovina.  

PubMed

Listeria monocytogenes is often present in meat and meat products that are sold in the area of northeast Bosnia and Herzegovina. The major objective of this study was to examine the virulence of L. monocytogenes strains isolated from these types of food in that geographic area. Polymerase chain reaction was used to detect eight genes responsible for virulence of this pathogen, namely, prfA, inlA, inlB, hly, plcA, plcB, actA, and mpl. All examined isolates were confirmed to possess the eight virulence genes. Ten different pulsed-field gel electrophoresis (PFGE) macrorestriction profiles were recognized among 19 L. monocytogenes strains after restriction with two different endonucleases (ApaI and AscI). The pathogenicity of three different PFGE types of L. monocytogenes was confirmed through in vivo tests, which were performed on female white mice (Pasteur strain), and it ranged from 3.55 10(8) LD50 to 1.58 10(10) LD50. All of the three different PFGE types of L. monocytogenes were regarded as moderately virulent in relation to the reference strain L. monocytogenes Scott A. This result might be one of the reasons for the absence of reported listeriosis in northeast Bosnia and Herzegovina, despite the high degree of food contamination with this pathogen. PMID:21612523

Hodi?, Snjezana; Huki?, Mirsada; Franciosa, Giovanna; Aureli, Paolo

2011-09-01

366

Use of Pulsed-Field Gel Electrophoresis and Flagellin Gene Typing in Identifying Clonal Groups of Campylobacter jejuni and Campylobacter coli in Farm and Clinical Environments  

PubMed Central

Although campylobacters have been isolated from a wide range of animal hosts, the association between campylobacters isolated from humans and animals in the farm environment is unclear. We used flagellin gene typing and pulsed-field gel electrophoresis (PFGE) to investigate the genetic diversity among isolates from animals (cattle, sheep, and turkey) in farm environments and sporadic cases of campylobacteriosis in the same geographical area. Forty-eight combined fla types were seen among the 315 Campylobacter isolates studied. Six were found in isolates from all four hosts and represented 50% of the total number of isolates. Seventy-one different SmaI PFGE macrorestriction profiles (mrps) were observed, with 86% of isolates assigned to one of 29 different mrps. Fifty-seven isolates from diverse hosts, times, and sources had an identical SmaI mrp and combined fla type. Conversely, a number of genotypes were unique to a particular host. We provide molecular evidence which suggests a link between campylobacters in the farm environment with those causing disease in the community. PMID:11282587

Fitzgerald, Collette; Stanley, Karen; Andrew, Sarah; Jones, Keith

2001-01-01

367

Improved Identification of Rapidly Growing Mycobacteria by a 16S23S Internal Transcribed Spacer Region PCR and Capillary Gel Electrophoresis  

PubMed Central

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

Gray, Timothy J.; Kong, Fanrong; Jelfs, Peter; Sintchenko, Vitali; Chen, Sharon C-A.

2014-01-01

368

A rapid method of species identification of wild chironomids (Diptera: Chironomidae) via electrophoresis of hemoglobin proteins in sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE).  

PubMed

Studying aquatic benthic macroinvertebrates (BMIs) in the field requires accurate taxonomic identification, which can be difficult and time consuming. Conventionally, head capsule morphology has been used to identify wild larvae of Chironomidae. However, due to the number of species and possible damage and/or deformity of their head capsules, another supporting approach for identification is needed. Here, we provide hemoglobin (Hb) protein in hemolymph of chironomids as a new biomarker that may help resolve some of the ambiguities and difficulties encountered during taxonomic identification. Chironomids collected from two locations in Maine and New Jersey, USA were identified to the genus level and in some cases to the species-level using head capsule and body morphologies. The head capsule for a particular individual was then associated with a corresponding Hb protein profile generated from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Distinct Hb profiles were observed from one group (Thienemannimyia) and four genera (Chironomus, Cricotopus, Dicrotendipes, and Glyptotendipes) of chironomids. Several species were polymorphic, having more than one Hb profile and/or having bands of the same size as those of other species. However, major bands and the combination of bands could distinguish individuals at the genus and sometimes species-level. Overall, this study showed that Hb profiles can be used in combination with head capsule morphology to identify wild chironomids. PMID:24923437

Oh, J T; Epler, J H; Bentivegna, C S

2014-10-01

369

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

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

Wang, Yu; Gong, Xueqin; Wang, Shumei; Chen, Lixue; Sun, Li

2014-01-01

370

Evaluation of the genotoxicity induced by the fungicide fenarimol in mammalian and plant cells by use of the single-cell gel electrophoresis assay.  

PubMed

Fenarimol, a systemic pyrimidine carbinol fungicide, is considered to be not genotoxic or weakly genotoxic, although the available toxicological data are controversial and incomplete. Our results obtained in vitro with leukocytes of two different rodent species (rat and mouse) show that fenarimol affects DNA, as detected by the single-cell gel electrophoresis (SCGE, Comet) assay. This fungicide is able to induce DNA damage in a dose-related manner, with significant effectiveness at 36 nM, but without significant interspecies differences. Simultaneous exposure of rat leukocytes to fenarimol (36-290 nM) and a model genotoxic compound (50 microg/ml bleomycin) produced a supra-additive cytotoxic and genotoxic effect. This supports previous findings suggesting possible co-toxic, co-mutagenic, cancer-promoting and co-carcinogenic potential of fenarimol, and modification of the effects of other xenobiotics found to be influenced by this agrotoxic chemical, with consequent different toxicological events. The potential for DNA strand breaks to act as a biomarker of genetic toxicity in plants in vivo was also considered, in view of the fact that higher plants represent reliable sensors in an ecosystem. Significant DNA breakage was observed in the nuclei of Impatiens balsamina leaves after in vivo treatment with fenarimol (145 nM, 1h). More than 50% of the cells showed such DNA damage. PMID:12972058

Poli, P; de Mello, M A; Buschini, A; de Castro, V L S S; Restivo, F M; Rossi, C; Zucchi, T M A D

2003-09-01

371

Genotypic characterization of Salmonella typhi by amplified fragment length polymorphism fingerprinting provides increased discrimination as compared to pulsed-field gel electrophoresis and ribotyping.  

PubMed

Amplified fragment length polymorphism (AFLP) is a recently developed, PCR-based high resolution fingerprinting method that is able to generate complex banding patterns which can be used to delineate intraspecific genetic relationships among bacteria. In the present study, AFLP was evaluated for its usefulness in the molecular typing of Salmonella typhi in comparison to ribotyping and pulsed-field gel electrophoresis (PFGE). Six S. typhi isolates from diverse geographic areas (Malaysia, Indonesia, India, Chile, Papua New Guinea and Switzerland) gave unique, heterogeneous profiles when typed by AFLP, a result which was consistent with ribotyping and PFGE analysis. In a further study of selected S. typhi isolates from Papua New Guinea which caused fatal and non-fatal disease previously shown to be clonally related by PFGE, AFLP discriminated between these isolates but did not indicate a linkage between genotype with virulence. We conclude that AFLP (discriminatory index=0.88) has a higher discriminatory power for strain differentiation among S. typhi than ribotyping (DI=0.63) and PFGE (DI=0.74). PMID:10856775

Nair, S; Schreiber, E; Thong, K L; Pang, T; Altwegg, M

2000-06-01

372

Molecular Epidemiology of Salmonella enterica Serovar Typhimurium Isolates Determined by Pulsed-Field Gel Electrophoresis: Comparison of Isolates from Avian Wildlife, Domestic Animals, and the Environment in Norway  

PubMed Central

The molecular epidemiology of 142 isolates of Salmonella enterica serovar Typhimurium from avian wildlife, domestic animals, and the environment in Norway was investigated using pulsed-field gel electrophoresis (PFGE) and computerized numerical analysis of the data. The bacterial isolates comprised 79 isolates from wild-living birds, including 46 small passerines and 26 gulls, and 63 isolates of nonavian origin, including 50 domestic animals and 13 environmental samples. Thirteen main clusters were discernible at the 90% similarity level. Most of the isolates (83%) were grouped into three main clusters. These were further divided into 20 subclusters at the 95% similarity level. Isolates from passerines, gulls, and pigeons dominated within five subclusters, whereas isolates from domestic animals and the environment belonged to many different subclusters with no predominance. The results support earlier results that passerines constitute an important source of infection to humans in Norway, whereas it is suggested that gulls and pigeons, based on PFGE analysis, represent only a minor source of human serovar Typhimurium infections. Passerines, gulls, and pigeons may also constitute a source of infection of domestic animals and feed plants or vice versa. Three isolates from cattle and a grain source, of which two were multiresistant, were confirmed as serovar Typhimurium phage type DT 104. These represent the first reported phage type DT 104 isolates from other sources than humans in Norway. PMID:12406755

Refsum, Thorbjrn; Heir, Even; Kapperud, Georg; Vardund, Traute; Holstad, Gudmund

2002-01-01

373

Characterisation of E. coli O157 isolates from bovine hide and beef trimming in Irish abattoirs by pulsed field gel electrophoresis.  

