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Silver Staining of 2D Electrophoresis Gels Thierry Rabilloud  

E-print Network

Silver 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 are discussed in this chapter, and optimized silver staining protocols are proposed. ii. Key Words: mass

Paris-Sud XI, Université de



E-print Network

. of Mathematics and Statistics, Baltimore, MD 21250, VGeorgetown University Medical Center, Dept. of Biostatistics differentially expressed pro- teins in simulated two-dimensional electrophoresis (2DE) gels us- ing spatial, Phoretix]. These methods rely on spot-by-spot analysis and are thus subject to the errors accumulated

Adali, Tulay


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


Protein profiling using two-dimensional difference gel electrophoresis (2-D DIGE).  


2-D DIGE relies on pre-electrophoretic labeling of samples with one of three spectrally distinct fluorescent dyes, followed by electrophoresis of all samples in one 2-D gel. The dye-labeled samples are then viewed individually by scanning the gel at different wavelengths, which circumvents problems with gel-to-gel variation and spot matching between gels. Image analysis programs are used to generate volume ratios for each spot, which essentially describe the intensity of a particular spot in each test sample, and thus enable protein abundance level changes to be identified and quantified. This unit describes the 2-D DIGE procedure including sample preparation from various cell types, labeling of proteins, and points to consider in the downstream processing of fluorescently labeled samples. PMID:24510675

Feret, Renata; Lilley, Kathryn S



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

PubMed Central

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

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



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


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



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.



Gel Electrophoresis  

NSDL National Science Digital Library

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



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

ERIC Educational Resources Information Center

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

Kim, Thomas D.; Craig, Paul A.



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



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

SciTech Connect

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

Liang, X.



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

PubMed Central

Background In the post-genome era, most research scientists working in the field of proteomics are confronted with difficulties in management of large volumes of data, which they are required to keep in formats suitable for subsequent data mining. Therefore, a well-developed open source laboratory information management system (LIMS) should be available for their proteomics research studies. Results We developed an open source LIMS appropriately customized for 2-D gel electrophoresis-based proteomics workflow. The main features of its design are compactness, flexibility and connectivity to public databases. It supports the handling of data imported from mass spectrometry software and 2-D gel image analysis software. The LIMS is equipped with the same input interface for 2-D gel information as a clickable map on public 2DPAGE databases. The LIMS allows researchers to follow their own experimental procedures by reviewing the illustrations of 2-D gel maps and well layouts on the digestion plates and MS sample plates. Conclusion Our new open source LIMS is now available as a basic model for proteome informatics, and is accessible for further improvement. We hope that many research scientists working in the field of proteomics will evaluate our LIMS and suggest ways in which it can be improved. PMID:17018156

Morisawa, Hiraku; Hirota, Mikako; Toda, Tosifusa



Cationic detergents enable the separation of membrane proteins of Plasmodium falciparum-infected erythrocytes by 2D gel electrophoresis.  


The intraerythrocytic stage of Plasmodium falciparum alters the characteristics of its host cell by exporting selected plasmodial proteins. Although it is clear that the physicochemical and immunobiological properties of the host cell are modulated during parasite development, the involved plasmodial proteins and their mode of action are not completely known. Using cetyltrimethylammonium bromide (CTAB) or benzyldimethyl-n-hexadecylammonium chloride (16-BAC) for the first dimension and SDS for the second dimension, we separated proteins from membranes of human erythrocytes and of erythrocytes infected with the malaria parasite P. falciparum. Protein spots were analyzed by MALDI-TOF/TOF MS and annotated in respective 2D master gels. By using the alternative 2D approach, characteristic host cell membrane proteins and, more importantly, membrane-associated and exported plasmodial proteins were identified that might play a role in parasite-induced host cell modulation. PMID:22539315

Philipp, Stephan; Jakoby, Thomas; Tholey, Andreas; Janssen, Ottmar; Leippe, Matthias; Gelhaus, Christoph



Comparison of the urinary protein patterns of athletes by 2D-gel electrophoresis and mass spectrometry-a pilot study.  


Urinary proteins and exercise-induced proteinuria have been the subject of much research. Proteinuria has been studied in depth after different running and cycling intensities and durations and the different mechanisms of glomerular filtration and tubular dysfunction have been elucidated. The present study was carried out to compare urinary protein profiles of athletes in different sport categories (endurance sport, team sport, strength sport). Doping-control urine samples obtained from in-competition testing and specimens derived from a control group were analysed by means of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and significantly deviating protein spots were enzymatically hydrolysed and identified by nanoflow liquid chromatography-orbitrap mass spectrometry. Endurance sport samples demonstrated a significant increase of mainly medium-sized urinary proteins such as transferrin, zinc alpha-2-glycoprotein and prostaglandin H2 D-isomerase (30-80 kDa) in 2D-PAGE experiments. Proteinuria was evident in all samples after protein concentration measurements (protein/creatinine > 15 mg/mmol). Alterations were also observed in strength sport samples, which showed an increase of low molecular weight proteins or protein fragments (<30 kDa, e.g., transthyretin, CD 59 antigen or an N-terminal transferrin fragment). In contrast, the concentration measurements did not imply proteinuria but total protein excretion was in a normal range. The study provides a first overview on 2D maps of the urinary proteome after different types of exercise. Future studies may lead to the establishment of urinary protein maps that are typical for a certain type of sport or even an individual athlete. These maps may complement the blood passport of athletes in doping control. PMID:20355218

Kohler, Maxie; Franz, Stefan; Regeniter, Axel; Ikonen, Anna; Walpurgis, Katja; Thomas, Andreas; Schnzer, Wilhelm; Thevis, Mario



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



Application of highly sensitive fluorescent dyes (CyDye DIGE Fluor saturation dyes) to laser microdissection and two-dimensional difference gel electrophoresis (2D-DIGE) for cancer proteomics  

Microsoft Academic Search

Proteome data combined with histopathological information provides important, novel clues for understanding cancer biology and reveals candidates for tumor markers and therapeutic targets. We have established an application of a highly sensitive fluorescent dye (CyDye DIGE Fluor saturation dye), developed for two-dimensional difference gel electrophoresis (2D-DIGE), to the labeling of proteins extracted from laser microdissected tissues. The use of the

Setsuo Hirohashi; Tadashi Kondo



Copolymers For Capillary Gel Electrophoresis  


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)



Gel Electrophoresis Lab: DNA Fingerprinting  

NSDL National Science Digital Library

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

Ehlers, Megan



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.



Difference gel electrophoresis.  


DIGE is a protein labelling and separation technique allowing quantitative proteomics of two or more samples by optical fluorescence detection of differentially labelled proteins that are electrophoretically separated on the same gel. DIGE is an alternative to quantitation by MS-based methodologies and can circumvent their analytical limitations in areas such as intact protein analysis, (linear) detection over a wide range of protein abundances and, theoretically, applications where extreme sensitivity is needed. Thus, in quantitative proteomics DIGE is usually complementary to MS-based quantitation and has some distinct advantages. This review describes the basics of DIGE and its unique properties and compares it to MS-based methods in quantitative protein expression analysis. PMID:19003860

Timms, John F; Cramer, Rainer



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

Kirschner, Marc W.


Fluorescence detection for gel and capillary electrophoresis  

SciTech Connect

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

Hogan, B.



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

EPA Science Inventory

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


Difference gel electrophoresis: a single gel method for detecting changes in protein extracts.  


We describe a modification of two-dimensional (2-D) polyacrylamide gel electrophoresis that requires only a single gel to reproducibly detect differences between two protein samples. This was accomplished by fluorescently tagging the two samples with two different dyes, running them on the same 2-D gel, post-run fluorescence imaging of the gel into two images, and superimposing the images. The amine reactive dyes were designed to insure that proteins common to both samples have the same relative mobility regardless of the dye used to tag them. Thus, this technique, called difference gel electrophoresis (DIGE), circumvents the need to compare several 2-D gels. DIGE is reproducible, sensitive, and can detect an exogenous difference between two Drosophila embryo extracts at nanogram levels. Moreover, an inducible protein from E. coli was detected after 15 min of induction and identified using DIGE preparatively. PMID:9420172

Unl, M; Morgan, M E; Minden, J S



Looking at proteins from two dimensions: a review on five decades of 2D electrophoresis.  


Abstract Separating proteins according to two different gel-electrophoretic methods not only increases the resolution power for highly complex samples when compared to one-dimensional separations, but is also a valuable tool for protein and protein complex characterization. There are a number of different electrophoresis methods which can be combined. The combination of isoelectric focusing under denaturing conditions and SDS polyacrylamide gel electrophoresis delivers the highest resolution of all bio-analytic techniques. This is a short review on the history and state of the art of two-dimensional electrophoresis methods, and contains some practical tips for high resolution 2D electrophoresis, which are based on several decades of experience with this method. PMID:25137570

Westermeier, Reiner



Two-dimensional Gel Electrophoresis (2DE)  

NASA Astrophysics Data System (ADS)

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

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


[Serum protein electrophoresis: comparison of capillary zone electrophoresis Capillarys (Sebia) and agarose gel electrophoresis Hydrasys (Sebia)].  


Since several years, serum proteins electrophoresis became a routine analysis, mainly performed by agarose gel electrophoresis, frequently semi-automated. We compared the new fully automated capillary electrophoresis system from Sebia, Capillarys, with our reference method, agarose gel electrophoresis Hydrasys (Sebia). This study focused on the evaluation of both the analytical performances and some practical aspects such as ease of use, rapidity, costs. It appears clearly from that study that both methods give similar results for the detection of monoclonal proteins. We notice that the capillary electrophoresis (Capillarys) displays higher sensitivity (97.2%) than the agarose gel electrophoresis Hydrasys (93.5%), however with a lower specificity (93.7 versus 98.9%). On the other hand, the Capillarys method displays obvious practical advantages such as full automation, ease of use and rapidity. PMID:14671753

Lissoir, B; Wallemacq, P; Maisin, D




SciTech Connect

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




Quantitation in two-dimensional fluorescence difference gel electrophoresis: effect of protein fixation.  


Analyzing a large number of unfixed gels in a 2-D fluorescence difference gel electrophoresis (2-DIGE) experiment presents a challenge of avoiding variable protein diffusion within and across the comparison groups. The characteristics of protein detection and quantitation in a 2-D differential in gel fluorescence experiment were compared for gels with and without protein fixation. The current study tests and concludes that when dealing with a large sample size with variable protein diffusion across the 2-D gel over a period of 2-4 days, it is a preferred choice to fix the gel without affecting the protein quantitation. PMID:16607608

Tannu, Nilesh; Hemby, Scott E



The Genetic Science Learning Center: Gel Electrophoresis  

NSDL National Science Digital Library

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



Gel electrophoresis of intact subcellular particles.  


The review describes the application of gel electrophoresis to the characterization and separation of viruses, ribosomes, vesicles and other subcellular particles. The preparation of the sample, the choice of the buffer, the gel medium, the apparatus and the detection of the particle (staining and scanning) as well as the necessary theory are discussed. This includes the mathematical evaluation of experimental data on the basis of Ferguson plots using the extended Ogston theory. Simple methods and sophisticated computer simulation techniques are described and exemplified in application to the determination of particle size and charge, the pore size of the gel (unpublished data) and the two-dimensional agarose electrophoresis (unpublished). It is shown that the nature of the particle (e.g. spherical or rod-shaped, pliable or rigid texture) determines the shape of the non-linear Ferguson plot. In addition, the review gives a number of practical applications of gel electrophoresis, isoelectric focusing, titration curves and immuno-electrophoresis to subcellular particles. Pros and cons are evaluated. A comparison with other analytical procedures is made. The review is concluded by a futuristic outlook. PMID:3305546

Tietz, D



Two-dimensional gel electrophoresis based technologies for potential biomarkers identification in amniotic fluid: a simple model.  


To assess the efficiency of two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and of two-dimensional electrophoresis and ammoniacal silver staining (2D-E), different amounts of soybean trypsin inhibitor and horse myoglobin were added to amniotic fluid. In this model, a minimum of 5 to 10 ng of "artificial" biomarkers was detected. PMID:17168815

Queloz, P-A; Crettaz, D; Thadikkaran, L; Sapin, V; Tissot, J-D



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.



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

PubMed Central

The intention of this review is to provide an overview of current methodologies employed in the rapidly developing field of ocular proteomics with emphasis on sample preparation, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Appropriate sample preparation for the diverse range of cells and tissues of the eye is essential to ensure reliable results. Current methods of protein staining for 2D-PAGE, protein labelling for two-dimensional difference gel electrophoresis, gel-based expression analysis and protein identification by MS are summarised. The uses of gel-free MS-based strategies (MuDPIT, iTRAQ, ICAT and SILAC) are also discussed. Proteomic technologies promise to shed new light onto ocular disease processes that could lead to the discovery of strong novel biomarkers and therapeutic targets useful in many ophthalmic conditions. PMID:21406065



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



Evaluation of protocols used in 2-d electrophoresis for proteome analysis of young rice caryopsis.  


In order to obtain a high-resolution electrophorogram of rice young panicle proteome, we evaluated various protocols commonly used in two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) of proteins, including gel staining protocol, pH range of immobilized pH gradient (IPG) strips and sample loading quantity. Results showed that a silver staining protocol using sensitized solution containing glacial acetic acid, sodium acetate and sodium thiosulfate (reported by Heukeshoven and Dernick in 1988) and a Coomassie Brilliant Blue staining method using solution containing G-250, ammonium sulfate and phosphoric acid (reported by Pink et al in 2010) demonstrated the superior staining effect. In addition, we also showed that higher resolution was achieved when IPG gel strip with pH range of 5-8 was used, compared to that with pH range of 4-7. Finally, the optimal loading quantity was determined as 130 ?g using the 17 cm-long nonlinear IPG strip with pH 5-8 in combination with the silver nitrate staining protocol. The evaluated results would be helpful in proteome analysis of young rice caryopsis. PMID:22289479

Liao, Jiang-Lin; Huang, Ying-Jin



Heterogeneous and Anisotropic Dynamics of a 2D Gel  

NASA Astrophysics Data System (ADS)

We report x-ray photon correlation spectroscopy (XPCS) results on bidimensional (2D) gels formed by a Langmuir monolayer of gold nanoparticles. The system allows an experimental determination of the fourth order time correlation function, which is compared to the usual second order correlation function and to the mechanical response measured on macroscopic scale. The observed dynamics is anisotropic, heterogeneous and superdiffusive on the nanoscale. Different time scales, associated with fast heterogeneous dynamics inside 2D cages and slower motion of larger parts of the film, can be identified from the correlation functions. The XPCS results are discussed in view of other experimental results and models of three-dimensional gel dynamics.

Orsi, D.; Cristofolini, L.; Baldi, G.; Madsen, A.



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



Multilevel thresholding of gel electrophoresis images using firefly algorithm  

Microsoft Academic Search

Gel electrophoresis (GE) is a process of DNA, RNA and protein molecules separation using electric field applied to a gel matrix. This paper describes the image processing techniques applied on GE image to segment the bands from their background. A few pre-processing steps are applied on the image prior to the segmentation technique for the purpose of removing noise in

M. H. Mohd Noor; A. R. Ahmad; Z. Hussain; K. A. Ahmad; A. R. Ainihayati



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.



Supramolecular gel electrophoresis of acidic native proteins.  