PubMed

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

Duffy, G; O'Brien, S B; Carney, E; Sheridan, J J; McDowell, D A; Blair, I S

2005-03-01

374

Single-cell gel electrophoresis assay in the ten spotted live-bearer fish, Cnesterodon decemmaculatus (Jenyns, 1842), as bioassay for agrochemical-induced genotoxicity.  

PubMed

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

Vera-Candioti, Josefina; Soloneski, Sonia; Larramendy, Marcelo L

2013-12-01

375

Molecular Fingerprinting by PCR-Denaturing Gradient Gel Electrophoresis Reveals Differences in the Levels of Microbial Diversity for Musty-Earthy Tainted Corks ?  

PubMed Central

The microbial community structure of cork with marked musty-earthy aromas was analyzed using denaturing gradient gel electrophoresis of amplified ribosomal DNA. Cork stoppers and discs were used for DNA extraction and were analyzed by using selective primers for bacteria and fungi. Stoppers clearly differed from discs harboring a different fungal community. Moreover, musty-earthy samples of both types were shown to have a specific microbiota. The fungi Penicillium glabrum and Neurospora spp. were present in all samples and were assumed to make only a small contribution to off-odor development. In contrast, Penicillium islandicum and Penicillium variabile were found almost exclusively in 2,4,6-trichloroanisole (TCA) tainted discs. Conversely, Rhodotorula minuta and Rhodotorula sloofiae were most common in cork stoppers, where only small amounts of TCA were detected. Alpha- and gammaproteobacteria were the most commonly found bacteria in either control or tainted cork stoppers. Specific Pseudomonas and Actinobacteria were detected in stoppers with low amounts of TCA and 2-methoxy-3,5-dimethylpyrazine. These results are discussed in terms of biological degradation of taint compounds by specific microorganisms. Reliable and straightforward microbial identification methods based on a molecular approach provided useful data to determine and evaluate the risk of taint formation in cork. PMID:19201983

Prat, Chantal; Ruiz-Rueda, Olaya; Trias, Rosalia; Antic, Enriqueta; Capone, Dimitra; Sefton, Mark; Baeras, Llus

2009-01-01

376

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)

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.

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

377

Antimicrobial susceptibility, pulsed-field gel electrophoresis, and multi-locus sequence typing of Campylobacter coli in swine before, during, and after the slaughter process.  

PubMed

The objective of this study was to determine persistence of clonal strains from farm to retail by assessing the clonal relatedness of Campylobacter coli isolated on farm, peri-harvest, and at processing from 11 individually identified pigs. Phenotypic (antimicrobial susceptibility) and genotypic (pulsed field gel electrophoresis [PFGE] and multi-locus sequence typing [MLST]) characterization of isolates was conducted. There was high genetic diversity of Campylobacter isolates from on-farm fecal samples. Campylobacter isolates from farm, post-evisceration, hide, and carcass samples showed similar phenotypes and belonged to the same genotypic clusters based on PFGE and sequence types (STs) based on MLST. Five STs that have not been previously reported were identified (ST-4083, ST-4084, ST-4085, ST-4086, ST-4087). Despite high genotypic diversity of C. coli on farm, retail meat products were consistently contaminated with isolates of the same STs, particularly ST854 and ST1056, as isolates collected from previous stages confirming persistence of strains from pre- to post-harvest. PMID:22663186

Abley, Melanie J; Wittum, Thomas E; Funk, Julie A; Gebreyes, Wondwossen A

2012-06-01

378

Genetic Characterization of Atypical Enteropathogenic Escherichia coli Isolates from Ewes' Milk, Sheep Farm Environments, and Humans by Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis  

PubMed Central

A collection of 81 isolates of enteropathogenic Escherichia coli (EPEC) was obtained from samples of bulk tank sheep milk (62 isolates), ovine feces (4 isolates), sheep farm environment (water, 4 isolates; air, 1 isolate), and human stool samples (9 isolates). The strains were considered atypical EPEC organisms, carrying the eae gene without harboring the pEAF plasmid. Multilocus sequence typing (MLST) was carried out with seven housekeeping genes and 19 sequence types (ST) were detected, with none of them having been previously reported for atypical EPEC. The most frequent ST included 41 strains isolated from milk and human stool samples. Genetic typing by pulsed-field gel electrophoresis (PFGE) resulted in 57 patterns which grouped in 24 clusters. Comparison of strains isolated from the different samples showed phylogenetic relationships between milk and human isolates and also between milk and water isolates. The results obtained show a possible risk for humans due to the presence of atypical EPEC in ewes' milk and suggest a transmission route for this emerging pathogen through contaminated water. PMID:23872571

Otero, Vernica; Rodrguez-Calleja, Jos-Mara; Otero, Andrs; Garca-Lpez, Mara-Luisa

2013-01-01

379

Stability of Related Human and Chicken Campylobacter jejuni Genotypes after Passage through Chick Intestine Studied by Pulsed-Field Gel Electrophoresis  

PubMed Central

The genomic stability of 12 Campylobacter jejuni strains consisting of two groups of human and chicken isolates was studied by analysis of their PFGE (pulsed-field gel electrophoresis) patterns after passage through newly hatched chicks intestines. The patterns of SmaI, SalI, and SacII digests remained stable after intestinal passage, except for those of two strains. One originally human strain, FB 6371, changed its genotype from II/A (SmaI/SacII) to I/B. Another strain, BTI, originally isolated from a chicken, changed its genotype from I/B to a new genotype. The genomic instability of the strains was further confirmed by SalI digestion and ribotyping of the HaeIII digests. In addition, heat-stable serotype 57 of strain FB 6371 changed to serotype 27 in all isolates with new genotypes but remained unchanged in an isolate with the original genotype. Serotype 27 of strain BTI remained stable. Our study suggests that during intestinal colonization, genomic rearrangement, as demonstrated by changed PFGE and ribopatterns, may occur. PMID:10224037

Hnninen, Marja-Liisa; Hakkinen, Marjaana; Rautelin, Hilpi

1999-01-01

380

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

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

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

381

Denaturing gradient gel electrophoresis for nonlethal detection of Aeromonas salmonicida in salmonid mucus and its potential for other bacterial fish pathogens.  

PubMed

Denaturing gradient gel electrophoresis (DGGE) of 16S rDNA was used to nonlethally detect Aeromonas salmonicida and other bacteria in salmonid skin mucus. Mucus samples from wild spawning coho salmon (Oncorhynchus kisutch) with endemic A. salmonicida and from cultured lake trout (Salvelinus namaycush) were tested by PCR-DGGE and were compared with mucus culture on Coomassie brilliant blue agar and internal organ culture. PCR-DGGE gave a highly reproducible 4-band pattern for 9 strains of typical A. salmonicida, which was different from other Aeromonas spp. Aeromonas salmonicida presence in mucus was evident as a band that comigrated with the bottom band of the A. salmonicida 4-band pattern and was verified by sequencing. PCR-DGGE found 36 of 52 coho salmon positive for A. salmonicida, compared with 31 positive by mucus culture and 16 by organ culture. Numerous other bacteria were detected in salmonid mucus, including Pseudomonas spp., Shewanella putrefaciens, Aeromonas hydrophila and other aeromonads. However, Yersinia ruckeri was not detected in mucus from 27 lake trout, but 1 fish had a sorbitol-positive Y. ruckeri isolated from organ culture. Yersinia ruckeri seeded into a mucus sample suggested that PCR-DGGE detection of this bacterium from mucus was possible. PCR-DGGE allows nonlethal detection of A. salmonicida in mucus and differentiation of some Aeromonas spp. and has the potential to allow simultaneous detection of other pathogens present in fish mucus. PMID:22506865

Quinn, Robert A; Stevenson, Roselynn M W

2012-05-01

382

Use of pulsed-field gel electrophoresis typing to study an outbreak of infection due to Serratia marcescens in a neonatal intensive care unit.  

PubMed Central

Serratia marcescens is a well-known cause of nosocomial infections and outbreaks, particularly in critically ill neonates and immunocompromised patients. Numerous methods have been proposed for typing. We used pulsed-field gel electrophoresis (PFGE) typing to analyze an outbreak in a neonatal intensive care unit (NICU). We included 23 patient isolates from an outbreak (March to July 1995), and 10 patient isolates from different wards during the same time period. PFGE of whole-cell DNA digested by SpeI was used as a marker of strain identity. The most common presentation of the infection was sepsis in 18 of 23 (78%) neonates. Only four different biotypes were identified; biotype A8d accounted for 84% of the strains. PFGE typing revealed two clones responsible for two different clonal strain dissemination outbreaks from March to July, with 24 patient isolates being pattern A and 4 patient isolates being pattern E. PFGE typing suggests cross transmission between patients in the NICU and other wards. The isolates from 5 other patients showed distinct PFGE patterns. Extensive investigation and cultures failed to identify any environmental or staff reservoir of S. marcescens. This is one of the first reports applying PFGE to the study of S. marcescens, and this method was a useful marker of strain identity. PFGE typing distinguished strains which appeared to be the same by biotyping. PMID:8940460

Miranda, G; Kelly, C; Solorzano, F; Leanos, B; Coria, R; Patterson, J E

1996-01-01

383

Comparative typing of Pseudomonas aeruginosa by random amplification of polymorphic DNA or pulsed-field gel electrophoresis of DNA macrorestriction fragments.  

PubMed Central

Eighty-seven strains of Pseudomonas aeruginosa were typed by random amplification of polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments. Stains were clustered on the basis of interpretative criteria as presented previously for the PFGE analysis. Clusters of strains were also defined on the basis of epidemiological data and subsequently reanalyzed by RAPD. It was found that in an RAPD assay employing the enterobacterial repetitive intergenic consensus sequence ERIC2 as a primer, single band differences can be ignored; in this case, clonally related strains could be grouped as effectively and reliably as with PFGE. These data could be corroborated by the use of other primer species. However, some primers either showed reduced resolution or, in contrast, identified DNA polymorphisms beyond epidemiologically and PFGE-defined limits. Apparently, different primers define different windows of genetic variation. It is suggested that criteria for interpretation of the ERIC2 PCR fingerprints can be simple and straightforward: when single band differences are ignored, RAPD-determined grouping of P. aeruginosa is congruent with that obtained by PFGE. Consequently, this implies that RAPD can be used with trust as a first screen in epidemiological characterization of P. aeruginosa. The ability to measure the rate of molecular evolution of the P. aeruginosa genome clearly depends on the choice of restriction enzyme or primer when RAPD or PFGE, respectively, is applied for the detection of DNA polymorphisms. PMID:8940470

Renders, N; Rmling, Y; Verbrugh, H; van Belkum, A

1996-01-01

384

Comprehensive analysis of collagen metabolism in vitro using (4(/sup 3/H))/(/sup 14/C)proline dual-labeling and polyacrylamide gel electrophoresis  

SciTech Connect

A method to simultaneously quantify the production, secretion, and prolyl hydroxylation of individual types of collagen in cell culture samples has been developed. Collagens were biosynthetically labeled with a mixture of (/sup 14/C)proline and (4-/sup 3/H)proline. The labeled collagens were isolated and their component alpha-chains were resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Migration of the collagen alpha-chains was determined by fluorography, and radioactivity in excised bands was quantified by scintillation counting. (/sup 14/C)Proline labeling of collagen chains was used to determine the production and secretion of the different types of collagen. The ratios of the component alpha 1(I) and alpha 2(I) chains of type I collagen were also determined in this way. Prolyl hydroxylation of collagen alpha-chains was readily determined by measurement of their /sup 3/H:/sup 14/C ratios. Following 4-hydroxylation, /sup 3/H was lost from the (4-3H)proline with alteration of this ratio. This dual-labeling method is suitable for the comprehensive analysis of collagen metabolism in multiple samples.

Bateman, J.F.; Harley, V.; Chan, D.; Cole, W.G.

1988-01-01

385

Comparison of Protein A Gene Sequencing with Pulsed-Field Gel Electrophoresis and Epidemiologic Data for Molecular Typing of Methicillin-Resistant Staphylococcus aureus  

PubMed Central

The epidemiologic relatedness of methicillin-resistant Staphylococcus aureus (MRSA) isolates is currently determined by analysis of chromosomal DNA restriction patterns by pulsed-field gel electrophoresis (PFGE). We have evaluated an alternative typing system (MicroSeq StaphTrack Kit; Perkin-Elmer Biosystems) based on the sequence analysis of the chromosomally encoded polymorphic repeat X region of the S. aureus protein A (spa) gene. A total of 69 clinical MRSA isolates were divided into 18 groups according to the number and nucleotide sequences of the spa repeats. Molecular typing results obtained both by spa sequencing and from the PFGE patterns were concordant except for one group, which contained 20 isolates recovered over a 2-year period from hospitalized patients at the Mayo Clinic. Although the spa typing patterns were indistinguishable for those isolates, PFGE analysis yielded seven related but distinguishable patterns. Further coagulase gene sequence analysis subtyped those 20 strains into four groups which followed distinct temporal and geographic distributions. During a 2-year epidemic period there were up to 7 fragment changes in PFGE patterns among epidemiologically related isolates, suggesting that PFGE may be unsuitable for long-term typing of strains involved in epidemics. Although more limited than PFGE in discriminatory power, spa sequencing analysis could be used as a screening method for typing of MRSA strains because of the shorter turnaround time, ease of use, and the inherent advantages of sequence analysis, storage, and sharing of information. PMID:10747105

Tang, Yi-Wei; Waddington, Michael G.; Smith, Douglas H.; Manahan, Janice M.; Kohner, Peggy C.; Highsmith, Leanne M.; Li, Haijing; Cockerill, Franklin R.; Thompson, Rodney L.; Montgomery, Stacy O.; Persing, David H.

2000-01-01

386

Culture-based and denaturing gradient gel electrophoresis analysis of the bacterial community structure from the intestinal tracts of earthworms(Eisenia fetida).  

PubMed

The bacterial communities in the intestinal tracts of earthworm were investigated by culture-dependent and - independent approaches. In total, 72 and 55 pure cultures were isolated from the intestinal tracts of earthworms under aerobic and anaerobic conditions, respectively. Aerobic bacteria were classified as Aeromonas (40%), Bacillus (37%), Photobacterium (10%), Pseudomonas (7%), and Shewanella (6%). Anaerobic bacteria were classified as Aeromonas (52%), Bacillus (27%), Shewanella (12%), Paenibacillus (5%), Clostridium (2%), and Cellulosimicrobium (2%). The dominant microorganisms were Aeromonas and Bacillus species under both aerobic and anaerobic conditions. In all, 39 DNA fragments were identified by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis. Aeromonas sp. was the dominant microorganism in feeds, intestinal tracts, and casts of earthworms. The DGGE band intensity of Aeromonas from feeds, intestinal tracts, and casts of earthworms was 12.8%, 14.7%, and 15.1%, respectively. The other strains identified were Bacillus, Clostridium, Enterobacter, Photobacterium, Pseudomonas, Shewanella, Streptomyces, uncultured Chloroflexi bacterium, and uncultured bacterium. These results suggest that PCR-DGGE analysis was more efficient than the culture-dependent approach for the investigation of bacterial diversity and the identification of unculturable microorganisms. PMID:21952364

Hong, Sung Wook; Kim, In Su; Lee, Ju Sam; Chung, Kun Sub

2011-09-01

387

Validation of Pulsed-Field Gel Electrophoresis and spa Typing for Long-Term, Nationwide Epidemiological Surveillance Studies of Staphylococcus aureus Infections?  

PubMed Central

Pulsed-field gel electrophoresis (PFGE) of genomic macrorestriction fragments has been used by the Belgian Reference Laboratory for Staphylococci for national hospital surveys of methicillin-resistant Staphylococcus aureus since 1992. The sequencing of the polymorphic X region of the protein A gene (spa typing) offers significant advantages over PFGE in terms of speed, ease of interpretation, and exportability. To validate its potential use for national surveillance, we evaluated the robustness of spa typing compared with that of PFGE based on a collection of 217 S. aureus strains representative of the Belgian S. aureus epidemiology during the last 13 years. spa typing and PFGE both showed high discriminatory power (discriminatory indexes of 0.98 and 0.96, respectively) and achieved high concordance (95.9%) in type classification. Both methods also showed good concordance with multilocus sequence typing (MLST) (95.5%). However, we observed occasional violations of MLST clonal complex assignment by spa typing. Our results suggest that both PFGE and spa typing are reliable methods for long-term, nationwide epidemiological surveillance studies. We suggest that spa typing, which is a single-locus-based method, should preferably be used in combination with additional markers, such as staphylococcal cassette chromosome mec typing or resistance or virulence gene detection. PMID:17093021

Hallin, M.; Deplano, A.; Denis, O.; De Mendona, R.; De Ryck, R.; Struelens, M. J.