Amphiphilic tris-urea molecules self-assemble into a supramolecular hydrogel in tris(hydroxymethyl)aminomethane-glycine buffer. The supramolecular hydrogel is used as a matrix for the electrophoresis of acidic native proteins, in which proteins are separated based on their isoelectric points rather than their molecular weights. The proteins remain in their native forms during migration, and their activities are retained after electrophoresis. Glucoside substituents on the amphiphilic tris-urea molecule allow for the affinity electrophoresis of a carbohydrate-binding protein to be performed. The proteins can be efficiently recovered from the supramolecular hydrogel using a simple procedure. This is a major advantage of using this noncovalent, self-assembled material. PMID:25147927

Munenobu, Kanako; Hase, Takayuki; Oyoshi, Takanori; Yamanaka, Masamichi



The instantaneous monitoring of polyacrylamide gels during electrophoresis.  

PubMed Central

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

Elliott, A



Extensions of Gel Electrophoresis with Proteins  

NSDL National Science Digital Library

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

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



Analysis of Drosophila chromosome 4 using pulsed field gel electrophoresis  

Microsoft Academic Search

Previous estimates of the size ofDrosophila melanogaster chromosome4 have indicated that it is 1% to 4% of the genome or ~6 Mb. We have used pulsed field gel electrophoresis (PFGE) to separate megabase-sized molecules ofD. melanogaster chromosomal DNA. Southern blots of these gels were probed with DNA fragments from thecubitus interruptus andzfh-2 genes, which are located on chromosome4. They each

John Locke; Heather E. McDermid



Agarose gel electrophoresis Solutions and reagents  

E-print Network

to 1L Adjust pH to 8.3 with NaOH or acetic acid Store at RT TBE (5X) Tris 54g EDTA 4.65g Boric Acid 24g ddH2O up to 1L Adjust pH to 8.3 with boric acid Store at RT - Ethidium bromide (EtBr), 10 mg/ml (20) -Electrophoresis buffer: TAE buffer (Tris, acetate, EDTA) 50X, 1L Tris 242g Acetic acid 57,1ml EDTA 100ml ddH2O up

Abou Elela, Sherif


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


Pulsed field gel electrophoresis (PFGE), the gold standard of molecular typing methods, has a major disadvantage of an unusually long electrophoretic time. From the original protocol of 6 days, it was modified to 3 days and subsequently to a single day. We describe the procedure of stacking five to six gels one on top of another in order to increase and maximize the output in a shorter time without compromising the resolution and reproducibility. All the variables that affect pulsed field gels during electrophoresis were taken into consideration. We firstly optimized the parameters to be used and secondly determined whether stacking of five to six gels had any effect on the molecular separation during electrophoresis in comparison with a single gel run. DNA preparation, restriction, electrophoresis, staining and gel documentation was carried out based on previously published methods. Gels were analysed using BioNumerics and dice coefficient and unweighted pair group methods were used to generate dendrograms based on 1.5% tolerance values. Identical band profiles and band resolution-separation were seen in the PFGE patterns with single gel and multiple stacking gels. Cluster analysis further strengthened the fact that results from stacking gels were reproducible and comparable with a single gel run. This method of stacking gels saves time and maximizes the output at the same time. The run time for a single gel was about 28 hours, but with six stacked gels the run time was 54 hours compared with 28 x 6 = 168 hours if they were run separately as single gels thus saving time of 67.86%. Beside the big factor of saving time, stacking gels save resources (electricity, reagents, water, chemicals and working time) by increasing the sample throughput in a shorter time without compromising on quality of data. But optimization of working parameters is vital depending on the PFGE system used. PMID:19384038

Heng, See Kah; Heng, Chua Kek; Puthucheary, S D



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


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.



Semiquantitative Proteomic Analysis of the Human Spliceosome via a Novel Two-Dimensional Gel Electrophoresis Method ?  

PubMed Central

More than 200 proteins associate with human spliceosomes, but little is known about their relative abundances in a given spliceosomal complex. Here we describe a novel two-dimensional (2D) electrophoresis method that allows separation of high-molecular-mass proteins without in-gel precipitation and thus without loss of protein. Using this system coupled with mass spectrometry, we identified 171 proteins altogether on 2D maps of stage-specific spliceosomal complexes. By staining with a fluorescent dye with a wide linear intensity range, we could quantitate and categorize proteins as present in high, moderate, or low abundance. Affinity-purified human B, Bact, and C complexes contained 69, 63, and 72 highly/moderately abundant proteins, respectively. The recruitment and release of spliceosomal proteins were followed based on their abundances in A, B, Bact, and C spliceosomal complexes. Staining with a phospho-specific dye revealed that approximately one-third of the proteins detected in human spliceosomal complexes by 2D gel analyses are phosphorylated. The 2D gel electrophoresis system described here allows for the first time an objective view of the relative abundances of proteins present in a particular spliceosomal complex and also sheds additional light on the spliceosome's compositional dynamics and the phosphorylation status of spliceosomal proteins at specific stages of splicing. PMID:21536652

Agafonov, Dmitry E.; Deckert, Jochen; Wolf, Elmar; Odenwalder, Peter; Bessonov, Sergey; Will, Cindy L.; Urlaub, Henning; Luhrmann, Reinhard



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

E-print Network

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

Chen, Xiaojia



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.



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.



Separation of long RNA by agarose-formaldehyde gel electrophoresis.  


We describe a method to facilitate electrophoretic separation of high-molecular-weight RNA species, such as ribosomal RNAs and their precursors, on agarose-formaldehyde gels. Two alternative "pK-matched" buffer systems were substituted for the traditionally used Mops-based conductive medium. The key advantages include shortened run times, a 5-fold reduction in formaldehyde concentration, a significantly improved resolution of long RNAs, and consistency in separation. The new procedure has a streamlined work flow that helps to minimize errors and is broadly applicable to agarose gel electrophoresis of RNA samples and their subsequent analysis by Northern blotting. PMID:23800830

Mansour, Farrah H; Pestov, Dimitri G



Separation of long RNA by agarose-formaldehyde gel electrophoresis  

PubMed Central

We describe a method to facilitate electrophoretic separation of high molecular weight RNA species, such as ribosomal RNAs and their precursors, on agarose-formaldehyde gels. Two alternative pK-matched buffer systems were substituted for the traditionally used MOPS-based conductive media. The key advantages include shortened run times, a five-fold reduction in formaldehyde concentration, a significantly improved resolution of long RNAs, and consistency in separation. The new procedure has a streamlined work flow that helps minimize errors and is broadly applicable to agarose gel electrophoresis of RNA samples and their subsequent analysis by Northern blotting. PMID:23800830

Mansour, Farrah H.; Pestov, Dimitri G.



Basics and recent advances of two dimensional- polyacrylamide gel electrophoresis  

PubMed Central

Gel- based proteomics is one of the most versatile methods for fractionating protein complexes. Among these methods, two dimensional- polyacrylamide gel electrophoresis (2-DE) represents a mainstay orthogonal approach, which is popularly used to simultaneously fractionate, identify, and quantify proteins when coupled with mass spectrometric identification or other immunological tests. Although 2-DE was first introduced more than three decades ago, several challenges and limitations to its utility still exist. This review discusses the principles of 2-DE as well as both recent methodological advances and new applications. PMID:24735559



[Improvement of two-dimensional gel electrophoresis of total proteins from rice anthers].  


This paper reported an improvement in 2-D gel electrophoresis of the proteome in Honglian cytoplasmic male sterile rice. An IPGphor unit with immobile pH gradient strips was used as the first dimension and SDS-PAGE as the second. The total anther proteins were extracted using TCA/acetone and then were washed 5-6 times with acetone till the proteins were white and clean, and then tributylphosphine and DTT were added into the rehydration buffer to improve the solubility of the proteins. The 2-D gel was stained by both methods of coomassie blue G-250 and silver. Extraction of proteins, pH of the strips and rehydration of the strips were optimized and compared. Higher repeatability and better separating protein pattern could be gained by this technique. PMID:17117559

Wen, Li; Liu, Gai; Wang, Kun; Peng, Xiao Jue; Wan, Cui Xiang; Li, Guo Min; Tao, Jun; Zhu, Ying Guo



Three methods of capillary electrophoresis compared with high-resolution agarose gel electrophoresis for serum protein electrophoresis.  


We assessed the BioFocus 2000 capillary electrophoresis instrument for use in a routine clinical laboratory. We examined 210 serum samples received for serum protein electrophoresis by four methods: (1) The Bio-Rad HR015EC high-resolution serum protein kit on the BioFocus; (2) the Jenkins-Guerin (JG) method on the Applied Biosystems 270A HT Capillary Electrophoresis System (JG-ABI); (3) the Jenkins-Guerin method using the BioFocus (JG-BF); and (4) the quantitation of monoclonal bands found in 76 of the 210 samples was assayed by Helena Titan Hi-Res agarose gel electrophoresis (HRAGE). The correlation coefficient between the three sets of capillary electrophoresis monoclonal band results and the Helena quantitation was 0.92 or better. Although the quantitative comparison of monoclonal bands by HR015EC was very good, the lack of sharpness of monoclonal bands using the HR015EC kit meant our preference was to use the JG method on either the ABI or on the Biofocus. PMID:9892066

Jenkins, M A



Improvement of two-dimensional gel electrophoresis proteome maps of the haloarchaeon Haloferax volcanii.  


Proteins of haloarchaea are remarkably unstable in low-ionic-strength solvents and tend to aggregate under standard two-dimensional (2-D) gel electrophoresis conditions, causing strong horizontal streaking. We have developed a new approach to generate 2-D maps of halophilic proteins which included washing cells with 1.5 M Tris-HCl buffer. In addition, proteins were precipitated with acetone, solubilized with urea and thiourea in the presence of the sulfobetaine detergent 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS), reduced with tributylphosphine (TBP), and separated with microrange strips of immobilized pH gradients (pH 3.9-5.1). This combination enabled the construction of highly reproducible 2-D maps of Haloferax volcanii proteins. PMID:15627962

Karadzic, Ivanka M; Maupin-Furlow, Julie A



Superoxide dismutase isozyme detection using two-dimensional gel electrophoresis zymograms.  


Superoxide dismutases (SODs) are ubiquitous antioxidant enzymes involved in cell protection from reactive oxygen species. Their antioxidant activities make them of interest to applied biotechnology industries and are usually sourced from plants. SODs are also involved in stress signaling responses in plants, and can be used as indicators of these responses. In this article, a suitable method for the separation of different SOD isoforms using two-dimensional-gel electrophoresis (2D-GE) zymograms is reported. The method was developed with a SOD standard from bovine erythrocytes and later applied to extracts from Stemona tuberosa. The first (non-denaturing isoelectric focusing) and second (denaturing sodium dodecylsulphate-polyacrylamide gel electrophoresis) dimensions of duplicate 2D-GE gels were stained with either Coomassie brilliant blue G-250 for total protein visualization, or SOD activity (zymogram) using riboflavin/nitroblue tetrazolium. For confirmation, putative SOD activity positive spots were subject to trypsin digestion and nano-liquid chromatography tandem mass spectrometry, followed by searching the MASCOT database for potential identification. The method could separate different SOD isoforms from a plant extract and at least partially maintain or allow renaturation to the native forms of the enzyme. Peptide sequencing of the 2D-GE suggested that the SODs were resolved correctly, identifying the control CuZn-SOD from bovine erythrocytes. The two SODs from S. tuberosa tubers were found to be likely homologous of a CuZn-SOD. SOD detection and isoform separation by 2D-GE zymograms was efficient and reliable. The method is likely applicable to SOD detection from plants or other organisms. Moreover, a similar approach could be developed for detection of other important enzymes in the future. PMID:24334192

Niyomploy, Ploypat; Srisomsap, Chantragan; Chokchaichamnankit, Daranee; Vinayavekhin, Nawaporn; Karnchanatat, Aphichart; Sangvanich, Polkit



Analysis of protein glycation using fluorescent phenylboronate gel electrophoresis  

PubMed Central

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

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




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



Rapid optimization of metal nanoparticle surface modification with high-throughput gel electrophoresis.  


The ability to effectively control and optimize surface modification of metal nanoparticles is paramount to the ability to employ metal nanoparticles as diagnostic and therapeutic agents in biology and medicine. Here we present a high-throughput two-dimensional-grid gel electrophoresis cell (2D-GEC)-based method, capable of optimizing the surface modification of as many as 96 samples of metal nanoparticles in approximately 1 h. The 2D-GEC method determines not only the average zeta-potential of the modified particles but also the homogeneity of the surface modification by measuring the distance between the front of the sample track and the area where the maximum optical density is achieved. The method was tested for optimizing pH and concentration of the modifiers (pM) for functionalizing gold nanorod thiol-containing acidic agents. PMID:24392839

Beskorovaynyy, Alexander V; Kopitsyn, Dmitry S; Novikov, Andrei A; Ziangirova, Maya; Skorikova, Galina S; Kotelev, Mikhail S; Gushchin, Pavel A; Ivanov, Evgeniy V; Getmansky, Michael D; Itzkan, Irving; Muradov, Alexander V; Vinokurov, Vladimir A; Perelman, Lev T



Analysis of the fibrin-polymerizing reaction using sodium dodecylsulfate-agarose gel electrophoresis.  


1. A new method of sodium dodecylsulfate gel electrophoresis, sodium dodecyl-sulfate-agarose gel electrophoresis, was developed. The new electrophoresis was shown to have a high and efficient resolving power for fibrin polymers and was also applicable to other proteins. 2. By sodium dodecylsulfate-agarose gel electrophoresis, the fibrin polymerizing reaction with activated fibrin stabilizing factor was analyzed in detail. The cross-linking between the gamma-chains was indicated to occur at an intermolecular level. The solubility of polymerized fibrin was suggested to be related mainly to the formation of the crosslinks between gamma-chains. PMID:1168501

Moroi, M; Inoue, N; Yamasaki, M



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



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.



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


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



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


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



Serial displacement chromatofocusing and its applications in multidimensional chromatography and gel electrophoresis: II. Experimental results.  


Part I of this study investigated the theory and basic characteristics of "serial displacement chromatofocusing" (SDC). In Part II of this study, SDC is applied to two prototype applications which have potential uses in proteomics and related areas involving the analysis of complex analyte mixtures. In the first application, SDC was used as a prefractionation method prior to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to separate a human prostate cancer cell lysate. It was observed that the resolution achieved in narrow-pI-range 2D-PAGE was improved when using SDC prefractionation, so that SDC may be useful as a low-cost, high-speed, and highly scalable alternative to electrophoretic prefractionation methods for 2D-PAGE. The second application involves the use of SDC as the first dimension, and reversed-phase chromatography as the second dimension, to produce a novel, fully automated, two-dimensional high-performance liquid chromatography technique. The method was shown to have performance advantages over one-dimensional reversed-phase chromatography for peptide separations. PMID:19128804

Shen, Hong; Li, Xiang; Bieberich, Charles; Frey, Douglas D



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

PubMed Central

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

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



Misincorporation during DNA synthesis, analyzed by gel electrophoresis.  

PubMed Central

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

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



Automated microdensitometry and quantification of lipoproteins by agarose gel electrophoresis.  


A major obstacle in the application of quantitative microelectrophoresis has been tedious manipulations and calculations. To overcome these difficulties, we have developed an automatic system for the microdensitometry and calculations as part of a quantitative agarose gel electrophoresis facility. Results are internally standardized by serum cholesterol and/or triglyceride measurements. The hardware consists of a densitometer, an analog to digital converter, a cathode ray tube terminal, a teleprinter, and a small computer. A program in 4K words allows sample coding, electrophoretic scan display, indexing, and systematic identification of each peak. Data are acquired from scans of electrophoretic patterns of serum alone or in combination with the 1.006 gm/ml VLDL top and/or bottom preparative lipoprotein fractions. As many as 30 scans can be stored in 4K words of memory and then sent via high-speed telephone line to a larger computer for remote processing. The analysis corrects for baseline drifts and pre-beta asymmetry and will properly identify and quantify the amount of VLDL, LDL, and HDL with corrections for "sinking pre-beta" and "floating beta" in LDL and VLDL, respectively. Results are given in milligrams per 100 milliliter as well as percentile rank and standard deviation score ranking of each lipoprotein class as compared to an appropriate normal reference population. The latter data are in a form more meaningful to the physician and patient and provide a quantitative dimension to lipoprotein phenotyping. PMID:194006

Wong, R A; Banchero, P G; Jensen, L C; Pan, S S; Adamson, G L; Lindgren, F T



Proteomic analysis of plasma proteins in diabetic rats by 2D electrophoresis and MALDI-TOF-MS.  