2007-01-01

388

Comparison of Staphylococcus aureus Isolates from Bovine and Human Skin, Milking Equipment, and Bovine Milk by Phage Typing, Pulsed-Field Gel Electrophoresis, and Binary Typing  

PubMed Central

Staphylococcus aureus isolates (n = 225) from bovine teat skin, human skin, milking equipment, and bovine milk were fingerprinted by pulsed-field gel electrophoresis (PFGE). Strains were compared to assess the role of skin and milking equipment as sources of S. aureus mastitis. PFGE of SmaI-digested genomic DNA identified 24 main types and 17 subtypes among isolates from 43 herds and discriminated between isolates from bovine teat skin and milk. Earlier, phage typing (L. K. Fox, M. Gershmann, D. D. Hancock, and C. T. Hutton, Cornell Vet. 81:183-193, 1991) had failed to discriminate between isolates from skin and milk. Skin isolates from humans belonged to the same pulsotypes as skin isolates from cows. Milking equipment harbored strains from skin as well as strains from milk. We conclude that S. aureus strains from skin and from milk can both be transmitted via the milking machine, but that skin strains are not an important source of intramammary S. aureus infections in dairy cows. A subset of 142 isolates was characterized by binary typing with DNA probes developed for typing of human S. aureus. Typeability and overall concordance with epidemiological data were lower for binary typing than for PFGE while discriminatory powers were similar. Within several PFGE types, binary typing discriminated between main types and subtypes and between isolates from different herds or sources. Thus, binary typing is not suitable as replacement for PFGE but may be useful in combination with PFGE to refine strain differentiation. PMID:12409348

Zadoks, R. N.; van Leeuwen, W. B.; Kreft, D.; Fox, L. K.; Barkema, H. W.; Schukken, Y. H.; van Belkum, A.

2002-01-01

389

Stage-specific analysis of plasma protein profiles in ovarian cancer: Difference in-gel electrophoresis analysis of pooled clinical samples  

PubMed Central

Introduction: Ovarian cancer is the leading cause of death from gynecological cancer. Non-specific symptoms early in disease and the lack of specific biomarkers hinder early diagnosis. Multi-marker blood screening tests have shown promise for improving identification of early stage disease; however, available tests lack sensitivity, and specificity. Materials and Methods: In this study, pooled deeply-depleted plasma from women with Stage 1, 2 or 3 ovarian cancer and healthy controls were used to compare the 2-dimensional gel electrophoresis (2-DE) protein profiles and identify potential novel markers of ovarian cancer progression. Results/Discussion: Stage-specific variation in biomarker expression was observed. For example, apolipoprotein A1 expression is relatively low in control and Stage 1, but shows a substantial increase in Stage 2 and 3, thus, potential of utility for disease confirmation rather than early detection. A better marker for early stage disease was tropomyosin 4 (TPM4). The expression of TPM4 increased by 2-fold in Stage 2 before returning to normal levels in Stage 3 disease. Multiple isoforms were also identified for some proteins and in some cases, displayed stage-specific expression. An interesting example was fibrinogen alpha, for which 8 isoforms were identified. Four displayed a moderate increase at Stage 1 and a substantial increase for Stages 2 and 3 while the other 4 showed only moderate increases. Conclusion: Herein is provided an improved summary of blood protein profiles for women with ovarian cancer stratified by stage. PMID:23858298

Bailey, Mark J.; Shield-Artin, Kristy L.; Oliva, Karen; Ayhan, Mustafa; Reisman, Simone; Rice, Gregory E.

2013-01-01

390

High Genetic Diversity of Enterococcus faecium and Enterococcus faecalis Clinical Isolates by Pulsed-Field Gel Electrophoresis and Multilocus Sequence Typing from a Hospital in Malaysia  

PubMed Central

Little is known on the genetic relatedness and potential dissemination of particular enterococcal clones in Malaysia. We studied the antibiotic susceptibility profiles of Enterococcus faecium and Enterococcus faecalis and subjected them to pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). E. faecium and E. faecalis displayed 27 and 30 pulsotypes, respectively, and 10 representative E. faecium and E. faecalis isolates (five each) yielded few different sequence types (STs): ST17 (2 isolates), ST78, ST203, and ST601 for E. faecium, and ST6, ST16, ST28, ST179, and ST399 for E. faecalis. Resistance to tazobactam-piperacillin and ampicillin amongst E. faecium isolates was highly observed as compared to E. faecalis isolates. All of the isolates were sensitive to vancomycin and teicoplanin. The presence of epidemic and nosocomial strains of selected E. faecium STs: 17, 78, and 203 and E. faecalis ST6 as well as high rates of resistance to multiple antibiotics amongst E. faecium isolates is of a particular concern. PMID:23819125

Weng, Poh Leng; Ramli, Ramliza; Shamsudin, Mariana Nor; Cheah, Yoke-Kqueen; Hamat, Rukman Awang

2013-01-01

391

Solid-phase microextraction coupled with capillary electrophoresis to determine ephedrine derivatives in water and urine using a sol-gel derived butyl methacrylate/silicone fiber.  

PubMed

A sensitive method for determination of ephedrine derivatives using headspace solid-phase microextraction (SPME) with a novel fiber followed by capillary electrophoresis has been developed. The co-poly(butyl methacrylate/hydroxy-terminated silicone oil) (BMA/OH-TSO) was used as stationary phases with the aid of gamma-methacryloxypropyltrimethoxysilane (KH-570) as bridge in SPME using sol-gel-coating method and cross-linking technology. It has high extraction efficiency for ephedrine derivatives in comparison with commercial poly(dimethylsiloxane) and poly(acrylate)-coated fiber. The coating exhibits good thermal and solvent stability as well as long lifetime. A simple and flexible device for desorption of analytes after headspace SPME was constructed. The effect of various experimental parameters for SPME (temperature, time, pH, ionic strength, desorption solvent, etc.) were discussed. Field amplified sample injection (FASI) was applied for on-line sample concentration and a sensitivity enhancement of two orders of magnitude was achieved. Linear ranges were found to be 20-5000 ng/ml. The detection limits for (1R,2S)-ephedrine, (1R,2R)-pseudoephedrine and (1S,2S)-pseudoephedrine were 3, 5 and 5ng/ml, respectively. Relative standard deviation (n=6) was found to be 4.96-7.57%. The method was successfully applied to the analysis of ephedrine derivatives in human urine. PMID:18970420

Fang, Huaifang; Liu, Mingming; Zeng, Zhaorui

2006-01-15

392

Combined Use of a Solid-Phase Hexapeptide Ligand Library with Liquid Chromatography and Two-Dimensional Difference Gel Electrophoresis for Intact Plasma Proteomics  

PubMed Central

The intact plasma proteome is of great interest in biomarker studies because intact proteins reflect posttranslational protein processing such as phosphorylation that may correspond to disease status. We examined the utility of a solid-phase hexapeptide ligand library in combination with conventional plasma proteomics modalities for comprehensive profiling of intact plasma proteins. Plasma proteins were sequentially fractionated using depletion columns for albumin and immunoglobulin, and separated using an anion-exchange column. Proteins in each fraction were treated with a solid-phase hexapeptide ligand library and compared to those without treatment. Two-dimensional difference gel electrophoresis demonstrated an increased number of protein spots in the treated samples. Mass spectrometric studies of these protein spots with unique intensity in the treated samples resulted in the identification of high- and medium-abundance proteins. Our results demonstrated the possible utility of a solid-phase hexapeptide ligand library to reveal greater number of intact plasma proteins. The characteristics of proteins with unique affinity to the library remain to be clarified by more extensive mass spectrometric protein identification, and optimized protocols should be established for large-scale plasma biomarker studies. PMID:22389768

Hagiwara, Tatsuo; Saito, Yumi; Nakamura, Yukiko; Tomonaga, Takeshi; Murakami, Yasufumi; Kondo, Tadashi

2011-01-01

393

Porcine salivary analysis by 2-dimensional gel electrophoresis in 3 models of acute stress: A pilot study  

PubMed Central

The purpose of this research was to study changes in the salivary proteome of healthy pigs in stressful situations to identify any potential new salivary biomarker of stress. Three groups of animals were subjected to 3 stress models: snaring restraint followed by simulated sampling of vena cava blood; brief transport by road; and restriction of movement in a digestibility cage. Saliva was obtained from each animal before and 15 and 30 min after the induction of stress. The samples from the animals that showed the greatest increase in salivary cortisol concentration were pooled and run on 2-dimensional gels. Coomassie Brilliant Blue R-250 was used for spot detection and mass spectrometry for spot identification. Statistical analyses showed that 2 proteins had significant differences in expression before and after the induction of stress. These proteins were identified as odorant-binding protein and fragments of albumin. Further studies will be necessary to confirm the value of using these proteins as salivary biomarkers of stress in pigs. PMID:24688174

Fuentes-Rubio, Mara; Cern, Jos J.; de Torre, Carlos; Escribano, Damin; Gutirrez, Ana M.; Tecles, Fernando

2014-01-01

394

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

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

GEORGE A. KOWALCHUK; JOHN R. STEPHEN; WIETSE DE BOER; JIM I. PROSSER; T. MARTIN EMBLEY; ANDJAN W. WOLDENDORP

1997-01-01

395

Proteomic analysis of Acinetobacter lwoffii K24 by 2-D gel electrophoresis and electrospray ionization quadrupole-time of flight mass spectrometry  

Microsoft Academic Search

The MS\\/MS analysis by Electrospray ionization quadrupole-time of flight mass spectrometry (ESI-Q-TOF MS) was applied to identify proteins in proteome analysis of bacteria whose genomes are not known. The protein identification by ESI-Q-TOF MS was performed sequentially by database search and then de novo sequencing using MS\\/MS spectra. Soil bacteria having unanalyzed genome, Acinetobacter lwoffii K24 is an aniline degrading

Eun-A Kim; Jin Young Kim; Soo-Jung Kim; Kyeong Ryang Park; Hae-Jin Chung; Sun-Hee Leem; Seung Il Kim

2004-01-01

396

DNA double-strand breaks in mammalian cells exposed to gamma-rays and very heavy ions. Fragment-size distributions determined by pulsed-field gel electrophoresis.  