Despite tremendous advances in our understanding of the molecular basis of diabetes mellitus, substantial gaps still remain in our understanding of disease pathogenesis and in the development of effective strategies for early diagnosis and treatment. The proteomic approach has offered many opportunities and challenges in identifying new marker proteins and therapeutic targets, i.e., using 2D-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. The differential protein expressions were analyzed in alloxan-induced diabetic rats treated with Cynodon dactylon leaf extract. The plant extract was administered for 15 days that resulted in a significant increase in plasma insulin and C-peptide levels. We have also identified four differentially expressed proteins from rat plasma. These four diabetes-associated proteins were broadly classified into three groups as per their function: (1) lipid metabolism-associated protein (Apo A-IV), (2) antioxidant activity-related proteins [preprohaptoglobin and heat shock proteins B8 (HspB8)], and (3) muscle function-related protein (TPM3). Apo A-IV, HspB8, and preprohaptoglobin may play a key role in the recovery of diabetes mellitus and also prevent the diabetes-associated complications such as prevention of oxidative stress due to free radical and free hemoglobin. These results show the value of proteomic approach in identifying the potential markers that may eventually serve as diagnostic markers or therapeutic targets. PMID:22258647

Karthik, D; Ilavenil, S; Kaleeswaran, B; Sunil, S; Ravikumar, S



Two dimensional gel electrophoresis analysis of DNA replication in Tetrahymena pyriformis  

E-print Network

Neutral/neutral two dimensional gel electrophoresis was used to study replication of the Tetrahymena pyriformis RDNA minichromosome. During vegetative growth RDNA is replicated exclusively from origin in the 5' nontranscribed spacer (NTS). Whereas...

Ma, Yue



Technical notes on acrylamide gel electrophoresis used for comparing isozymes of mosquito larvae.  


Various slight modifications of electrophoretic techniques were tried to obtain more reproducible results for comparing several isozymes with individual mosquito larvae. Horizontal electrophoresis was done using polyacrylamide gel plates of 1 mm thickness on a simple cooling system in which ice-cold water was circulated. This made it possible to carry out electrophoresis effectively even during field work in tropical areas. Gel concentration in routine work is 5% for usual enzymes, but for esterase, 6% gels have been adopted. Gel concentrations, running distance, running time, visualization of isozyme bands, etc., have especially been considered. PMID:6148783

Tsukamoto, M



Rheological and mechanical behavior of polyacrylamide hydrogels chemically crosslinked with allyl agarose for two-dimensional gel electrophoresis.  


Two-dimensional (2-D) gel electrophoresis currently represents one of the most standard techniques for protein separation. In addition to the most commonly employed polyacrylamide crosslinked hydrogels, acrylamide-agarose copolymers have been proposed as promising systems for separation matrices in 2-D electrophoresis, because of the good resolution of both high and low molecular mass proteins made possible by careful control and optimization of the hydrogel pore structure. As a matter of fact, a thorough understanding of the nature of the hydrogel pore structure as well as of the parameters by which it is influenced is crucial for the design of hydrogel systems with optimal sieving properties. In this work, a series of acrylamide-based hydrogels covalently crosslinked with different concentrations of allyl agarose (0.2-1%) is prepared and characterized by creep-recovery measurements, dynamic rheology and tensile tests, in the attempt to gain a clearer understanding of structure-property relationships in crosslinked polyacrylamide-based hydrogels. The rheological and mechanical properties of crosslinked acrylamide-agarose hydrogels are found to be greatly affected by crosslinker concentration. Dynamic rheological tests show that hydrogels with a percentage of allyl agarose between 0.2% and 0.6% have a low density of elastically effective crosslinks, explaining the good separation of high molecular mass proteins in 2-D gel electrophoresis. Over the same range of crosslinker concentration, creep-recovery measurements reveal the presence of non-permanent crosslinks in the hydrogel network that justifies the good resolution of low molecular mass proteins as well. In tensile tests, the hydrogel crosslinked with 0.4% of allyl agarose exhibits the best results in terms of mechanical strength and toughness. Our results show how the control of the viscoelastic and the mechanical properties of these materials allow the design of mechanically stable hydrogels with improved sieving ability in protein electrophoresis over a wide range of molecular masses. PMID:24368174

Suriano, R; Griffini, G; Chiari, M; Levi, M; Turri, S



P216-T Beyond Differential In-Gel Electrophoresis: Top-down Multiplexed Analysis in Two-dimensional Gels Using an Isobaric Mass Tagging Approach  

PubMed Central

Large sets of biological samples are commonly encountered in biomedical research, and a rigorous, reliable top-down quantification method for proteins from multiple samples is required. With differential in-gel electrophoresis (DIGE), Cy3 and Cy5 dyes are used to fluorescently label two different protein samples prior to running them on the same 2D gel. Often, Cy2 dye is used as an internal standard as well. This approach permits analysis of two to three samples under identical conditions, eliminating the need to register and match the images as in traditional 2D gels. Though conceptually seductive, problems plague DIGE, especially when attempting to perform complex quantitative proteomics studies. Since the DIGE images are generated with two to three different dyes of differing molar extinction coefficients, quantum yields, and physicochemical properties, absolute quantification of small intensity differences is challenging. Also, DIGE is overly simplistic in its basic assumption that all information required in an experiment can be obtained from a single 2D gel using an easy control-vs.-perturbed state experimental design. Multi-point time-course experiments and drug dose-response experiments require many more than the two to three sample capacity of DIGE. An isobaric mass tagging strategy is presented wherein seven different samples are simultaneously fractionated on a single 2D gel and protein abundances subsequently quantified by mass spectrometry. Workflows that replace or alternatively augment DIGE are described. Reproducibility of conventional DIGE vs. the isobaric mass tagging strategy demonstrates higher accuracy quantification obtained with the latter approach.

Xie, B.; Hekking, B.; Lisoukov, H.; Wang, Y.; Parman, C.; Patton, W. F.



Non-denaturing polyacrylamide gradient gel electrophoresis for the diagnosis of dysbetalipoproteinemia  

Microsoft Academic Search

Dysbetalipoproteinemia, an uncommon but highly atherogenic mixed hyperlipidemia due to the accumulation of remnants of triglyceride-rich lipoproteins, is characterized by cholesterol-enriched VLDL that migrates in the ? -position on agarose gels. The demonstration of a broad ? -band on agarose gel electrophoresis of plasma is an insensitive method and ultracentrifugation is an impractical method of diagnosing this condition. Non-denaturing polyacrylamide

Dirk J. Blom; Pamela Byrnes; Sheena Jones; A. David Marais



The fractionation of high-molecular-weight ribonucleic acid by polyacrylamide-gel electrophoresis  

PubMed Central

1. Gels were prepared with recrystallized acrylamide and bisacrylamide. Electrophoresis was in trissodium acetateEDTA buffer for 05 to 3hr. Gels were scanned at 280 or 265m?. Techniques are described for slicing and radioactive counting. 2. The mobility of RNA was inversely related to the sedimentation coefficient and varied with gel concentration. Electrophoresis in 2226% gels gives a fractionation similar to density-gradient centrifugation. It shows the two ribosomal RNA components, the 45s precursor, transfer RNA and minor components. In 5% and 75% gels, 4s and 5s RNA separated and ribosomal RNA was excluded. 3. The resolution is greater and more detailed than by centrifugation, and many samples can be analysed simultaneously and rapidly. PMID:5339944

Loening, U. E.



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

PubMed Central

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

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



On the limiting pore size of hydrophilic gels for electrophoresis and isoelectric focusing.  


The maximum pore diameter that can be obtained in hydrophilic gels, either highly diluted agarose (0.16%C) or highly cross-linked polyacrylamide (45%CBis or 60%C DHEBA) is around 500 nm. An empirical equation has been derived linking the mean pore diameter (mean p) to gel concentration (C) in dilute agarose gels: mean p = 140.7 x C(-0.7). It is suggested that other equations hold for concentrated gels and for highly cross-linked polyacrylamides, since the matrix structure is different. Most of the cross linkers for polymerizing polyacrylamide gels have been tabulated and their properties studied. A new gel matrix is described: a highly cross-linked N,N'-(1,2-dihydroxyethylene)bisacrylamide gel, which is hydrophilic, highly porous and can be conveniently used for electrophoresis in horizontal, ultrathin layers cast on silanized glass surfaces. PMID:7252045

Righetti, P G; Brost, B C; Snyder, R S



Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots  


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

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



Multilocus enzyme analysis in aerobic and anaerobic bacteria using gel electrophoresis-nitrocellulose blotting.  


An optimized multilocus enzyme electrophoresis method, which involves polyacrylamide-agarose gel electrophoresis followed by electrophoretic transfers on nitrocellulose sheets, was developed for the analysis of enzyme polymorphism in several aerobic and anaerobic bacterial species including Staphylococcus aureus, Streptococcus pneumoniae, S. agalactiae, Klebsiella pneumoniae and K. oxytoca, Clostridium bifermentans and C. sordellii, and Prevotella bivia. Serial electrophoretic transfers (during 5-15 min each) from a single polyacrylamide gel could be achieved for most enzymes studied, and allowed an increased definition of enzyme bands on nitrocellulose as compared to migration gels. Four enzymes, which could not be blotted in such conditions, could still be stained in gels after blotting. Thus, the method allowed the combined analysis of several enzymes after a single gel electrophoresis separation. The analysis of enzyme polymorphism in the various species studied raised the interest of polymorphic loci such as esterase or glutamic-oxaloacetic transaminase for epidemiologic studies. The method characterized a genetic diversity of enzyme loci of S. pneumoniae higher than previously reported, and is thus convenient for the analysis of genetic relationships between related isolates. Since the present method reduces the tediousness of multilocus enzyme electrophoresis and requires experimental conditions that are not specific for the bacterial population studied, it may be proposed for rapid population genetics analysis of a wide variety of bacteria. PMID:10754243

Combe, M; Lemeland, J; Pestel-Caron, M; Pons, J



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

PubMed Central

Background The endemic seagrass Posidonia oceanica (L.) Delile colonizes soft bottoms producing highly productive meadows that play a crucial role in coastal ecosystems dynamics. Human activities and natural events are responsible for a widespread meadows regression; to date the identification of "diagnostic" tools to monitor conservation status is a critical issue. In this study the feasibility of a novel tool to evaluate ecological impacts on Posidonia meadows has been tested. Quantification of a putative stress indicator, i.e. phenols content, has been coupled to 2-D electrophoretic protein analysis of rhizome samples. Results The overall expression pattern from Posidonia rhizome was determined using a preliminary proteomic approach, 437 protein spots were characterized by pI and molecular weight. We found that protein expression differs in samples belonging to sites with high or low phenols: 22 unique protein spots are peculiar of "low phenols" and 27 other spots characterize "high phenols" samples. Conclusion Posidonia showed phenols variations within the meadow, that probably reflect the heterogeneity of environmental pressures. In addition, comparison of the 2-D electrophoresis patterns allowed to highlight qualitative protein expression differences in response to these pressures. These differences may account for changes in metabolic/physiological pathways as adaptation to stress. A combined approach, based on phenols content determination and 2-D electrophoresis protein pattern, seems a promising tool to monitor Posidonia meadows health state. PMID:17663776

Migliore, Luciana; Rotini, Alice; Randazzo, Davide; Albanese, Nadia N; Giallongo, Agata



Subtyping of Haemophilus influenzae Strains by Pulsed-Field Gel Electrophoresis  

Microsoft Academic Search

A total of 200 isolates of Haemophilus influenzae were analyzed by serotyping, biotyping, and pulsed-field gel electrophoresis (PFGE). A total of 178 epidemiologically unrelated strains of H. influenzae demonstrated a variety of genome patterns by PFGE, and 165 genotypes were thus obtained in this study. PFGE typing proved to have a much stronger discriminatory power than either serotyping or biotyping.




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

E-print Network

On-Chip Native Gel Electrophoresis-Based Immunoassays for Tetanus Antibody and Toxin Amy E. Herr was developed for detection of tetanus antibodies in buffer as well as diluted serum samples. After an off was 0.68 nM. A competitive immunoassay was also developed for tetanus toxin C-fragment by allowing un

Herr, Amy E.


A simple monolithic column electroelution for protein recovery from gel electrophoresis.  


Protein recovery from gel electrophoresis plays an important role in functional genomics and proteomics but faces a series of issues (e.g., complex procedure, low recovery, long experimental time). In this study, a monolithic column electroelution (MCE) was developed for protein recovery from gel electrophoresis. With the model proteins of bovine serum albumin (BSA), hemoglobin (Hb), and myoglobin (Mb), the developed device and method were compared with common electroelution procedures in agarose gel electrophoresis (AGE). The comparative experiments revealed that (i) the protein recovery achieved with the developed device was greater than 83%, much higher than the 41% to 50% achieved with the common devices; (ii) the running time to obtain 70% recovery was approximately 15 min, evidently shorter than the 240 min with the common devices; and (iii) the device and procedure were simple and less time-consuming as compared with those of the common devices. It was observed that the serum protein bands cut from polyacrylamide gel electrophoresis could be transferred into solution in 15 to 30 min with 82% yield. The device, along with its relevant procedure, has potential use in protein extraction and proteomics as well as in DNA studies. PMID:22800655

Li, Guo-Qing; Shao, Jing; Guo, Chen-Gang; Dong, Jing-Yu; Fan, Liu-Yin; Cao, Cheng-Xi



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



Pulsed-field gel electrophoresis for isolation of full-length phytoplasma chromosomes from plants.  


Pulsed-field gel electrophoresis (PFGE) is a powerful technique for genomic studies of unculturable plant-pathogenic phytoplasmas, which enables separation of full-length phytoplasma chromosomes from contaminating host plant nucleic acids. The PFGE method described here involves isolation of phytoplasmal DNA from high-titer phytoplasma-infected herbaceous plants using a phytoplasma enrichment procedure, embedding of phytoplasma chromosomes in agarose blocks, and separation of entire phytoplasma chromosomes from contaminating host plant nucleic acids by electrophoresis. Full-length phytoplasma chromosomes are resolved as single, discrete bands in the gel. The identity of these bands can be confirmed by Southern blot hybridization using a ribosomal DNA fragment as a probe. The method does not utilize gamma-irradiation to linearize phytoplasma chromosomes prior to electrophoresis. PMID:22987433

Marcone, Carmine



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.



Antioxidant effects of carnitine supplementation on 14-3-3 protein isoforms in the aged rat hippocampus detected using fully automated two-dimensional chip gel electrophoresis.  


Abstract We here described the antioxidant effects of carnitine supplementation on 14-3-3 protein isoforms in the aged rat hippocampus detected using the fully automated two-dimensional chip gel electrophoresis system (Auto2D). This system was easy and convenient to use, and the resolution obtained was more sensitive and higher than that of conventional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). We separated and identified five isoforms of the 14-3-3 protein (beta/alpha, gamma, epsilon, zeta/delta, and eta) using the Auto2D system. We then examined the antioxidant effects of carnitine supplementation on the protein profiles of the cytosolic fraction in the aged rat hippocampus, demonstrating that carnitine supplementation suppressed the oxidation of methionine residues in these isoforms. Since methionine residues are easily oxidized to methionine sulfoxide, the convenient and high-resolution 2-D PAGE system can be available to analyze methionine oxidation avoiding artifactual oxidation. We showed here that the Auto2D system was a very useful tool for studying antioxidant effects through proteomic analysis of protein oxidation. PMID:25179439

Iwamoto, M; Miura, Y; Tsumoto, H; Tanaka, Y; Morisawa, H; Endo, T; Toda, T



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


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



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.



Trapping of megabase-sized DNA molecules during agarose gel electrophoresis  

PubMed Central

Megabase DNA molecules become trapped in agarose gels during electrophoresis if the electric field exceeds a few volts per cm. Fluorescence microscopy reveals that these molecules invariably arrest in U-shaped conformations. The field-vs.-size dependence for trapping indicates that a critical molecular tension is required for trapping. The size of unligated ?-ladders, sheared during gel electrophoresis at a given field, coincides with the size of molecules trapped at that field, suggesting that both processes occur through nick melting near the vertex of the U-shape. Consistently, molecules nicked by exposure to UV radiation trap more readily than unexposed ones. The critical trapping tension at the vertex is estimated to be 15 pN, a force sufficient to melt nicks bent around gel fibers, and, according to our model, trap a molecule. Strategies to reduce molecular tension and avoid trapping are discussed. PMID:9892654

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



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.



Genetic distances in the tribe Bovini studied by gel electrophoresis  

E-print Network

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

Butts, Kelley Elaine McRae



Aligning goals, assessments, and activities: an approach to teaching PCR and gel electrophoresis.  


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

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



Continuous monitoring of enzymatic activity within native electrophoresis gels: Application to mitochondrial oxidative phosphorylation complexes  

PubMed Central

Native gel electrophoresis allows the separation of very small amounts of protein complexes while retaining aspects of their activity. In-gel enzymatic assays are usually performed by using reaction-dependent deposition of chromophores or light scattering precipitates quantified at fixed time points after gel removal and fixation, limiting the ability to analyze enzyme reaction kinetics. Herein, we describe a custom reaction chamber with reaction media recirculation and filtering and an imaging system that permits the continuous monitoring of in-gel enzymatic activity even in the presence of turbidity. Images were continuously collected using time-lapse high resolution digital imaging, and processing routines were developed to obtain kinetic traces of the in-gel activities and analyze reaction time courses. This system also permitted the evaluation of enzymatic activity topology within the protein bands of the gel. This approach was used to analyze the reaction kinetics of two mitochondrial complexes in native gels. Complex IV kinetics showed a short initial linear phase where catalytic rates could be calculated, whereas Complex V activity revealed a significant lag phase followed by two linear phases. The utility of monitoring the entire kinetic behavior of these reactions in native gels, as well as the general application of this approach, is discussed. PMID:22975200

Covian, Raul; Chess, David; Balaban, Robert S.



Diffusion of DNA during gel electrophoresis; a predictive function spanning the relevant regimes  

NASA Astrophysics Data System (ADS)

Gel electrophoresis is used extensively to separate DNA. Diffusion of the DNA bands during electrophoresis is an important phenomenon which reduces the resolution obtained. As with DNA mobility, the diffusion of DNA can be split into several different regimes, each described by relevant theory. Unfortunately, until recently there was no single formula for DNA mobility or diffusion that could be used in more than one regime. However, Van Winkle and co workers [Van Winkle DH, Beheshti A, Rill RL, ELECTROPHORESIS 23 (1): 15-19 JAN 2002] have successfully developed an analytical function to analyze DNA mobility data, throughout the relevant regimes. We present the development of a complementary function for the analysis of DNA diffusion. This function should be very useful both in analyzing DNA electrophoretic data, and as a predictive tool.

McCormick, Laurette; Slater, Gary



Detection of cation-specific conformational changes in ribosomal RNA by gel electrophoresis.  


Electrophoresis of ribosomal RNA in polyacrylamide-agarose composite gels separates 16S and 23S species into multiple bands. These bands of RNA represent multiple conformational forms of the molecules as judged by oligonucleotide analysis of the 16S RNA. Gel electrophoresis was used to test for cation-specific conformational changes in ribosomal RNA. Relative to magnesium-equilibrated RNA, barium ion and putrescine induced alterations in the electrophoretic behavior of ribosomal RNA while calcium ion produced no change. Exchange of a critical level of bound magnesium ion for barium or putrescine was necessary for these changes to take place. The alterations in electrophoretic behavior were unaffected by simply restoring magnesium ion, but in addition required heating for reversal. We suggest that these conformational changes are a result of interaction at a specific class of cation binding sites previously observed with intact ribosomes. PMID:10793686

Morris, D R; Dahlberg, J E; Dahlberg, A E



Detection of cation-specific conformational changes in ribosomal RNA by gel electrophoresis  

PubMed Central

Electrophoresis of ribosomal RNA in polyacrylamide-agarose composite gels separates 16S and 23S species into multiple bands. These bands of RNA represent multiple conformational forms of the molecules as judged by oligonucleotide analysis of the 16S RNA. Gel electrophoresis was used to test for cation-specific conformational changes in ribosomal RNA. Relative to magnesium-equilibrated RNA, barium ion and putrescine induced alterations in the electrophoretic behavior of ribosomal RNA while calcium ion produced no change. Exchange of a critical level of bound magnesium ion for barium or putrescine was necessary for these changes to take place. The alterations in electrophoretic behavior were unaffected by simply restoring magnesium ion, but in addition required heating for reversal. We suggest that these conformational changes are a result of interaction at a specific class of cation binding sites previously observed with intact ribosomes. Images PMID:10793686

Morris, David R.; Dahlberg, James E.; Dahlberg, Albert E.



Rapid Discrimination of Listeria monocytogenes Strains by Microtemperature Gradient Gel Electrophoresis  

Microsoft Academic Search

Microtemperature gradient gel electrophoresis (-TGGE) was examined for use for the rapid subtyping of Listeria monocytogenes strains. Comparison of genomes between L. monocytogenes strains F2365 and H7858 identified a sequence encoding a portion of the PRT\\/PTS system IIA 2 protein domain as appropriate for -TGGE analysis. Thirty-one strains belonging to 10 different serovar types were tested by PCR, and sequence

Tatsuya Tominaga



Two-dimensional gel electrophoresis; better than a poke in the ICAT?  

Microsoft Academic Search

To date, the most widely used technology for conducting proteomic studies has been two-dimensional gel electrophoresis (2DGE), but this approach does have drawbacks. Isotope-coded affinity tagging (ICAT) is starting to challenge 2DGE as a new proteomic tool for the analysis of proteins in complex biological specimens. An appraisal of these two methodologies reveals that neither ICAT nor 2DGE provide comprehensive

Wayne F Patton; Birte Schulenberg; Thomas H Steinberg



Assessment of Microbial Populations Dynamics in a Blue Cheese by Culturing and Denaturing Gradient Gel Electrophoresis  

Microsoft Academic Search

The composition and development of microbial population during the manufacture and ripening of two batches of a blue-veined\\u000a cheese was examined by culturing and polymerase chain reaction (PCR) denaturing gradient gel electrophoresis (DGGE) (PCRDGGE).\\u000a Nine selective and\\/or differential media were used to track the cultivable populations of total and indicator microbial groups.\\u000a For PCRDGGE, the V3 hyper variable region of

ngel Alegra; Renata Gonzlez; Mario Daz; Baltasar Mayo



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

Microsoft Academic Search

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

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



Single-color laser-induced fluorescence detection and capillary gel electrophoresis for DNA sequencing  

NASA Astrophysics Data System (ADS)

Low zeptomole (1 zmol equals 10-21 mol = 600 molecules) detection limits are produced for DNA sequencing by capillary gel electrophoresis. A 750 (mu) w green helium-neon laser ((lambda) equals 543.5 nm) is used to excite tetramethylrhodamine-labeled DNA fragments in a sheath-flow cuvette. A cooled photomultiplier tube is used to detect fluorescence in a single spectral channel. Sequencing data is generated at a rate of about 70 bases/hour.

Chen, Da Yong; Swerdlow, Harold; Harke, Heather; Zhang, Jian Z.; Dovichi, Norman J.



Single-color laser-induced fluorescence detection and capillary gel electrophoresis for DNA sequencing  

Microsoft Academic Search

Low zeptomole (1 zmol equals 10-21 mol = 600 molecules) detection limits are produced for DNA sequencing by capillary gel electrophoresis. A 750 (mu) w green helium-neon laser ((lambda) equals 543.5 nm) is used to excite tetramethylrhodamine-labeled DNA fragments in a sheath-flow cuvette. A cooled photomultiplier tube is used to detect fluorescence in a single spectral channel. Sequencing data is

Da Yong Chen; H. Swerdlow; Heather Harke; Jian Z. Zhang; Norman J. Dovichi



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


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



Proteome analysis of glycoforms: a review of strategies for the microcharacterisation of glycoproteins separated by two-dimensional polyacrylamide gel electrophoresis.  


Preparative two-dimensional polyacrylamide electrophoresis (2-D PAGE) is a method of separation which for the first time allows protein isoforms to be readily purified for subsequent analysis. The profile of the 2-D separation of the protein complement (proteome) of eukaryotic cells and tissues typically contains obvious 'trains' of spots which differ in pI and/or apparent molecular mass. These are usually isoforms of the same protein and result from post-translational modifications. There is growing evidence that alterations to the glycosylation and/or phosphorylation of a protein can be correlated with developmental and pathological changes; these changes can be visualised on the 2-D separation. It is not clear, however, how these modifications alter the structural properties of the protein and affect their migration in this mode of separation. Strategies need to be developed to obtain a more detailed understanding of the reason for the appearance of isoforms as discrete spots on 2-D PAGE. Standard proteins, fetuin and ovalbumin, were used to monitor the effect of the removal of glycans and phosphates on the migration of the glycoproteins in the 2-D system. The isoforms were not simply explained by the presence or absence of a single modification. To further investigate the reasons for the different migration of the isoforms it is necessary to characterise the modifications in more detail. Unlike protein analysis, until recently the available methodology for the analysis of the glycans attached to proteins has not been sensitive enough to allow analysis of single spots in gels or blots resulting from 2-D electrophoresis. In this paper we review current and future strategies for characterisation of protein modifications using single spots from 2-D gels. PMID:9150924

Packer, N H; Pawlak, A; Kett, W C; Gooley, A A; Redmond, J W; Williams, K L



DNA electrophoresis in agarose gels: A new mobility vs. DNA length dependence  

NASA Astrophysics Data System (ADS)

Separations were performed on double stranded DNA (dsDNA) using electrophoresis. Electrophoresis is the steady transport of particles under the influence of an external electric field. Double stranded DNA fragments ranging in length from 200 base pairs (bp) to 194,000 bp (0.34 nm = 1 bp) were electrophoresed at agarose gel concentrations T = 0.4%--1.5%. The electric field was varied from 0.62 V/cm to 6.21 V/cm. A wide range of electric fields and gel concentrations were used to study the usefulness of a new interpolation equation, 1mL =1mL-( 1mL-1 ms)e-L/g , where mL,ms , and g are independent free fitting parameters. The long length mobility limit is interpreted as mL , the short length mobility limit is ms , and g is the crossover between the long length limit and the short length limit. This exponential relation fit very well (chi2 ? 0.999) when there are two smooth transitions observed in the "reptation plots" (plotting 3mL/m? vs. L) (J. Rousseau, G. Drouin, and G. W. Slater, Phys Rev Lett. 1997, 79, 1945--1948). Fits deviate from the data when three different slopes were observed in the reptation plots. Reptation plots were used to determine a phase diagram for dsDNA migration regimes. The phase diagrams define different regions where mechanisms for molecular transport affect the migration of dsDNA in agarose gels during electrophoresis. The parameters from the equation have also been interpreted to provide a physical description of the structure of the agarose gel by calculating the pore sizes. The relations between the values for the pore sizes and the phase diagrams are interpreted to better understand the migration of the DNA through agarose gels.

Beheshti, Afshin



Comparative fluorescence two-dimensional gel electrophoresis using a gel strip sandwich assembly for the simultaneous on-gel generation of a reference protein spot grid.  


The comparison of proteins separated on 2DE is difficult due to gel-to-gel variability. Here, a method named comparative fluorescence gel electrophoresis (CoFGE) is presented, which allows the generation of an artificial protein grid in parallel to the separation of an analytical sample on the same gel. Different fluorescent stains are used to distinguish sample and marker on the gel. The technology combines elements of 1DE and 2DE. Special gel combs with V-shaped wells are placed in a stacking gel above the pI strip. Proteins separated on the pI strip are electrophoresed at the same time as marker proteins (commercially available purified protein of different molecular weight) placed in V-wells. In that way, grids providing approximately 100 nodes as landmarks for the determination of protein spot coordinates are generated. Data analysis is possible with commercial 2DE software capable of warping. The method improves comparability of 2DE protein gels, because they are generated in combination with regular in-gel anchor points formed by protein standards. This was shown here for two comparative experiments with three gels each using Escherichia coli lysate. For a set of 47 well-defined samples spots, the deviation of the coordinates was improved from 7% to less than 1% applying warping using the marker grid. Conclusively, as long as the same protein markers, the same size of pI-strips and the same technology are used, gel matching is reproducibly possible. This is an important advancement for projects involving comparison of 2DE-gels produced over several years and in different laboratories. PMID:22648808

Ackermann, Doreen; Wang, Weiqun; Streipert, Benjamin; Geib, Birgit; Grn, Lothar; Knig, Simone



Caged amphibian tadpoles and in situ genotoxicity monitoring of aquatic environments with the alkaline single cell gel electrophoresis (comet) assay  

Microsoft Academic Search

In previous studies we demonstrated that indigenous amphibian tadpoles are suitable organisms for monitoring small bodies of water (e.g., creeks, ponds, and drainage ditches) using the alkaline single cell gel electrophoresis (SCG) or `comet' assay. This approach involves detection, under alkaline conditions, of cell DNA fragments which on electrophoresis migrate from the nuclear core, resulting in a `comet with tail'

Steven Ralph; Michael Petras



[Effects of the Radix Ranuncoli Ternati extracts on Mycobacterium tuberculosis proteome profiling revealed by 2D electrophoresis].  


Radix Ranuncoli Ternati is clinically effective traditional Chinese medicine for multidrug resistant tuberculosis. Its active components and mechanism of action remain unsolved. Two dimensional gel electrophoresis (2-DE) was employed to address this problem. Globlal proteome of Mycobacterium tuberculosis untreated and treated with Radix Ranuncoli Ternati were compared, and 22 protein spots were found to be expressed differentially. 3 protein spots which remarkably decreased in Mycobacterium tuberculosis treated with Radix Ranuncoli Ternati were subjected to matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis. The data obtained from peptide mass finger printing were used for database search. The 3 protein spots in gel were identified as cysA2 (thiosulfate sulfurtransferase), tsf (elongation factor EF-Ts) and hspX (heat shock protein X). These data provide insights into the changed global protein patterns of Mycobacterium tuberculosis treated with Radix Ranuncoli Ternati and may prove useful for further study in the mechanism in how Radix Ranuncoli Ternati influence the life of Mycobacterium tuberculosis. The differentially expressed proteins may be potential novel antituberculosis drug targets. PMID:16496699

He, Ying; Yue, Jun; Hu, Chang-Hua; Wang, Hong-Hai; Xie, Jian-Ping; Tang, Cui; Wei, Xiu-Li; Liu, Xue-Mei; Song, Jie



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


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

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



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

SciTech Connect

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

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



Dispersion functions and factors that determine resolution for DNA sequencing by gel electrophoresis  

SciTech Connect

The number of bases that can be read in a single run by a DNA sequencing instrument that detects fluorophore labeled DNA arriving at a ``finish-line`` located a fixed distance from the starting wells is influenced by numerous parameters. Strategies for improving the length-of-read of a DNA sequencer can be based on quantitative models of the separation of DNA by gel electrophoresis. The dispersion function of the electrophoretic system--the relationship between molecular contour length and time of arrival at the detector--is useful in characterizing the performance of a DNA sequencer. We adapted analytical representations of dispersion functions, originally developed for snapshot imaging of DNA gels, (samples electrophoresed for constant time), to finish-line imaging, and demonstrated that a logistic-type function with non-integral exponent is required to describe the experimental data. We use this dispersion function to determine the resolution length and resolving power of a LI-COR DNA sequencing system and a custom built capillary gel electrophoresis system, and discuss the factors that presently limit the number of bases that can be determined reliably in a single sequencing run.