PubMed

The spatial distribution of DNA double-strand breaks (DSB) was assessed after treatment of mammalian cells (V79) with densely ionizing radiation. Cells were exposed to beams of heavy charged particles (calcium ions: 6.9 MeV/u, 2.1.10(3) keV/microm; uranium ions: 9.0 MeV/u, 1.4.10(4) keV/microm) at the linear accelerator UNILAC of GSI, Darmstadt. DNA was isolated in agarose plugs and subjected to pulsed-field gel electrophoresis under conditions that separated DNA fragments of size 50 kbp to 5 Mbp. The measured fragment distributions were compared to those obtained after gamma-irradiation and were analyzed by means of a convolution and a deconvolution technique. In contrast to the finding for gamma-radiation, the distributions produced by heavy ions do not correspond to the random breakage model. Their marked overdispersion and the observed excess of short fragments reflect spatial clustering of DSB that extends over large regions of the DNA, up to several mega base pairs (Mbp). At fluences of 0.75 and 1.5/microm2, calcium ions produce nearly the same shape of fragment spectrum, merely with a difference in the amount of DNA entering the gel; this suggests that the DNA is fragmented by individual calcium ions. At a fluence of 0.8/microm2 uranium ions produce a profile that is shifted to smaller fragment sizes in comparison to the profile obtained at a fluence of 0.4/microm2; this suggests cumulative action of two separate ions in the formation of fragments. These observations are not consistent with the expectation that the uranium ions, with their much larger LET, should be more likely to produce single particle action than the calcium ions. However, a consideration of the greater lateral extension of the tracks of the faster uranium ions explains the observed differences; it suggests that the DNA is closely coiled so that even DNA locations several Mbp apart are usually not separated by less than 0. 1 or 0.2 microm. PMID:9728743

Kraxenberger, F; Weber, K J; Friedl, A A; Eckardt-Schupp, F; Flentje, M; Quicken, P; Kellerer, A M

1998-07-01

397

Bacterioplankton community structure in a maritime antarctic oligotrophic lake during a period of holomixis, as determined by denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH).  

PubMed

The bacterioplankton community structure in Moss Lake, a maritime Antarctic oligotrophic lake, was determined with vertical depth in the water column, during the ice-free period on Signy Island in the South Orkney Islands. Bacterioplankton community structure was determined using a combination of direct counting of 4',6-diamidino-2-phenylindole (DAPI) stained cells, PCR amplification of 16S rRNA gene fragments, denaturing gradient gel electrophoresis (DGGE) and in situ hybridization with group-specific, fluorescently labeled oligonucleotide probes. Using PCR amplification of 16S rRNA gene fragments and DGGE, the bacterioplankton community composition was shown to be constant with vertical depth in the water column. Specific bacterioplankton species identified through cloning and sequencing the DGGE products obtained were Flavobacterium xinjiangensis (a Flavobacterium), Leptothrix discophora (a beta-Proteobacterium), and a number of uncultured groups: two beta-Proteobacteria, an unclassified Proteobacterium, three sequences from Actinobacteria, and a Cyanobacterium. Fluorescence in situ hybridization (FISH), however, demonstrated that there were minor but significant fluctuations in different groups of bacteria with vertical depth in the water column. It showed that the beta-Proteobacteria accounted for between 26.4 and 71.5%, the alpha-Proteobacteria 2.3-10.6%, the gamma-Proteobacteria 0-29.6%, and the Cytophaga-Flavobacterium group 1.8-23.5% of cells hybridizing to a universal probe. This study reports the first description of the community structure of an oligotrophic Antarctic freshwater lake as determined by PCR-dependent and PCR-independent molecular techniques. It also suggests that the bacterioplankton community of Moss Lake contains classes of bacteria known to be important in freshwater systems elsewhere in the world. PMID:12739078

Pearce, D A

2003-07-01

398

Analysis of ventilator-associated pneumonia infection route by genome macrorestriction-pulsed-field gel electrophoresis and its prevention with combined nursing strategies  

PubMed Central

The aim of the present study was to explore the infection route of ventilator-associated pneumonia (VAP) and assess the effectiveness of a combined nursing strategy to prevent VAP in intensive care units. Bacteria from the gastric juice and drainage from the hypolarynx and lower respiratory tracts of patients with VAP were analyzed using genome macrorestriction-pulsed-field gel electrophoresis (GM-PFGE). A total of 124 patients with tracheal intubation were placed in the intervention group and were treated with a combined nursing strategy, comprising mosapride (gastric motility stimulant) administration and semi-reclining positioning. A total of 112 intubated patients were placed in the control group and received routine nursing care. The incidence rate of VAP, days of ventilation and mortality rate of patients were compared between the two groups. The GM-PFGE fingerprinting results of three strains of Pseudomonas aeruginosa from the gastric juice, subglottic secretion drainage and drainage of the lower respiratory tract in patients with VAP were similar across groups. The number of days spent on a ventilator by patients in the intervention group (7.375.32 days) was lower compared with that by patients in the control group (12.344.98 days) (P<0.05). The incidence rate of VAP was reduced from 40.81 to 21.25% following intervention with the combined nursing strategy (P<0.05); furthermore, the mortality rate of intubated patients in the intervention group was 29.46%, a significant reduction compared with the 41.94% mortality rate observed in the control group (P<0.05). Gastroesophageal reflux (GER) was confirmed as one of the infection routes for VAP. The combined nursing strategy of gastric motility stimulant administration and the adoption of a semi-reclining position was effective in preventing VAP by reducing the occurrence of GER. PMID:25371757

WANG, XIAODONG; WANG, JUNPING; LI, JING; WANG, JING

2014-01-01

399

Simultaneous analysis of multiple enzymes increases accuracy of pulsed-field gel electrophoresis in assigning genetic relationships among homogeneous Salmonella strains.  

PubMed

Due to a highly homogeneous genetic composition, the subtyping of Salmonella enterica serovar Enteritidis strains to an epidemiologically relevant level remains intangible for pulsed-field gel electrophoresis (PFGE). We reported previously on a highly discriminatory PFGE-based subtyping scheme for S. enterica serovar Enteritidis that relies on a single combined cluster analysis of multiple restriction enzymes. However, the ability of a subtyping method to correctly infer genetic relatedness among outbreak strains is also essential for effective molecular epidemiological traceback. In this study, genetic and phylogenetic analyses were performed to assess whether concatenated enzyme methods can cluster closely related salmonellae into epidemiologically relevant hierarchies. PFGE profiles were generated by use of six restriction enzymes (XbaI, BlnI, SpeI, SfiI, PacI, and NotI) for 74 strains each of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium. Correlation analysis of Dice similarity coefficients for all pairwise strain comparisons underscored the importance of combining multiple enzymes for the accurate assignment of genetic relatedness among Salmonella strains. The mean correlation increased from 81% and 41% for single-enzyme PFGE up to 99% and 96% for five-enzyme combined PFGE for S. enterica serovar Enteritidis and S. enterica serovar Typhimurium strains, respectively. Data regressions approached 100% correlation among Dice similarities for S. enterica serovar Enteritidis and S. enterica serovar Typhimurium strains when a minimum of six enzymes were concatenated. Phylogenetic congruence measures singled out XbaI, BlnI, SfiI, and PacI as most concordant for S. enterica serovar Enteritidis, while XbaI, BlnI, and SpeI were most concordant among S. enterica serovar Typhimurium strains. Together, these data indicate that PFGE coupled with sufficient enzyme numbers and combinations is capable of discerning accurate genetic relationships among Salmonella serovars comprising highly homogeneous strain complexes. PMID:20980570

Zheng, Jie; Keys, Christine E; Zhao, Shaohua; Ahmed, Rafiq; Meng, Jianghong; Brown, Eric W

2011-01-01

400

Detection and characterization of fungal infections of Ammophila arenaria (marram grass) roots by denaturing gradient gel electrophoresis of specifically amplified 18s rDNA.  