Sutherland, J.C.; Reynolds, K.J.; Fisk, D.J.



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



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

SciTech Connect

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

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



Separation of rainbow trout (Salmo gairdneri) growth hormone by gel electrophoresis.  


Pituitaries from immature (n = 12) and mature female (n = 15) rainbow trout were cultured separately in vitro and subjected to polyacrylamide gel electrophoresis. Four protein bands were identified from the immature rainbow trout and three from the adults. The material from the immature trout was used to raise antisera. Three of the bands, including those with the highest (0.74) and lowest (0.27) Rf values, produced antibodies. Immunocytochemical studies revealed that all of the antisera bound strongly to the growth hormone cells and weakly, if at all, to prolactin cells in pituitary sections from rainbow trout. PMID:2127033

Skarphedinsson, O; Power, D M; Ingleton, P M



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


Blue native gel electrophoresis (BN-PAGE) is used extensively for characterization of mitochondrial respiratory complexes and uses the binding of Coomassie brilliant blue G-250 to visualize proteins. Oxidative modification of sulfhydryl groups of such proteins can be evaluated by labeling with iodoacetamide conjugated to biotin (BIAM) and detected with streptavidin peroxidase on Western blots following BN-PAGE. However, dissolving BIAM in dimethylformamide, a recommended solvent, reduces Coomassie blue G staining to proteins during BN-PAGE. This interference is prevented by dissolving BIAM in dimethyl sulfoxide. Precautions in the use of the dye for protein staining subsequent to BIAM labeling are discussed. PMID:24662748

Raghupathy, V; Oommen, Anna; Ramachandran, Anup



Application of denaturing gradient gel electrophoresis to detect DNA sequence differences encoding apolipoprotein E isoforms  

SciTech Connect

Apolipoprotein E (apoE) plays an important role in plasma lipid metabolism. Three common isoforms of this protein have been identified by the isoelectric focusing method. In this report the authors describe a new method for distinguishing these isoforms. Their method employs PCR amplification of the DNA sequence of exon 4 in the apoE gene followed by denaturing gradient gel electrophoresis (DGGE) to distinguish its different melting characteristics. Identification of the ApoE isoforms through DNA melting behavior rather than protein charge differences eliminates the problems associated with isoelectric focusing and facilitates screening for additional mutations at the apoE locus. 12 refs., 2 figs.

Parker, S.; Angelico, M.C.; Laffel, L.; Krolewski, A.S. (Joslin Diabetes Center, Boston, MA (United States) Harvard Medical School, Boston, MA (United States))



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.



Detection of cation-specific conformational changes in ribosomal RNA by gel electrophoresis.  


Electrophoresis of ribosomal RNA in polyacrylamide-agrose composite gels separates 16S and 23S species into multiple bands. These bands of RNA represent multiple conformational forms of the molecules as judged by oligonucleotide analysis of the 16S RNA. Gel elctrophoresis was used to test for cation-specific conformational changes in ribosomal RNA. Relative to magnesium-equilibrated RNA, barium ion and putrescine induced alterations in the electrophoretic behavior of ribosomal RNA while calcium ion produced no change. Exchange of a critical level of bound magnesium ion for barium or putrescine was necessary for these changes to take place. The alterations in electrophoretic behavior were unaffected by simply restoring magnesium ion, but in addition required heating for reversal. We suggest that these conformational changes are a result of interaction at a specific class of cation binding sites previously observed with intact ribosomes. PMID:1094419

Morris, D R; Dahlberg, J E; Dahlberg, A E



Detection of cation-specific conformational changes in ribosomal RNA by gel electrophoresis.  

PubMed Central

Electrophoresis of ribosomal RNA in polyacrylamide-agrose composite gels separates 16S and 23S species into multiple bands. These bands of RNA represent multiple conformational forms of the molecules as judged by oligonucleotide analysis of the 16S RNA. Gel elctrophoresis was used to test for cation-specific conformational changes in ribosomal RNA. Relative to magnesium-equilibrated RNA, barium ion and putrescine induced alterations in the electrophoretic behavior of ribosomal RNA while calcium ion produced no change. Exchange of a critical level of bound magnesium ion for barium or putrescine was necessary for these changes to take place. The alterations in electrophoretic behavior were unaffected by simply restoring magnesium ion, but in addition required heating for reversal. We suggest that these conformational changes are a result of interaction at a specific class of cation binding sites previously observed with intact ribosomes. Images PMID:1094419

Morris, D R; Dahlberg, J E; Dahlberg, A E



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.



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


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

Zhu, Xiaoming; Mason, Thomas G



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

NASA Astrophysics Data System (ADS)

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

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



Analysis of simian virus 40 wild-type and mutant virions by agarose gel electrophoresis.  


Intact wild-type simian virus 40 particles can be separated and resolved from a temperature-sensitive mutant and from a number of other viruses by agarose gel electrophoresis. The relative mobilities of the viruses appear to be a function of both virion size and surface composition. The virions of a temperature-sensitive strain of simian virus 40, tsB204, have significantly greater mobility than those of wild-type simian virus 40, when electrophoresis was conducted toward the cathode at pH 5.0. When electrophoresis was performed toward the anode at pH 7.0, TSB204 viruses have a slightly slower mobility as compared with that of the wild type. The data indicated that the virions of tsB204 have a greater positive charge at their surface than those of wild-type particles. No differences were detected in the finger print patterns of the tryptic peptides of VP1 and VP3 of these two virus strains. Although it was not possible to identify the structural polypeptide directly affected by the tsB204 mutation, we have shown that this mutation affects charge distribution on the surface of the virion. PMID:176451

Zweig, M; Barban, S; Salzman, N P



Evaluation of denaturing gradient gel electrophoresis to differentiate Escherichia coli populations in secondary environments.  


The development of methodology to differentiate mixed populations of Escherichia coli in the secondary habitat might improve monitoring of fecal pollution indicators and facilitate the development of strategies to mitigate bacterial pollution. The objective of this study was to determine the ability of denaturing gradient gel electrophoresis (DGGE) to differentiate mixed assemblages of E. coli in the natural environment. After confirming the identity of 184 environmental bacterial isolates as E. coli, each was subjected to polymerase chain reaction (PCR) of the beta-glucuronidase gene (uidA) followed by DGGE fingerprinting. The ability of DGGE to discriminate individual isolates at the strain level was determined by comparing fingerprints to those resulting from a standard, library-dependent fingerprinting method, BOX-PCR. Computerized analysis of fingerprints indicated that DGGE and BOX-PCR identified 15 and 21 unique phylotypes respectively. Rank-abundance plots comparing the numerical distribution of unique E. coli phylotypes detected by both methods revealed no difference in resolution at the population level. In water and sediment samples from two beaches, DGGE effectively distinguished indigenous E. coli populations with an average rate of correct classification (site-based) of 83%. Denaturing gradient gel electrophoresis of uidA genes isolated and PCR-amplified from environmental samples appears to be an effective tool to differentiate unique E. coli populations and should be useful to characterize E. coli dynamics in the secondary environment. PMID:16958751

Sigler, Von; Pasutti, Lauren



Use of pulsed-field gel electrophoresis to measure DNA damage and repair  

SciTech Connect

A method is described here for the analysis of single-strand break formation and repair in genomic DNA. The procedure involves exposing cells to a DNA-damaging agent, allowing time for recovery, and embedding the cells in agarose. After lysis and digestion with a protease, the DNA, which remains in the agarose plug, is denatured with glyoxal and separated by pulsed-field gel electrophoresis. The DNA in the gel is then transferred to a support membrane and quantitated with a radioanalytic imaging system to determine the average size of the DNA at each time point of recovery. The results indicate that the repair of methyl-induced breaks in total genomic DNA is approximately 80% complete in 48 hr in CHO B11 and ARL 14 cells exposed to dimethyl sulfate. These results are in agreement with those obtained by using other techniques like alkaline sucrose sedimentation. The method developed and described here has several advantages over existing techniques for repair measurements: It can be used to monitor genotoxic agents that nick DNA, to study the removal of breaks from genomic DNA, and to test for repair of damage in specific domains of chromatin that would be too large to examine by conventional electrophoresis.

Scicchitano, D.A. (American Health Foundation, Valhalla, NY (United States) New York Univ., New York (United States))



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.



Temperature gradient gel electrophoresis analysis of the beta-NGF gene in schizophrenia.  


Methods for localizing functional polymorphisms in candidate genes are important for the elucidation of pathogenesis in complex diseases such as schizophrenia and manic depression. Temperature gradient gel electrophoresis (TGGE), a variant of denaturing gradient gel electrophoresis (DGGE), can detect single-base mutations in a specified region of double-stranded DNA. This technique has been evaluated for use with polymerase chain reaction (PCR)-generated DNA fragments containing either transitional (A to G) or transversional (T to A) mutations. Single-base mutations of both types are detectable in PCR fragments up to 500 bp long. This method was then used to examine the coding region of the beta-nerve growth factor (NGF) gene for polymorphisms in PCR-generated DNA fragments derived from lymphocyte DNA of subjects with schizophrenia and normal subjects. No single-base mutations in sequence coding for the mature beta-NGF peptide were found in any of the subjects who were examined. If DNA sequence information is available for PCR primer design, TGGE detection of DNA polymorphisms can be used to rapidly determine whether or not a defect in a gene of interest contributes to the pathophysiology of the illness. PMID:7786881

Khan, A S; Freedman, R; Byerley, W; Leonard, S



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 (more)

Hofle, Michael David



Application of an improved system of electrophoresis in acrylamide gel to studies on the sera of different species.  


Polyacrylamide gels constitute a generally better matrix for routine electrophoresis of serum proteins than other media commonly employed, but the immunoglobulin fractions of the largest size may not migrate into gels in which the acrylamide concentration exceeds 5%. To facilitate adequate separation, slabs containing a semi-solid layer were prepared from discontinuous gels consisting of 2 to 8% acrylamide. Serum samples were subjected to electrophoresis, under carefully controlled conditions at pH 9.0, by means of a pulsed constant power supply. The method provided a rapid, reproducible, and relatively simple technique for the study of human serum proteins, either by electrophoresis or by immunoelectrophoresis, and for differentiation of serum samples from various animal species. PMID:5043376

Hyslop, N S



Buffer optimization for high resolution of human lung cancer tissue proteins by two-dimensional gel electrophoresis.  


A problem in proteomic analysis of lung cancer tissue is the presence of complex components of different histological backgrounds (squamous cell carcinoma, small cell lung carcinoma, and adenocarcinoma). The efficient solubilization of protein components before two-dimensional electrophoresis (2-DE) is a very critical. Poor solubilization has been associated with a failure to detect proteins and diffuse, streaked and/or trailing protein spots. Here, we have optimized the solubilization of human lung cancer tissue to increase protein resolution. Isoelectric focusing (IEF) rehydration buffer containing a thiourea-urea mixture provided superior resolution, whereas a buffer without thiourea yielded consistently poor results. In addition, IEF rehydration buffers containing CHAPS and DTT gave superior resolution, whereas buffers containing Nonidet P-40 (NP-40) and/or Triton X-100 did not. A tributylphosphine-containing buffer gave consistently poor results. Using optimized conditions, we used 2-D gel analysis of human lung cancer tissue to identify 11 differentially-expressed protein spots by MALDI-mass spectrometry. This study provides a methodological tool to study the complex mammalian proteomes. PMID:18800191

Lee, Kibeom; Pi, Kyungbae; Lee, Keeman



Measurement of protein sulfhydryls in response to cellular oxidative stress using gel electrophoresis and multiplexed fluorescent imaging analysis.  


The significance of free radicals in biology has been established by numerous investigations spanning a period of over 40 years. Whereas there are many intracellular targets for these radical species, the importance of cysteine thiol posttranslational modification has received considerable attention. The current studies present a highly sensitive method for measurement of the posttranslational modification of protein thiols. This method is based on labeling of proteins with monofunctional maleimide dyes followed by 2D gel electrophoresis to separate proteins and multiplexed fluorescent imaging analysis. The method correctly interrogates the thiol/disulfide ratio present in commercially available proteins. Exposure of pulmonary airway epithelial cells to high concentrations of menadione or t-butyl hydroperoxide resulted in the modification of cysteines in more than 141 proteins of which 60 were subsequently identified by MALDI-TOF/TOF MS. Although some proteins were modified similarly by these two oxidants, several showed detectably different maleimide ratios in response to these two agents. Proteins that were modified by one or both oxidants include those involved in transcription, protein synthesis and folding, and cell death/growth. In conclusion, these studies provide a novel procedure for measuring the redox status of cysteine thiols on individual proteins with a clearly demonstrated applicability to interactions of chemicals with pulmonary epithelial cells. PMID:18416539

Spiess, Page C; Morin, Dexter; Jewell, William T; Buckpitt, Alan R



Data-base techniques for multiple two-dimensional polyacrylamide gel electrophoresis analyses.  


Two-dimensional protein electrophoresis can benefit from a powerful set of computer-supported image processing and data structure/management procedures. Detection of quantitative differences is complicated by local inhomogeneities in the polyacrylamide base; biochemical changes and variations in temperature and preparative technique also make the between-gel density and x-y coordinate correspondences quite imprecise. The program presented here provides local alignment and computer-controlled variable "flicker" rates for multiple gels, with use of an interactive display system. Manual spot densitometry, referred to a National Bureau of Standards density wedge, can be complemented by a set of automatic densitometry routines for previously established lists of spots. The ability to establish a set of local landmarks, either by included standards or user identification, provides a basis for automatic n-way gel comparison for subsets or for the entire set of spots. Automatic segmentatin algorithms allow isolatin of spots and separation of touching and partially overlapping regions. Various analytical and statistical facilities are part of the user's access to the interactively developed data base. The data-structure and image-manipulation techniques developed here allow for user-directed and heuristic comparisons with online presentation of intermediate and final results. PMID:6996868

Lipkin, L E; Lemkin, P F



Altered Protein Expression of Streptococcus oralis Cultured at Low pH Revealed by Two-Dimensional Gel Electrophoresis  

PubMed Central

Streptococcus oralis is the predominant aciduric nonmutans streptococcus isolated from the human dentition, but the role of this organism in the initiation and progression of dental caries has yet to be established. To identify proteins that are differentially expressed by S. oralis growing under conditions of low pH, soluble cellular proteins extracted from bacteria grown in batch culture at pH 5.2 or 7.0 were analyzed by two-dimensional (2-D) gel electrophoresis. Thirty-nine proteins had altered expression at low pH; these were excised, digested with trypsin using an in-gel protocol, and further analyzed by peptide mass fingerprinting using matrix-assisted laser desorption ionization mass spectrometry. The resulting fingerprints were compared with the genomic database for Streptococcus pneumoniae, an organism that is phylogenetically closely related to S. oralis, and putative functions for the majority of these proteins were determined on the basis of functional homology. Twenty-eight proteins were up-regulated following growth at pH 5.2; these included enzymes of the glycolytic pathway (glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase), the polypeptide chains comprising ATP synthase, and proteins that are considered to play a role in the general stress response of bacteria, including the 60-kDa chaperone, Hsp33, and superoxide dismutase, and three distinct ABC transporters. These data identify, for the first time, gene products that may be important in the survival and proliferation of nonmutans aciduric S. oralis under conditions of low pH that are likely to be encountered by this organism in vivo. PMID:11472910

Wilkins, Joanna C.; Homer, Karen A.; Beighton, David



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.



AFLP Analysis of Gel Electrophoresis Images Using JelMarker Image Reader and GeneMarkerFragment Analysis Software  

Microsoft Academic Search

, T-RFLP, SNP discovery, fingerprinting, footprinting, etc. (1-3). Often the gel images are overcrowded and tedious to analyze and with the advent of capillary electrophoresis, software development to analyze these gel images has fallen by the wayside. SoftGenetics has recently developed software to fill this gap - JelMarker. JelMarker uses advanced image reading algorithms and specific band scoring techniques

Tamela Serensits; Zhang Xuanhe; Ni Shouyong; Jonathan Liu


Immunoblots and plasmid fingerprints compared with serotyping and polyacrylamide gel electrophoresis for typing Clostridium difficile.  

PubMed Central

Two new methods for typing Clostridium difficile, immunoblotting and plasmid fingerprinting, were compared with serotyping and polyacrylamide gel electrophoresis (PAGE). Of these methods, immunoblotting was found to be the most valuable for use in a comprehensive typing system. More groups could be distinguished by immunoblotting than by serotyping or PAGE. Immunoblotting results were also more reproducible and distinctive than results by PAGE. Plasmid fingerprinting was an excellent marker for plasmid-bearing strains, but it had limited use because many isolates lacked plasmids. A unique plasmid profile observed for one group of isolates correlated with differences in phenotypic characteristics resolved by immunoblot analysis but not by serotyping or PAGE. Preliminary attempts to correlate typing results with pathogenicity of isolates were not successful but underscored the need for future studies to include careful assessment of the clinical significance of isolates. Images PMID:3343314

Mulligan, M E; Peterson, L R; Kwok, R Y; Clabots, C R; Gerding, D N



Globin chain separation by SDS polyacrylamide gel electrophoresis. Simple screening method for elongated hemoglobin chains.  


A simple method for the separation of hemoglobin chains from hemolysate or globin, by sodium dodecyl sulfate polyacrylamide gel electrophoresis, is described. The alpha, beta, and gamma chains can be clearly separated from each other. The alpha chain has the highest mobility, the beta chain has a slower mobility than the gamma chain, while the delta chain has about the same mobility as the beta chain. Hemoglobins with elongated chains can easily be detected by this method. Tak-beta, elongated by 11 residues, moves much more slowly than betaA but is much faster than alpha Constant Spring which is elongated by 31 residues. Screening of several individuals with slow-moving hemoglobins using this method led to the finding of a case with Hb Tak-beta thalassemia and other carriers of Hb Tak. PMID:659552

Lie-Injo, L E; Solai, A; Ganesan, J



Phosphoprotein staining for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using fluorescent reagent morin hydrate.  


A fluorescence-based stain with 3,5,7,2',4'-pentahydroxyflavone (morin hydrate, MH) was designed to stain phosphoproteins in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Al(3+) was applied as a "fixed bridge," providing an efficient energy transfer channel between phosphoprotein and MH, to produce a strong fluorescent complex for the determination of phosphoprotein. As little as 62.5ng of ?-casein (7 or 8 phosphates) and ?-casein (5 phosphates), 125ng of ovalbumin (2 phosphates), and ?-casein (1 phosphate) could be visualized with a wide linear dynamic range. In comparison with conventional methods, MH stain is a time-saving method that takes just 90min. It also has good compatibility with routine protein stainings such as Coomassie Brilliant Blue R (CBBR) and SYPRO Ruby for total protein analysis. PMID:23274386

Wang, Xu; Hwang, Sun-Young; Cong, Wei-Tao; Jin, Li-Tai; Choi, Jung-Kap



Mapping of Escherichia Coli Chromosomal Tn5 and F Insertions by Pulsed Field Gel Electrophoresis  

PubMed Central

A low resolution Not I physical map of Escherichia coli was recently constructed. In this report we demonstrated that this map can be used to map Tn5 and F insertions physically. The transposon, Tn5, contains Not I recognition sequences in its IS50 sequences. F plasmid contains an unmapped Not I site. Hence, the location of Tn5 and F in the chromosome can be mapped by identifying the location of the introduced Not I sites using pulsed field gel electrophoresis. The physical mapping of genetically mapped Tn5 insertions confirm the previously constructed Not I map and helps align the E. coli physical and genetic maps. The use of Tn5 can assist the construction of both physical and genetic maps for microorganisms lacking such maps. Variations on this approach will facilitate physical mapping with a wide variety of organisms, enzymes, and genetic elements. PMID:2840334

Smith, C. L.; Kolodner, R. D.



Microchip capillary gel electrophoresis with electrochemical detection for the analysis of known SNPs.  


A novel microchip-based single nucleotide polymorphism (SNP) screening system has been developed. The system utilizes capillary gel electrophoresis (CGE) with electrochemical detection in a chip-based format to accomplish rapid scoring of a mock SNP site. The accuracy of the thermostable polymerase and the advantages of coupling this technique to microfluidics are demonstrated. An electrochemically labeled chain terminator is used in the single base extension (SBE) reaction, in which the terminator is incorporated only when its Watson-Crick complementary base is present at the mock SNP site. The resulting electrochemically active extension product is subsequently separated from any excess terminator by CGE and detected by sinusoidal voltammetry. Although no attempts at optimization have been made, the analysis is performed in less than 4 min. The technique presented could lead to a fast, simple, and cost effective SNP scoring system. PMID:15007453

Hebert, Nicole E; Brazill, Sara A



Dispersion coefficients of lambda DNA in agarose gels during electrophoresis by fringe recovery after photobleaching.  


By combining an electrophoretic cell with a fringe recovery after photobleaching setup, we have measured both the electrophoretic mobility mu and the dispersion coefficient D of double-stranded lambda-DNA in agarose gel as a function of the applied electric field E. Besides the determination of the mobilities, the analysis of the exponential decay of the signal gives, for the first time, experimental values of the longitudinal Dx and transverse Dy dispersion coefficients (field E perpendicular and parallel to the light fringes, respectively). Time dispersion measurements are complicated by the existence of a bleached area drift time. We present an analysis of their different contributions. Both dispersion coefficients increase linearly with increasing field E and, in the range of field we used, Dx is larger than Dy. Our results are consistent with the recent theoretical predictions of Slater (Electrophoresis 1993, 14, 1-7) and Duke et al. (Biopolymers 1994, 94, 239-247). PMID:8957172

Tinland, B



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.



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


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

Shi, Nan; Ugaz, Victor M



Miconazole Oral Gel Increases Exposure to Oral Oxycodone by Inhibition of CYP2D6 and CYP3A4?  

PubMed Central

Our aim was to assess the effect of miconazole oral gel on the pharmacokinetics of oral oxycodone. In an open crossover study with two phases, 12 healthy volunteers took a single oral dose of 10 mg of immediate-release oxycodone with or without thrice-daily 85-mg miconazole oral gel treatment. The plasma concentrations of oxycodone and its oxidative metabolites were measured for 48 h. Pharmacological effects of oxycodone were recorded for 12 h. Pharmacokinetic parameters were compared by use of the geometric mean ratios (GMRs) and their 90% confidence interval (CIs). Pretreatment with miconazole oral gel caused a strong inhibition of the CYP2D6-dependent metabolism and moderate inhibition of the CYP3A4-dependent metabolism of oxycodone. The mean area under the concentration-time curve (AUC) from time zero to infinity (AUC0-?; GMR, 1.63; 90% CI, 1.48 to 1.79) and the peak concentration of oxycodone (GMR, 1.31; 90% CI, 1.19 to 1.44) were increased. The AUC of the CYP2D6-dependent metabolite oxymorphone was greatly decreased (GMR, 0.17; 90% CI, 0.09 to 0.31) by miconazole gel, whereas that of the CYP3A4-dependent metabolite noroxycodone was increased (GMR, 1.30; 90% CI, 1.15 to 1.47) by miconazole gel. Differences in the pharmacological response to oxycodone between phases were insignificant. Miconazole oral gel increases the exposure to oral oxycodone, but the clinical relevance of the interaction is moderate. Miconazole oral gel produces a rather strong inhibitory effect on CYP2D6, which deserves further study. PMID:21173180

Grnlund, Juha; Saari, Teijo I.; Hagelberg, Nora; Neuvonen, Pertti J.; Olkkola, Klaus T.; Laine, Kari



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


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



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



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




Microplate array diagonal gel electrophoresis (MADGE), CpG-PCR and temporal thermal ramp-MADGE (Melt-MADGE) for single nucleotide analyses in populations  

Microsoft Academic Search

Important requirements for molecular genetic epidemiological studies are economy, sample parallelism, convenience of setup and accessibility, goals inadequately met by existent approaches. We invented microplate array diagonal gel electrophoresis (MADGE) to gain simultaneously the advantages of simple setup, 96-well microplate compatibility, horizontal electrophoresis, and the resolution of polyacrylamide. At essentially no equipment cost (one simple plastic gel former), 10100-fold savings

Ian N. M. Day; Sandra D. ODell; Emmanuel Spanakis; Glenn P. Weavind



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.



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.



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



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


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



Nested PCR-denaturing gradient gel electrophoresis analysis of human skin microbial diversity with age.  


To determine whether the composition and structure of skin microbiota differ with age, cutaneous bacteria were isolated from the axillary fossa of 37 healthy human adults in two age groups (old people and young adults). Bacterial genomic DNA was extracted and characterized by nested PCR-denaturing gradient gel electrophoresis (PCR-DGGE) with primers specifically targeting V3 region of the 16S rRNA gene. The excised gel bands were sequenced to identify bacterial categories. The total bacteria, Staphylococcus spp., Staphylococcus epidermidis and Corynebacterium spp. were further enumerated by quantitative PCR. There were no significant differences in the species diversity profiles between age groups. The similarity index was lower across age groups than that it was intra-group. This indicates that the composition of skin flora is more similar to others of the same age than across age groups. While Staphylococcus spp. and Corynebacterium spp. were the dominant bacteria in both groups, sequencing and quantitative PCR revealed that skin bacterial composition differed by age. The copy number of total bacteria and Corynebacterium spp. were significantly lower in younger subjects, whereas there were no statistical differences in the quantity of Staphylococcus spp. and Staphylococcus epidermidis. These results suggest that the skin flora undergo both quantitative and qualitative changes related to aging. PMID:24656938

Li, Wei; Han, Lei; Yu, Pengbo; Ma, Chaofeng; Wu, Xiaokang; Xu, Jiru



Low-cost, high-sensitivity laser-induced fluorescence detection for DNA sequencing by capillary gel electrophoresis.  


A low cost, 0.75-mW helium neon laser, operating in the green region at 534.5 nm, is used to excite fluorescence from tetramethylrhodamine isothiocyanate-labelled DNA fragments that have been separated by capillary gel electrophoresis. The detection limit (3 sigma) for the dye is 500 ymol [1 yoctomole (1 ymol) = 10(-24) mol] or 300 analyte molecules in capillary zone electrophoresis; the detection limit for labeled primer separated by capillary gel electrophoresis is 2 zmol [1 zeptomole (1 zmol) = 10(-21) mol]. The Richardson-Tabor peak-height encoded sequencing technique is used to prepare DNA sequencing samples. In 6% T, 5% C acrylamide, 7 M urea gels, sequencing rates of 300 bases/hour are produced at an electric field strength of 200 V/cm; unfortunately, the data are plagued by compressions. These compressions are eliminated with addition of 20% formamide to the sequencing gel; the gel runs slowly and sequencing data are generated at a rate of about 70 bases/hour. PMID:1761625

Chen, D Y; Swerdlow, H P; Harke, H R; Zhang, J Z; Dovichi, N J



[Determination of antisense phosphorothioate oligonucleotide drug Cantide by capillary gel electrophoresis].  


Cantide is a 20-mer antisense phosphorothioate oligonucleotide that inhibits telomerase catalytic subunit hTERT. A capillary gel electrophoresis (CGE) method with internal standard was used for the determination of Cantide. Cantide and the phosphorothioate internal standard were prepared on a solid-phase DNA synthesizer and purified by preparative strong anion-exchange chromatography and reversed-phase high performance liquid chromatography. Cantide and the internal standard had approximately equal percentage of base composition. Cantide was determined with capillary electrophoresis instrument and ssDNA kit. The size of the capillary column was 31 cm x 100 microm i.d. with an effective length of 20 cm. Samples were electrokinetically injected using -10 kV voltage for a duration of 1 s. The column temperature and sample storage temperature was 40 degrees C and 30 degrees C, respectively. The detection wavelength was 254 nm. The running buffer was a mixture of 7 mol/L urea-tris-boric acid (pH 8.5). The calibration curve was linear in the range of 12.5-800 mg/L with correlation coefficient of 0.99997. The limit of detection of Cantide was 0.220 mg/L. Intra-day and inter-day relative standard deviations (RSDs) for Cantide were 0.449%-1.89%, and 1.01%-1.48%, respectively. The average recovery was 96.95% with RSD of 6.88%. The assay validation study and the characterization of CGE revealed that the method can be used in quantitative analysis of Cantide for pharmacokinetic characterization, and the results are accurate and reproducible. PMID:15712897

Yang, Binghu; Sun, Oujun; Zhang, Minli; Wang, Shengqi



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



Pulsed-Field Gel Electrophoresis Is More Discriminating Than Multilocus Enzyme Electrophoresis and Random Amplified Polymorphic DNA Analysis for Typing Pyogenic Streptococci  

Microsoft Academic Search

. The SmaI restriction endonuclease digestion\\u000a \\u000a patterns of chromosomal DNAs from 99 pyogenic streptococci belonging to\\u000a \\u000a Lancefield group A (41 Streptococcus pyogenes), group C (seven S.\\u000a \\u000a dysgalactiae, 11 \\\\QS. equisimilis\\\\W, three S. equi, eight\\u000a \\u000a S. zooepidemicus) and group G (25 human group G\\u000a \\u000a Streptococcus, four S. canis) were analyzed by pulsed-field\\u000a \\u000a gel electrophoresis (PFGE), and the results were compared with

Frdric Bert; Catherine Branger; Nicole Lambert-Zechovsky



Identification of 2D-gel proteins : a comparison of MALDI/TOF peptide mass mapping to {mu} LC-ESI tandem mass spectrometry.  

SciTech Connect

A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and mu LC-MS/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by mu LC-MS/MS while 97% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTip(C18) and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using mu LC-MS/MS, 50% of the spots analyzed were identified as containing multiple proteins. mu LC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by mu LC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50% for approximately 70% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.

Lim, H.; Hays, L. G.; Eng, J.; Tollaksen, S. L.; Giometti, C. S.; Holden, J. F.; Adams, M. W. W.; Reich, C. I.; Olsen, G. J.; Yates, J. R.; Biosciences Division; The Scripps Research Inst.; Univ. of Georgia; Univ. of Illinois



Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their relation with semen freezability.  