PubMed Central

Marram grass (Ammophila arenaria L.), a sand-stabilizing plant species in coastal dune areas, is affected by a specific pathosystem thought to include both plant-pathogenic fungi and nematodes. To study the fungal component of this pathosystem, we developed a method for the cultivation-independent detection and characterization of fungi infecting plant roots based on denaturing gradient gel electrophoresis (DGGE) of specifically amplified DNA fragments coding for 18S rRNA (rDNA). A nested PCR strategy was employed to amplify a 569-bp region of the 18S rRNA gene, with the addition of a 36-bp GC clamp, from fungal isolates, from roots of test plants infected in the laboratory, and from field samples of marram grass roots from both healthy and degenerating stands from coastal dunes in The Netherlands. PCR products from fungal isolates were subjected to DGGE to examine the variation seen both between different fungal taxa and within a single species. DGGE of the 18S rDNA fragments could resolve species differences from fungi used in this study yet was unable to discriminate between strains of a single species. The 18S rRNA genes from 20 isolates of fungal species previously recovered from A. arenaria roots were cloned and partially sequenced to aid in the interpretation of DGGE data. DGGE patterns recovered from laboratory plants showed that this technique could reliably identify known plant-infecting fungi. Amplification products from field A. arenaria roots also were analyzed by DGGE, and the major bands were excised, reamplified, sequenced, and subjected to phylogenetic analysis. Some recovered 18S rDNA sequences allowed for phylogenetic placement to the genus level, whereas other sequences were not closely related to known fungal 18S rDNA sequences. The molecular data presented here reveal fungal diversity not detected in previous culture-based surveys. PMID:9327549

Kowalchuk, G A; Gerards, S; Woldendorp, J W

1997-01-01

401

Bacterial Community Composition in Central European Running Waters Examined by Temperature Gradient Gel Electrophoresis and Sequence Analysis of 16S rRNA Genes?  

PubMed Central

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

Beier, Sara; Witzel, Karl-Paul; Marxsen, Jrgen

2008-01-01

402

Detection of in vivo genotoxicity of haloalkanes and haloalkenes carcinogenic to rodents by the alkaline single cell gel electrophoresis (comet) assay in multiple mouse organs.  

PubMed

The micronucleus test is widely used to assess in vivo clastogenicity because of its convenience, but it is not appropriate for some carcinogenic chemical classes. Halogenated compounds, for example, are inconsistent micronucleus inducers. We assessed the genotoxicity of 7 haloalkanes and haloalkenes carcinogenic to rodents in 7 mouse organs-stomach, liver, kidney, bladder, lung, brain, and bone marrow-using the alkaline single cell gel electrophoresis (SCG) assay. The carcinogens we studied were 1, 2-dibromo-3-chloropropane (DBCP), 1,3-dichloropropene (mixture of cis and trans) (DCP), 1,2-dibromoethane (EDB), 1,2-dichloroethane (EDC), vinyl bromide, dichloromethane, and carbon tetrachloride; only DBCP induces micronuclei in mouse bone marrow. Except for carbon tetrachloride, halocompounds studied are mutagenic to Salmonella typhimurium. Mice were sacrificed 3 or 24 h after carcinogen administration. DCP and EDC induced DNA damage in all of the organs studied. Vinyl bromide yielded DNA damage in all of the organs except for bone marrow. DBCP induced DNA damage in the stomach, liver, kidney, lung, and bone marrow; EDB in the stomach, liver, kidney, bladder, and lung; and dichloromethane in the liver and lung. Since no deaths, morbidity, clinical signs, organ pathology, or microscopic signs of necrosis were observed, the DNA damage was not attributable to cytotoxicity. On the other hand, the positive response in the liver induced by carbon tetrachloride, which was accompanied by necrosis, was considered to be a false positive response. We suggest that the alkaline SCG assay can be used in multiple organs to detect in vivo genotoxicity that is not expressed in bone marrow cells in mice given non-necrogenic doses of halocompounds. PMID:9804871

Sasaki, Y F; Saga, A; Akasaka, M; Ishibashi, S; Yoshida, K; Su, Y Q; Matsusaka, N; Tsuda, S

1998-11-01

403

A study of salivary lactate dehydrogenase isoenzyme levels in patients with oral leukoplakia and squamous cell carcinoma by gel electrophoresis method  

PubMed Central

Context: The enzyme lactate dehydrogenase (LDH), which is found in almost all the cells of body tissues, can be separated into five fractions and the isoenzyme pattern is believed to vary according to the metabolic requirement of each tissue. LDH concentration in saliva, as an expression of cellular necrosis, could be considered to be a specific indicator for oral lesions that affect the integrity of the oral mucosa. Aim: The present study was designed to evaluate salivary LDH isoenzyme pattern in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) and to correlate between LDH isoenzyme levels and histopathologic grading in selected cases of OL and OSCC. Materials and Methods: Clinically diagnosed 30 cases each of OL and OSCC were selected for the study and 30 healthy individuals of comparable age served as control. Unstimulated whole saliva was aseptically collected and was processed immediately for LDH isoenzymes measurement by agarose gel electrophoresis. Biopsy specimen obtained was processed and stained by hematoxylin and eosin. Sections of OL and OSCC cases were scrutinized histopathologically and appropriately graded for epithelial dysplasia and differentiation of carcinoma respectively. Statistical Analysis Used: Two sample t test for testing the significance of difference between two group means was used. Results and Conclusion: The present salivary analysis for LDH isoenzyme reveals an overall increased salivary LDH isoenzyme level in OL and OSCC cases and a significant correlation between levels of salivary LDH isoenzymes and histopathologic grades of dysplasia in OL and OSCC. Salivary analysis of LDH will definitely provide the clinician and/or the patient himself with an efficient, non invasive and friendly new tool for diagnosis and monitoring of oral precancer and cancer. PMID:25364177

Joshi, Priya Shirish; Golgire, Someshwar

2014-01-01

404

Distribution of Campylobacter jejuni isolates from turkey farms and different stages at slaughter using pulsed-field gel electrophoresis and flaA-short variable region sequencing.  

PubMed

The aim of this study was to assess the diversity of thermotolerant Campylobacter spp. isolated from turkey flocks at six rearing farms 1-2 weeks prior to slaughter (360 faecal swab samples) and from 11 different stages at the slaughterhouse (636 caecal, environmental, neck skin and meat samples). A total of 121 Campylobacter isolates were identified to species level using a multiplex PCR assay and were typed by pulsed-field gel electrophoresis (PFGE) and flaA-short variable region (SVR) sequencing. All Campylobacter isolates were identified as Campylobacter jejuni. PFGE analysis with KpnI restriction enzyme resulted in 11 PFGE types (I-XI) and flaA SVR typing yielded in nine flaA-SVR alleles. The Campylobacter-positive turkey flocks A, C and E were colonized by a limited number of Campylobacter clones at the farm and slaughter. The present study confirms the traceability of flock-specific strains (PFGE types I, V and IX; flaA types 21, 36 and 161) from the farm along the entire processing line to meat cuts. It seems that stress factors such as high temperature of the defeathering water (54-56 C), drying of the carcass skin during air chilling (24 h at 2 C), and oxygen in the air could not eliminate Campylobacter completely. Campylobacter-negative flocks became contaminated during processing by the same subtypes of Campylobacter introduced into the slaughter house by preceeding positive flocks even if they were slaughtered on subsequent days. Proper and efficient cleaning and disinfection of slaughter and processing premises are needed to avoid cross-contamination, especially in countries with a low prevalence of Campylobacter spp. The majority of flaA SVR alleles displayed a distinct association with a specific PFGE type. However, a linear relationship for all strains among both typing methods could not be established. To specify genetic relatedness of strains, a combination of different genotyping methods, is needed. PMID:21824338

Perko-Mkel, P; Alter, T; Isohanni, P; Zimmermann, S; Lyhs, U

2011-09-01

405

Application of PCR-denaturing-gradient gel electrophoresis (DGGE) method to examine microbial community structure in asparagus fields with growth inhibition due to continuous cropping.  