The objective was to evaluate protein profiles of equine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and to determine whether any of these proteins were related to semen freezability. Seminal plasma was collected from 10 stallions, of high and low semen freezability, housed at the State Stud of Lower Saxony, and routinely used in AI programs. Twenty-five protein spots were identified from the two-dimensional gel (12%), seven of which were present in all samples (all proteins were identified by MALDI-MS). Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used to generate ion images of samples in one or more mass-to-charge (m/z) values, providing the capability of mapping specific molecules to two-dimensional coordinates of the original sample. Of the 25 proteins identified, two spots had greater relative content (P < 0.05) in seminal plasma samples collected from stallions with high semen freezability: spot 5 (80-85 kDa, isoelectric point [pI] 7.54), identified as CRISP-3; and spot 45 (18.2 kDa, pI 5.0-5.2), identified as HSP-2. Conversely, protein content was greater (P < 0.05) in seminal plasma samples from stallions with low semen freezability: spot 7 (75.4 kDa, pI 6.9-7.4), identified as lactoferrin; spot 15 (26.7 kDa, pI 5.51), identified as kallikrein; spot 25 (25 kDa, pI 7.54), identified as CRISP-3; and spot 35 (13.9 kDa, pI 3.8-4.2), identified as HSP-1. In conclusion, there were differences in the seminal plasma protein profile from stallions with high and low semen freezability. Furthermore, CRISP-3 and HSP-2 were potential seminal plasma markers of high semen freezability. PMID:21601917

Jobim, M I M; Trein, C; Zirkler, H; Gregory, R M; Sieme, H; Mattos, R C



Genetic variability among Chlamydia trachomatis reference and clinical strains analyzed by pulsed-field gel electrophoresis.  


Pulsed-field gel electrophoresis (PFGE) was applied to Chlamydia trachomatis reference strains representing each of the 18 serovars and to 29 clinical isolates from genital specimens collected in Bordeaux, France, or Malm, Sweden. Comparison of the fingerprint patterns of the reference strains revealed a high level of polymorphism of the total DNA when SmaI was used (14 profiles), whereas the other enzymes, Sse8387I and ApaI, showed fewer differences. Some serovars, considered to be closely related on the basis of their antigenic determinants located on the major outer membrane protein (MOMP), such as D and Da or I and Ia, were shown to be different after PFGE of their genomic DNAs. However, serovars B and Ba and serovars L2 and L2a had identical patterns after analysis with the three endonucleases. When applied to clinical isolates, which were typed by restriction fragment length polymorphism analysis of the MOMP gene, PFGE allowed the detection of intragenotype polymorphisms and showed the identity of two strains successively isolated from the same patient. This technique seems to be an efficient tool for epidemiological studies when used in addition to serotyping or genotyping by restriction fragment length polymorphism analysis of the MOMP gene. PMID:7883878

Rodriguez, P; Allardet-Servent, A; de Barbeyrac, B; Ramuz, M; Bebear, C



Assessment of microbial populations in methyl ethyl ketone degrading biofilters by denaturing gradient gel electrophoresis.  


Denaturing gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction-amplified genes coding for 16S rRNA was used to assess differences in bacterial community structure as a function of spatial location along the height of two biofilters used to treat a model waste gas stream containing methyl ethyl ketone (MEK). One of the laboratory-scale biofilters was operated as a conventional continuous-flow biofilter (CFB) and the other was operated as a sequencing batch biofilter (SBB). Both biofilters, inoculated with an identical starting culture and operated over a period lasting more than 300 days, received the same influent MEK concentration and same mass of MEK on a daily basis. The systems differed, however, in terms of the fraction of time during which contaminated air was supplied and the overall operating strategy employed. DGGE analysis indicated that microbial community structures differed as a function of height in each of the biofilters. The DGGE banding patterns also differed between the two biofilters, suggesting that operating strategies imposed on the biofilters imparted a sufficiently large selective pressure to influence microbial community structures. This may explain, in part, the superior performance of the SBB over the CFB during model transient loading conditions, and it may open new possibilities for purposely manipulating the microbial populations in biofilters treating gas-phase contaminants in a manner that leads to more favorable treatment performance. PMID:14735321

Li, C; Moe, W M



Characterization of protistan assemblages in the Ross Sea, Antarctica, by denaturing gradient gel electrophoresis.  


The diversity of protistan assemblages has traditionally been studied using microscopy and morphological characterization, but these methods are often inadequate for ecological studies of these communities because most small protists inherently lack adequate taxonomic characters to facilitate their identification at the species level and many protistan species also do not preserve well. We have therefore used a culture-independent approach (denaturing gradient gel electrophoresis [DGGE]) to obtain an assessment of the genetic composition and distribution of protists within different microhabitats (seawater, meltwater or slush on sea-ice floes, and ice) of the Ross Sea, Antarctica. Samples of the same type (e.g., water) shared more of the same bands than samples of different types (e.g., ice versus water), despite being collected from different sites. These findings imply that samples from the same environment have a similar protistan species composition and that the type of microenvironment significantly influences the protistan species composition of these Antarctic assemblages. It should be noted that a large number of bands among the samples within each microhabitat were distinct, indicating the potential presence of significant genetic diversity within each microenvironment. Sequence analysis of selected DGGE bands revealed sequences that represent diatoms, dinoflagellates, ciliates, flagellates, and several unidentified eukaryotes. PMID:15066793

Gast, Rebecca J; Dennett, Mark R; Caron, David A



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

E-print Network

Abstract: FLT3 (fms-related tyrosine kinase 3) is a receptor tyrosine kinase class III that is expressed on by early hematopoietic progenitor cells and plays an important role in hematopoietic stem cell proliferation, differentiation and survival. FLT3 is also expressed on leukemia blasts in most cases of acute myeloid leukemia (AML). In order to determine the frequency of FLT3 oncogene mutations, we analyzed genomic DNA of adult de novo acute myeloid leukemia (AML). Polymerase chain reaction (PCR) and conformation-sensitive gel electrophoresis (CSGE) were used for FLT3 exons 11, 14, and 15, followed by direct DNA sequencing. Two different types of functionally important FLT 3 mutations have been identified. Those mutations were unique to patients with inv(16), t(15:17) or t(8;21) and comprised fifteen cases with internal tandem duplication (ITD) mutation in the juxtamembrane domain and eleven cases with point mutation (exon 20, Asp835Tyr). The high frequency of the flt3 proto-oncogene mutations in acute myeloid leukemia AML suggests a key role for the receptor function. The association ofInt. J. Mol. Sci. 2008, 9

Mamdooh Gari; Adel Abuzenadah; Adeel Chaudhary; Mohammed Al-qahtani; Waseem Ahmad; Fatin Al-sayes; Sahira Lary; Ghazi Damanhouri



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

PubMed Central

Genetic relationships among 46 isolates of Mycobacterium avium recovered from 37 patients in a 2,500-bed hospital from 1993 to 1998 were assessed by pulsed-field gel electrophoresis (PFGE) and PCR amplification of genomic sequences located between the repetitive elements IS1245 and IS1311. Each technique enabled the identification of 27 to 32 different patterns among the 46 isolates, confirming that the genetic heterogeneity of M. avium strains is high in a given community. Furthermore, this retrospective analysis of sporadic isolates allowed us (i) to suggest the existence of two remanent strains in our region, (ii) to raise the question of the possibility of nosocomial acquisition of M. avium strains, and (iii) to document laboratory contamination. The methods applied in the present study were found to be useful for the typing of M. avium isolates. In general, both methods yielded similar results for both related and unrelated isolates. However, the isolates in five of the six PCR clusters were distributed among two to three PFGE patterns, suggesting that this PCR-based method may have limitations for the analysis of strains with low insertion sequence copy numbers or for resolution of extended epidemiologic relationships. PMID:10405383

Pestel-Caron, Martine; Graff, Gabriel; Berthelot, Gilles; Pons, Jean-Louis; Lemeland, Jean-Francois



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


Isolates of Pseudomonas aeruginosa from clinical infections in mink were subjected to serotyping and pulsed-field gel electrophoresis (PFGE) using SpeI. A total of 212 isolates of P. aeruginosa from the year 1998 to 2001 were included in this study: 168 isolates from mink obtained from 74 farm outbreaks of haemorrhagic pneumonia. Isolates from mink were separated into 34 distinct clones by PFGE subtyping. All isolates from mink infected during the same farm outbreak were identical, except in one case where two different strains were isolated from mink obtained from the same farm outbreak. P. aeruginosa of specific PFGE types were found to cause clusters of outbreaks on several farms within a few weeks of each other. However, PFGE types of strains causing clusters of farm outbreaks changed from year to year. These results suggest that some outbreaks of haemorrhagic pneumonia are caused by pathogenic strains of P. aeruginosa spread between farms and animals either mechanically, or through feed or water from a common source, rather than by random nosocomial infections with strains from the farm environment. PMID:12814891

Hammer, Anne Sofie; Pedersen, Karl; Andersen, Thomas Holmen; Jrgensen, Jens Christian; Dietz, Hans Henrik



Assessment of microbial populations dynamics in a blue cheese by culturing and denaturing gradient gel electrophoresis.  


The composition and development of microbial population during the manufacture and ripening of two batches of a blue-veined cheese was examined by culturing and polymerase chain reaction (PCR) denaturing gradient gel electrophoresis (DGGE) (PCR-DGGE). Nine selective and/or differential media were used to track the cultivable populations of total and indicator microbial groups. For PCR-DGGE, the V3 hyper variable region of the bacterial 16S rRNA gene and the eukaryotic D1 domain of 28S rDNA were amplified with universal primers, specific for prokaryotes and eukaryotes, respectively. Similarities and differences between the results obtained by the culturing and the molecular method were recorded for some populations. Culturing analysis allows minority microbial groups (coliforms, staphylococci) to be monitored, although in this study PCR-DGGE identified a population of Streptococcus thermophilus that went undetected by culturing. These results show that the characterization of the microbial populations interacting and evolving during the cheese-making process is improved by combining culturing and molecular methods. PMID:21046392

Alegra, Angel; Gonzlez, Renata; Daz, Mario; Mayo, Baltasar



Fluctuations in the velocity of individual DNA Molecules during agarose gel electrophoresis  

PubMed Central

The velocity of the center of mass of individual T4 DNA molecules during agarose gel electrophoresis, computed from digitized video-microscopic images, fluctuated between 0 and 4.5 ?m/s after a field E = 5 V/cm was applied; the amplitude of the velocity peaks was twice the averaged steady-state velocity. The velocity fluctuations correlated with changes in molecular configuration. The mean velocity (10 molecules) showed a sharp rise in less than 0.2 s, followed by a shallow minimum and a broad peak, before reaching a plateau. The much smaller amplitude of these oscillatory features demonstrated that the velocity fluctuations of individual molecules were largely, but not entirely, uncorrelated with the onset of the field. The components of the shape tensor S of individual chains, which are a measure of the extension of the chains, were also determined for each image sequence. Only the principal component in the direction of E, Sxx, increased. ImagesFIGURE 4FIGURE 5 PMID:19431863

Howard, Timothy D.; Holzwarth, G.



On the effects of intercalators in DNA condensation: a force spectroscopy and gel electrophoresis study.  


In this work we have characterized the effects of the intercalator ethidium bromide (EtBr) on the DNA condensation process by using force spectroscopy and gel electrophoresis. We have tested two condensing agents: spermine (spm(4+)), a tetravalent cationic amine which promotes cation-induced DNA condensation, and poly(ethylene glycol) (PEG), a neutral polymer which promotes DNA ?-condensation. Two different types of experiments were performed. In the first type, bare DNA molecules disperse in solution are first treated with EtBr for intercalation, and then the condensing agent is added to the sample with the purpose of verifying the effects of the intercalator in hindering DNA condensation. In the second experiment type, the bare DNA molecules are first condensed, and then the intercalator is added to the sample in order to verify its influence on the previously condensed DNA. The results obtained with the two different experimental techniques used agree very well, indicating that previously intercalated EtBr can hinder both cation-induced and ?-condensation, being more efficient in the first case. On the other hand, EtBr has little effect on the previously formed cation-induced condensates, but is efficient in unfolding the ?-condensates. PMID:24720756

Rocha, M S; Cavalcante, A G; Silva, R; Ramos, E B



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

SciTech Connect

Thin-layer chromatography (TLC) is a broadly applicable separation technique. It offers many advantages over high performance liquid chromatography (HPLC), such as easily adapted for two-dimensional separation, for whole-column'' detection and for handling multiple samples, etc. However, due to its draggy development of detection techniques comparing with HPLC, TLC has not received the attention it deserves. Therefore, exploring new detection techniques is very important to the development of TLC. It is the principal of this dissertation to present a new detection method for TLC -- indirect fluorometric detection method. This detection technique is universal sensitive, nondestructive, and simple. This will be described in detail from Sections 1 through Section 5. Section 1 and 3 describe the indirect fluorometric detection of anions and nonelectrolytes in TLC. In Section 2, a detection method for cations based on fluorescence quenching of ethidium bromide is presented. In Section 4, a simple and interesting TLC experiment is designed, three different fluorescence detection principles are used for the determination of caffeine, saccharin and sodium benzoate in beverages. A laser-based indirect fluorometric detection technique in TLC is developed in Section 5. Section 6 is totally different from Sections 1 through 5. An ultrasonic effect on the separation of DNA fragments in agarose gel electrophoresis is investigated. 262 refs.

Yinfa, Ma.



Differential Silver-Staining Sodium Dodecyl SulfatePolyacrylamide Gel Electrophoresis: A Nonisotopic Method for Characterizing Gel-Separated HistoneDNA Complexes  

Microsoft Academic Search

Some nonspecific, DNA-binding proteins, like the linker histones, precipitate DNA upon binding. This is a poorly understood process that limits analysis of such nucleoprotein complexes using standard gel electrophoresis. To circumvent this problem, low concentrations of glutaraldehyde were used to crosslink the linker histones to DNA; then the partially crosslinked complexes were solubilized in SDS2and separated by SDSPAGE. Differential detection

George John Carter; Kensal van Holde



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



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


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

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



A computer program allows the separation of a wide range of chromosome sizes by pulsed field gel electrophoresis.  

PubMed Central

The introduction of Pulsed Field Gel Electrophoresis techniques, which allow the separation of DNA molecules of molecular weights as high as chromosomes of lower eukaryotes, has given a powerful tool to geneticists. The resolution expected from these techniques is dependent on numerous parameters, among them pulse time and field strength. A given set of these parameters allows only a limited range of molecular weights to be resolved. To allow the separation of a broader molecular weight range on a single gel, we designed a computer program, driving a simple switching device, to take care of switching electrodes and power supplies in OFAGE migrations. This program has been designed to be used with any technique calling for periodic switching or inversion of the electric field, and/or variation of the electric field applied during electrophoresis. As an example, we show the results obtained with yeast genera in which chromosome sizes range from 260 to 9,000 kilobase pairs. Images PMID:3387211

Sor, F



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

Microsoft Academic Search

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

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



Random Amplified Polymorphic DNA Typing versus Pulsed-Field Gel Electrophoresis for Epidemiological Typing of Vancomycin-Resistant Enterococci  

Microsoft Academic Search

Sixty vancomycin-resistant vanA mutant Enterococcus faecium (VRE) isolates, collected during a 40-month period from 48 patients hospitalized in a French Cancer Referral Center, were typed by using random amplified polymorphic DNA (RAPD), and the results were compared with those previously obtained by typing withSmaI pulsed-field gel electrophoresis (PFGE), which is currently recognized as the ''gold standard.'' The discriminating power of




[Screening for mutation variants in exons 5, 7, 12 of phenylalanine hydroxylase gene using denaturing gradient gel-electrophoresis].  