PubMed

Growth inhibition due to continuous cropping of asparagus is a major problem; the yield of asparagus in replanted fields is low compared to that in new fields, and missing plants occur among young seedlings. Although soil-borne disease and allelochemicals are considered to be involved in this effect, this is still controversial. We aimed to develop a technique for the biological field diagnosis of growth inhibition due to continuous cropping. Therefore, in this study, fungal community structure and Fusarium community structure in continuously cropped fields of asparagus were analyzed by polymerase chain reaction/denaturing-gradient gel electrophoresis (PCR-DGGE). Soil samples were collected from the Aizu region of Fukushima Prefecture, Japan. Soil samples were taken from both continuously cropped fields of asparagus with growth inhibition and healthy neighboring fields of asparagus. The soil samples were collected from the fields of 5 sets in 2008 and 4 sets in 2009. We were able to distinguish between pathogenic and non-pathogenic Fusarium by using Alfie1 and Alfie2GC as the second PCR primers and PCR-DGGE. Fungal community structure was not greatly involved in the growth inhibition of asparagus due to continuous cropping. By contrast, the band ratios of Fusarium oxysporum f. sp. asparagi in growth-inhibited fields were higher than those in neighboring healthy fields. In addition, there was a positive correlation between the band ratios of Fusarium oxysporum f. sp. asparagi and the ratios of missing asparagus plants. We showed the potential of biological field diagnosis of growth inhibition due to continuous cropping of asparagus using PCR-DGGE. PMID:22200640

Urashima, Yasufumi; Sonoda, Takahiro; Fujita, Yuko; Uragami, Atsuko

2012-01-01

406

Application of PCR-Denaturing-Gradient Gel Electrophoresis (DGGE) Method to Examine Microbial Community Structure in Asparagus Fields with Growth Inhibition due to Continuous Cropping  

PubMed Central

Growth inhibition due to continuous cropping of asparagus is a major problem; the yield of asparagus in replanted fields is low compared to that in new fields, and missing plants occur among young seedlings. Although soil-borne disease and allelochemicals are considered to be involved in this effect, this is still controversial. We aimed to develop a technique for the biological field diagnosis of growth inhibition due to continuous cropping. Therefore, in this study, fungal community structure and Fusarium community structure in continuously cropped fields of asparagus were analyzed by polymerase chain reaction/denaturing-gradient gel electrophoresis (PCR-DGGE). Soil samples were collected from the Aizu region of Fukushima Prefecture, Japan. Soil samples were taken from both continuously cropped fields of asparagus with growth inhibition and healthy neighboring fields of asparagus. The soil samples were collected from the fields of 5 sets in 2008 and 4 sets in 2009. We were able to distinguish between pathogenic and non-pathogenic Fusarium by using Alfie1 and Alfie2GC as the second PCR primers and PCR-DGGE. Fungal community structure was not greatly involved in the growth inhibition of asparagus due to continuous cropping. By contrast, the band ratios of Fusarium oxysporum f. sp. asparagi in growth-inhibited fields were higher than those in neighboring healthy fields. In addition, there was a positive correlation between the band ratios of Fusarium oxysporum f. sp. asparagi and the ratios of missing asparagus plants. We showed the potential of biological field diagnosis of growth inhibition due to continuous cropping of asparagus using PCR-DGGE. PMID:22200640

Urashima, Yasufumi; Sonoda, Takahiro; Fujita, Yuko; Uragami, Atsuko

2012-01-01

407

Identification of bacteria in a biodegraded wall painting by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA.  

PubMed Central

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

Rlleke, S; Muyzer, G; Wawer, C; Wanner, G; Lubitz, W

1996-01-01

408

Development and Validation of PCR Primers To Assess the Diversity of Clostridium spp. in Cheese by Temporal Temperature Gradient Gel Electrophoresis  

PubMed Central

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 PCRs performed with DNAs from cluster I and non-cluster I species as the templates. TTGE profiles of the PCR products, comprising the V5-V6 region of the 16S rRNA gene, allowed us to distinguish the majority of cluster I species. PCR-TTGE was applied to analyze commercial cheeses with defects. All cheeses gave a signal after nested PCR, and on the basis of band comigration with TTGE profiles of reference strains, all the bands could be assigned to a clostridial species. The direct identification of Clostridium spp. was confirmed by sequencing of excised bands. C. tyrobutyricum and C. beijerinckii contaminated 15 and 14 of the 20 cheese samples tested, respectively, and C. butyricum and C. sporogenes were detected in one cheese sample. Most-probable-number counts and volatile fatty acid were determined for comparison purposes. Results obtained were in agreement, but only two species, C. tyrobutyricum and C. sporogenes, could be isolated by the plating method. In all cheeses with a high amount of butyric acid (>100 mg/100 g), the presence of C. tyrobutyricum DNA was confirmed by PCR-TTGE, suggesting the involvement of this species in butyric acid fermentation. These results demonstrated the efficacy of the PCR-TTGE method to identify Clostridium in cheeses. The sensitivity of the method was estimated to be 100 CFU/g. PMID:15640166

Le Bourhis, Anne-Galle; Saunier, Katiana; Dor, Jol; Carlier, Jean-Philippe; Chamba, Jean-Franois; Popoff, Michel-Robert; Tholozan, Jean-Luc

2005-01-01

409

Use of Antibiotic Susceptibility Patterns and Pulsed-Field Gel Electrophoresis To Compare Historic and Contemporary Isolates of Multi-Drug-Resistant Salmonella enterica subsp. enterica Serovar Newport  

PubMed Central

Recently, multi-drug-resistant (MDR) Salmonella enterica subspecies enterica serovar Newport reemerged as a public and animal health problem. The antibiotic resistance of 198 isolates and the pulsed-field gel electrophoresis patterns (PFGE) of 139 isolates were determined. Serovar Newport isolates collected between 1988 and 2001 were included in the study. One hundred seventy-eight isolates were collected from the San Joaquin valley in California and came from dairy cattle clinical samples, human clinical samples, bulk tank milk samples, fecal samples from preweaned calves, and waterways. Twenty clinical isolates from humans from various regions of the United States were also included in the study. Resistance to 18 antibiotics was determined using a disk diffusion assay. PFGE patterns were determined using a single enzyme (XbaI). The PFGE and antibiogram patterns were described using cluster analysis. Although the antibiotic resistance patterns of historic (1988 to 1995) and contemporary (1999 to 2001) isolates were similar, the contemporary isolates differed from the historic isolates by being resistant to cephalosporins and florfenicol and in their general sensitivity to kanamycin and neomycin. With few exceptions, the contemporary isolates clustered together and were clearly separated from the historic isolates. One PFGE-antibiogram cluster combination was predominant for the recent isolates, which were taken from human samples from all parts of the United States, as well as in the isolates from California, indicating a rapid dissemination of this phenotypic strain. The data are consistent with the hypothesis that the reemergence of MDR serovar Newport is not simply an acquisition of further antibiotic resistance genes by the historic isolates but reflects a different genetic lineage. PMID:14711658

Berge, Anna Catharina B.; Adaska, John M.; Sischo, William M.

2004-01-01

410

Analysis of the Dynamics of Bacterial Communities in the Rhizosphere of the Chrysanthemum via Denaturing Gradient Gel Electrophoresis and Substrate Utilization Patterns  

PubMed Central

In order to gain a better understanding of the spatial and temporal dynamics of bacterial communities of the rhizosphere of the chrysanthemum, two complementary methods were used: a molecular bacterial community profiling method, i.e., 16S rRNA gene-based PCR followed by denaturing gradient gel electrophoresis (DGGE), and an agar plate method in which 11 sole-carbon-source utilization tests were used. The DGGE patterns showed that the bacterial communities as determined from direct rhizosphere DNA extracts were largely stable along developing roots of the chrysanthemum, with very little change over time or between root parts of different ages. The patterns were also similar to those produced with DNA extracts obtained from bulk soil samples. The DGGE patterns obtained by using microbial colonies from dilution plates as the source of target DNA were different from those found with the direct DNA extracts. Moreover, these patterns showed differences among plant replicates but also among replicate plates. Results obtained with the sole-carbon-source utilization tests indicated that the metabolic profile of the bacterial communities in the rhizosphere of the root tip did not change substantially during plant growth. This suggests selective development of specific bacterial populations by the presence of a root tip. On the other hand, the metabolic profile of bacterial communities in the rhizosphere of the root base changed during plant growth. With eight sole-carbon-source utilization tests, a significant effect of the development stage of the plant on the number of bacteria which were able to grow on these carbon sources was observed. PMID:9835588

Duineveld, Bernadette M.; Rosado, Alexandre S.; van Elsas, Jan Dirk; van Veen, Johannes A.

1998-01-01

411

Boron bridging of rhamnogalacturonan-II, monitored by gel electrophoresis, occurs during polysaccharide synthesis and secretion but not post-secretion  

PubMed Central

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

Chormova, Dimitra; Messenger, David J; Fry, Stephen C

2014-01-01

412

Determination of genome size, macrorestriction pattern polymorphism, and nonpigmentation-specific deletion in Yersinia pestis by pulsed-field gel electrophoresis.  

PubMed Central

Of 16 restriction endonucleases known to hydrolyze rare 6- or 8-base recognition sequences that were tested, only SpeI, NotI, AscI, and SfiI generated fragments of chromosomal DNA from Yersinia pestis, the causative agent of bubonic plague, of sufficient length to permit physical analysis by pulsed-field gel electrophoresis (PFGE). Of the individual bands detected after single-dimensional PFGE of these digests, the largest sum was obtained with SpeI (3,575.6 +/- 114.6 kb). Of these 41 bands, 3 were found to contain comigrating fragments, as judged by the results of two-dimensional SpeI-ApaI PFGE; addition of these fragments and the three plasmids of the species yielded a refined estimate of 4,397.9 +/- 134.6 kb for the genome. This size was similar for eight strains of diverse geographical origin that exhibited distinct DNA macrorestriction patterns closely related to their biotypes. The high-frequency chromosomal deletion known to exist in nonpigmented mutants (unable to assimilate Fe3+ at 37 degrees C or store hemin at 26 degrees C) was shown by two-dimensional PFGE analysis with SpeI and ApaI or with SfiI and SpeI to be 92.5 and 106 kb in size, respectively. The endpoints of this deletion were precise, and its size was more than sufficient to encode the eight known peptides reported to be absent in nonpigmented mutants. This deletion had not occurred (but was able to do so) in a rare mutant capable of hemin storage but not iron transport. Images PMID:1551830

Lucier, T S; Brubaker, R R

1992-01-01

413

Use of two-dimensional gel electrophoresis to differentiate morphospecies of Alexandrium minutum, a paralytic shellfish poisoning toxin-producing dinoflagellate of harmful algal blooms.  

PubMed

Contamination of shellfish with paralytic shellfish poisoning toxins (PST) produced by toxic harmful algal blooms (HABs) have been negatively affecting the shellfish and aquaculture industries worldwide. Therefore, accurate and early identification of toxic phytoplankton species is crucial in HABs surveillance programs that allow fish-farmers to take appropriate preventive measures in shellfish harvesting and other aquaculture activities to overcome the negative impacts of HABs on human health. The identification of toxic dinoflagellates present in the water is currently a time-consuming operation since it requires skillful taxonomists and toxicologists equipped with optical and scanning electron microscopes as well as sophisticated equipment, for example, high-performance liquid chromotography-fluorescence detection. In this paper, a two-dimensional gel electrophoresis (2-DE)-based proteomic approach was applied to discriminate between toxic and nontoxic strains of Alexandrium minutum. Variation in morphological features between toxic and nontoxic strains was minimal and not significant. Also, variation in 2-DE protein patterns within either toxic or nontoxic strains was low, but pronounced differences were detected between toxic and nontoxic strains. The most notable differences between these strains were several abundant proteins with pIs ranging from 4.8 to 5.3 and apparent molecular masses between 17.5 and 21.5 kDa. Groups of proteins, namely NT1, NT2, NT3, and NT4, were consistently found in all nontoxic strains, while T1 and T2 were prominent in the toxic strains. These specific protein spots characteristic for toxic and nontoxic strains remained clearly distinguishable irrespective of the various growth conditions tested. Therefore, they have the potential to serve as "taxonomic markers" to distinguish toxic and nontoxic strains within A. minutum. Initial studies revealed that the expression pattern of T1 was tightly correlated to toxin biosynthesis in the examined alga and may be used to serve as a potential toxin indicator. PMID:15800974

Chan, Leo Lai; Hodgkiss, Ivor John; Lam, Paul Kwan-Sing; Wan, Jennifer Man-Fan; Chou, Hong-Nong; Lum, John Hon-Kei; Lo, Maria Gar-Yee; Mak, Abby Sin-Chi; Sit, Wai-Hung; Lo, Samuel Chun-Lap

2005-04-01

414

Epidemiologic typing and delineation of genetic relatedness of methicillin-resistant Staphylococcus aureus by macrorestriction analysis of genomic DNA by using pulsed-field gel electrophoresis.  

PubMed Central

To evaluate the usefulness of phenotypic and genotypic analyses for the epidemiologic typing of methicillin-resistant Staphylococcus aureus (MRSA), we characterized 64 epidemic MRSA isolates and 10 sporadic methicillin-susceptible S. aureus isolates from a university hospital and 18 MRSA isolates from hospitals in different geographical areas. Chromosomal DNA macrorestriction analysis with SstII was resolved by pulsed-field gel electrophoresis and compared with antibiotype analysis, phage type analysis, and standard genomic DNA restriction analysis with BglII. Indices of the discriminatory ability of these methods were 0.982, 0.959, 0.947, and 0.959, respectively. Macrorestriction patterns of 94% of MRSA isolates from patients, personnel, and the environment associated with a nosocomial outbreak were closely related (similarity coefficient, 85 to 100%). In contrast, methicillin-susceptible S. aureus isolates showed a marked diversity of macrorestriction patterns (median similarity, 41%). MRSA isolates from other geographical areas showed diverse macrorestriction patterns, with the exception of four isolates displaying identical or closely related patterns; these isolates were associated with concurrent outbreaks in four other Belgian hospitals. A concordance of genomic DNA macrorestriction typing with phenotypic methods was observed for 60 to 65% of MRSA isolates, and a concordance with standard DNA restriction analysis was found for 79 to 98% of these isolates. In conclusion, genomic DNA macrorestriction analysis was a useful complement to phenotypic methods for delineating epidemic isolates of MRSA, for identifying their nosocomial reservoirs, and for tracing their intra- and interhospital spread. The genetic relatedness of MRSA isolates, as estimated by this technique, appeared to correlate with their space-time clustering. Images PMID:1328279

Struelens, M J; Deplano, A; Godard, C; Maes, N; Serruys, E

1992-01-01

415

Analysis of ventilator-associated pneumonia infection route by genome macrorestriction-pulsed-field gel electrophoresis and its prevention with combined nursing strategies.  

PubMed

The aim of the present study was to explore the infection route of ventilator-associated pneumonia (VAP) and assess the effectiveness of a combined nursing strategy to prevent VAP in intensive care units. Bacteria from the gastric juice and drainage from the hypolarynx and lower respiratory tracts of patients with VAP were analyzed using genome macrorestriction-pulsed-field gel electrophoresis (GM-PFGE). A total of 124 patients with tracheal intubation were placed in the intervention group and were treated with a combined nursing strategy, comprising mosapride (gastric motility stimulant) administration and semi-reclining positioning. A total of 112 intubated patients were placed in the control group and received routine nursing care. The incidence rate of VAP, days of ventilation and mortality rate of patients were compared between the two groups. The GM-PFGE fingerprinting results of three strains of Pseudomonas aeruginosa from the gastric juice, subglottic secretion drainage and drainage of the lower respiratory tract in patients with VAP were similar across groups. The number of days spent on a ventilator by patients in the intervention group (7.375.32 days) was lower compared with that by patients in the control group (12.344.98 days) (P<0.05). The incidence rate of VAP was reduced from 40.81 to 21.25% following intervention with the combined nursing strategy (P<0.05); furthermore, the mortality rate of intubated patients in the intervention group was 29.46%, a significant reduction compared with the 41.94% mortality rate observed in the control group (P<0.05). Gastroesophageal reflux (GER) was confirmed as one of the infection routes for VAP. The combined nursing strategy of gastric motility stimulant administration and the adoption of a semi-reclining position was effective in preventing VAP by reducing the occurrence of GER. PMID:25371757

Wang, Xiaodong; Wang, Junping; Li, Jing; Wang, Jing

2014-12-01

416

Diversity of pulsed-field gel electrophoresis pulsotypes, serovars, and antibiotic resistance among Salmonella isolates from wild amphibians and reptiles in the California Central Coast.  

PubMed

A survey of cold-blooded vertebrates and associated surface waters in a produce-growing region on the Central California Coast was done between May and September 2011 to determine the diversity of Salmonella. Samples from 460 amphibians and reptiles and 119 water samples were collected and cultured for Salmonella. Animals sampled were frogs (n=331), lizards (n=59), newts (n=5), salamanders (n=6), snakes (n=39), and toads (n=20). Salmonella was isolated from 37 individual animals, including frogs, lizards, snakes, and toads. Snakes were the most likely to contain Salmonella, with 59% testing positive followed by 15.3% of lizards, 5% of toads, and 1.2% of frogs. Fifteen water samples (12.6%) were positive. Twenty-two different serovars were identified, and the majority of isolates were S. enterica subsp. IIIb, with subsp. I, II, and IIIa also found. The serovar isolated most frequently was S. enterica subsp. IIIb 16:z??:e,n,x,z??, from snakes and frogs in five different locations. S. enterica subsp. I serovar Typhimurium and the monophasic I 6,8:d:- were isolated from water, and subspecies I Duisburg and its variants were found in animals and water. Some samples contained more than one type of Salmonella. Analysis of pulsed-field gel electrophoresis pulsotypes indicated that some strains persisted in animals and water collected from the same location. Sixty-six isolates displayed antibiotic resistance,