The assays for analysis of the most frequent mutations of PAH gene in Ukraine (R158Q, R408W, Y414C, P281L, R252W, R261Q) for RKU patients and healthy people using denaturing gradient gel-electrophoresis (DGGE) were developed. The study of spectrum of mutations in exons 5,7,12 PAH gene using DGGE technique and further sequencing of unidentified mutant variants was held. PMID:19938643

Solov?ov, O O; Livshyts', L A



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

Microsoft Academic Search

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

Cristina Andreoli; Paola Leopardi; Riccardo Crebelli



Mapping of seminal plasma proteins by two-dimensional gel electrophoresis in men with normal and impaired spermatogenesis  

Microsoft Academic Search

The present study analyses differential polypeptide expression of seminal plasma from fertile and infertile men by two-dimensional gel electrophoresis. Optimization of solubilization of seminal plasma was obtained by using (3-(3-(cholamidopropyl) dimethyl-ammonio)-1-propane sulphonate) and chaotropic agent mixture in lysis buffer before separation in immobilized pH gradient for isoelectric focusing. A two-dimensional map of seminal plasma from a fertile man allowed the

M. Starita-Geribaldi; S. Poggioli; M. Zucchini; J. Garin; D. Chevallier; P. Fenichel; G. Pointis



Standardization and Interlaboratory Reproducibility Assessment of Pulsed-Field Gel Electrophoresis-Generated Fingerprints of Acinetobacter baumannii  

Microsoft Academic Search

A standard procedure for pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments of Acineto- bacter baumannii was set up and validated for its interlaboratory reproducibility and its potential for use in the construction of an Internet-based database for international monitoring of epidemic strains. The PFGE fingerprints of strains were generated at three different laboratories with ApaI as the restriction enzyme and

Harald Seifert; Lucilla Dolzani; Raffaela Bressan; Tanny van der Reijden; Beppie van Strijen; Danuta Stefanik; Herre Heersma; Lenie Dijkshoorn


Standardization and Interlaboratory Reproducibility Assessment of Pulsed-Field Gel Electrophoresis-Generated Fingerprints of Acinetobacter baumannii  

Microsoft Academic Search

A standard procedure for pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments of Acineto- bacter baumannii was set up and validated for its interlaboratory reproducibility and its potential for use in the construction of an Internet-based database for international monitoring of epidemic strains. The PFGE fingerprints of strains were generated at three different laboratories with ApaI as the restriction enzyme and

Harald Seifert; Lucilla Dolzani; Raffaela Bressan; Tanny van der Reijden; Beppie van Strijen; Danuta Stefanik; Herre Heersma; Lenie Dijkshoorn



PCR-Denaturing Gradient Gel Electrophoresis and Two Feces Antigen Tests for Detection of Helicobacter pylori in Mice  

Microsoft Academic Search

PCR-denaturing Gradient Gel Electrophoresis (PCR-DGGE), a method suitable for the detection of microbial species in complex ecosystems, was evaluated for the detection and identification of Helicobacter spp. in feces and stomach tissue of mice. Two commercially available stool antigen tests for clinical diagnostics in humans were also evaluated in the C57B1\\/6 mouse model of H. pylori infection. PCR-DGGE detected only

Hkan Sjunnesson; Tobias Flt; Erik Sturegrd; Waleed Abu Al-Soud; sa Ljungh; Torkel Wadstrm



Ribosomal ribonucleic acids of cultured cells: A preliminary survey of differences among mammalian species detectable by polyacrylamide gel electrophoresis  

Microsoft Academic Search

SummaryRibosomal ribonucleic acids (rRNA's) of cultured cells from various species were compared by polyacrylamide gel electrophoresis.\\u000a Electrophoretic mobility of the 28 S RNA component varied according to species. Human cell 28 S rRNA was distinguishable from\\u000a that of monkey cells. The mobility of chimpanzee 28 S rRNA was identical to that from human cells, but different from that\\u000a of monkey

M. E. Soergel; F. L. Schaffer



Protein expression profiles in pancreatic adenocarcinoma compared with normal pancreatic tissue and tissue affected by pancreatitis as detected by two-dimensional gel electrophoresis and mass spectrometry.  


Pancreatic cancer is a rapidly fatal disease, and there is an urgent need for early detection markers and novel therapeutic targets. The current study has used a proteomic approach of two-dimensional (2D) gel electrophoresis and mass spectrometry (MS) to identify differentially expressed proteins in six cases of pancreatic adenocarcinoma, two normal adjacent tissues, seven cases of pancreatitis, and six normal pancreatic tissues. Protein extracts of individual sample and pooled samples of each type of tissues were separated on 2D gels using two different pH ranges. Differentially expressed protein spots were in-gel digested and identified by MS. Forty proteins were identified, of which five [i.e., alpha-amylase; copper zinc superoxide dismutase; protein disulfide isomerase, pancreatic; tropomyosin 2 (TM2); and galectin-1] had been associated previously with pancreatic disease in gene expression studies. The identified proteins include antioxidant enzymes, chaperones and/or chaperone-like proteins, calcium-binding proteins, proteases, signal transduction proteins, and extracellular matrix proteins. Among these proteins, annexin A4, cyclophilin A, cathepsin D, galectin-1, 14-3-3zeta, alpha-enolase, peroxiredoxin I, TM2, and S100A8 were specifically overexpressed in tumors compared with normal and pancreatitis tissues. Differential expression of some of the identified proteins was further confirmed by Western blot analyses and/or immunohistochemical analysis. These results show the value of a proteomic approach in identifying potential markers for early diagnosis and therapeutic manipulation. The newly identified proteins in pancreatic tumors may eventually serve as diagnostic markers or therapeutic targets. PMID:15604267

Shen, Jianjun; Person, Maria D; Zhu, Jijiang; Abbruzzese, James L; Li, Donghui



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


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



Study on the Complex of Soluble Proteins in the Cells of Clostridium Perfringens by Electrophoresis in Polyacrylanide Gel (O Komplekse Rastvorimykh Belkov, Soderzhashchikhsya V Kletke Clostridium Pereringens).  

National Technical Information Service (NTIS)

The method of electrophoresis in polyacrylamide gel is suitable for separating soluble acid cell proteins. The proteins are separated into a great many sharply demarcated zones and the resultant 'protein spectra' are analyzed. As a rule, the strains of C....

S. A. Nikolaeva, V. I. Safonov



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



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


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



Application of pulsed field gel electrophoresis to the 1993 epidemic of whooping cough in the UK.  

PubMed Central

The purpose of this study was to DNA fingerprint the majority (64%) of isolates received at the Pertussis Reference Laboratory during the 1993 whooping cough epidemic by pulsed field gel electrophoresis of Xba I-generated restriction digests. Two DNA restriction patterns, types 1 and 3, predominated (40% and 23%, respectively, of 180 isolates) but type 2, identified in a previous study was notably absent. Twenty-one new DNA types occurred (24% of isolates), some being atypical as bands 155-230 kb were no longer conserved, but there was no statistically significant difference in their incidence in the upswing (June-September) compared to the downswing (October-December) phase of the epidemic. There was a relatively high proportion of new types, compared to type 1, at the peak (September). About 50% of isolates received were from the North Western Region, where 44% of isolates were DNA type 1. Whereas only 1 out of 10 isolates from Scotland were of this type, suggesting some geographic variation. Statistically significant findings included a higher proportion of isolates from female patients (P < 0.01), most marked in the 12-24 months age group (P < 0.05); a higher proportion of infants under 12 months requiring hospital admission compared to older children (P < 0.05); and a greater number of isolates from unvaccinated children (P < 0.01). Analysis of serotype according to four age groups (under 3 months, 3-12 months, 12-24 months and above 2 years) showed statistically significant differences (P < 0.05) with a noticeably lower proportion (38%) of serotype 1,3 in 3-12 months age group and higher prevalence (74%) of serotype 1,3 in the 12-24 months age group. There was no correlation between DNA type and serotype. Images Fig. 2 PMID:7641824

Syedabubakar, S. N.; Matthews, R. C.; Preston, N. W.; Owen, D.; Hillier, V.



Molecular analysis of Salmonella enteritidis by pulsed-field gel electrophoresis and ribotyping.  

PubMed Central

A total of 61 isolates of Salmonella enteritidis were analyzed by the techniques of pulsed-field gel electrophoresis (PFGE) and ribotyping. Twenty-three of the isolates were from Zurich, Switzerland, and 38 isolates were from the University Hospital, Kuala Lumpur, Malaysia. Five of the Malaysian isolates were hospital-related outbreak strains and were shown to be indistinguishable by PFGE analysis following digestion with three different restriction endonucleases, XbaI (5'-TCTAGA-3'), SpeI (5'-ACTAGT-3'), and AvrII (5'-CCTAGG-3'). The PFGE pattern of an isolate from a suspected carrier staff nurse was found to be identical to those of the hospital outbreak isolates. These isolates were also indistinguishable by ribotyping with SmaI and SphI. The same single PFGE pattern was also detected in 29 of 32 sporadic isolates of S. enteritidis. Four closely related ribotypes were detected among these 29 isolates. Similarly, outbreak-related strains from Switzerland showed close genetic identity by PFGE and ribotyping. Strains obtained from poultry showed more variations in their PFGE patterns and ribotypes, although the patterns were still closely related. In addition, SphI ribotypes A and D among the Swiss strains correlated with phage types 4 and 8, respectively. No correlation of phage types with PFGE pattern was noted. Both PFGE and ribotyping indicate that the S. enteritidis strains circulating in Malaysia and Switzerland are very similar and may be clonally related. Comparison of the PFGE patterns with the ribotypes for 23 Swiss and 16 Malaysian isolates showed that there was a 69% concordance in the grouping of isolates.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7615707

Thong, K L; Ngeow, Y F; Altwegg, M; Navaratnam, P; Pang, T



A PCR-denaturing gradient gel electrophoresis approach to assess Fusarium diversity in asparagus.  


In North America, asparagus (Asparagus officinalis) production suffers from a crown and root rot disease mainly caused by Fusarium oxysporum f. sp. asparagi and F. proliferatum. Many other Fusarium species are also found in asparagus fields, whereas accurate detection and identification of these organisms, especially when processing numerous samples, is usually difficult and time consuming. In this study, a PCR-denaturing gradient gel electrophoresis (DGGE) method was developed to assess Fusarium species diversity in asparagus plant samples. Fusarium-specific PCR primers targeting a partial region of the translation elongation factor-1 alpha (EF-1 alpha) gene were designed, and their specificity was tested against genomic DNA extracted from a large collection of closely and distantly related organisms isolated from multiple environments. Amplicons of 450 bp were obtained from all Fusarium isolates, while no PCR product was obtained from non-Fusarium organisms. The ability of DGGE to discriminate between Fusarium taxa was tested over 19 different Fusarium species represented by 39 isolates, including most species previously reported from asparagus fields worldwide. The technique was effective to visually discriminate between the majority of Fusarium species and/or isolates tested in pure culture, while a further sequencing step permitted to distinguish between the few species showing similar migration patterns. Total genomic DNA was extracted from field-grown asparagus plants naturally infested with different Fusarium species, submitted to PCR amplification, DGGE analysis and sequencing. The two to four bands observed for each plant sample were all affiliated with F. oxysporum, F. proliferatum or F. solani, clearly supporting the reliability, sensitivity and specificity of this approach for the study of Fusarium diversity from asparagus plants samples. PMID:15590089

Yergeau, E; Filion, M; Vujanovic, V; St-Arnaud, M



Gradient polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate: a practical approach to muscle contractile and regulatory proteins.  


Two gradient systems for polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) are described, with emphasis on improvements accumulated over two decades of studies on contractile proteins and regulatory enzymes from smooth muscle. The first "big slab" system utilizes 18 x 20 x 0.1 cm3 gels and a 10-18% acrylamide gradient, optimized for a high resolution of 10 to 500 kDa polypeptides. Eight (or more) gels are cast simultaneously with a gradient formation from "bottom to top" and 20% glycerol is added to the 18% acrylamide solution. The second "minislab" system represents an improved version of the system of Matsudaira and Burgess (Anal. Biochem. 1978, 87, 386-396), with 8 x 10 x 0.05 cm3 gels and 5-15% or 9-18% acrylamide gradient ranges. They are cast from "top to bottom" in 28-piece batches also with the addition of glycerol for improved gradient formation. Both types of gels can also be cast individually using a specially designed pestle-type gradient maker. For gel destaining, a convenient continuous hydrodynamic destainer is also described. PMID:7859701

Sobieszek, A



Exactly solvable Ogston model of gel electrophoresis. IX. Generalizing the lattice model to treat high field intensities  

NASA Astrophysics Data System (ADS)

Traditionally, the Ogston regime is studied solely in the limit of low field intensities. This explains why the theoretical discussion has focused until now on the relative roles of the fractional volume available to the analyte and the subtleties of the gel architecture. Over the past several years, we have developed a lattice model of gel electrophoresis that has allowed us to revisit the fundamental assumptions of the standard Ogston model. In particular, we demonstrated that the fractional free volume is not the relevant parameter for gel sieving. In this article, we continue the development of this model and we generalize our mathematical approach to treat nonvanishing electric field intensities. To do so, we must revisit the way biased random walks are normally modeled by stochastic processes. Straightforward generalizations based on standard Metropolis-like schemes fail at high field intensities. Moreover, our generalization requires the complete decoupling of the spatial directions parallel and perpendicular to the field direction. We show that our novel theoretical approach makes it possible to calculate exact mobilities in the presence of lattice obstacles. Several two-dimensional examples are then studied, including one that includes topological dead ends that act like traps. In the latter case, we recover results very similar to those reported by Serwer et al. [Biopolymers 29, 1863 (1990)] on the trapping electrophoresis of charged spheres in agarose gels. In the absence of such traps, the mobility is shown to be a very weak function of the electric field, thus validating the historical neglect of the field intensity in the development of obstruction models for the Ogston sieving regime of small analytes. Finally, we describe how the present model could be improved to treat more realistic cases and we discuss the problem of the field dependence of the diffusion coefficient during electrophoresis.

Gauthier, Michel G.; Slater, Gary W.



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


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



Photo-initiated cross-linked polyacrylamide gels for microdevice electrophoresis  

E-print Network

complex micro-channel network. The rate of photo-initiation in the free radical gel polymerization reaction, however, can exert a strong influence on the resulting gel structure. Experimental data on separation resolution of single stranded DNA (ss...

Agrawal, Shilpa



Agarose and Polyacrylamide Gel Electrophoresis Methods for Molecular Mass Analysis of 5500 kDa Hyaluronan  

PubMed Central

Agarose and polyacrylamide gel electrophoresis systems for the molecular mass-dependent separation of hyaluronan (HA) in the size range of approximately 5500 kDa have been investigated. For agarose-based systems, the suitability of different agarose types, agarose concentrations, and buffers systems were determined. Using chemoenzymatically synthesized HA standards of low polydispersity, the molecular mass range was determined for each gel composition, over which the relationship between HA mobility and logarithm of the molecular mass was linear. Excellent linear calibration was obtained for HA molecular mass as low as approximately 9 kDa in agarose gels. For higher resolution separation, and for extension to molecular masses as low as approximately 5 kDa, gradient polyacrylamide gels were superior. Densitometric scanning of stained gels allowed analysis of the range of molecular masses present in a sample, and calculation of weight-average and number-average values. The methods were validated for polydisperse HA samples with viscosity-average molecular masses of 112, 59, 37, and 22 kDa, at sample loads of 0.5 g (for polyacrylamide) to 2.5 g (for agarose). Use of the methods for electrophoretic mobility shift assays was demonstrated for binding of the HA-binding region of aggrecan (recombinant human aggrecan G1-IGD-G2 domains) to a 150 kDa HA standard. PMID:21684248

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



Use of pulsed-field gel electrophoresis of conserved XbaI fragments for identification of swine Salmonella serotypes.  


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

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



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


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



Comparison and genomic sizing of Escherichia coli O157:H7 isolates by pulsed-field gel electrophoresis.  

PubMed Central

Genomic DNAs of Escherichia coli O157:H7 strains isolated from patients and food samples were analyzed by pulsed-field gel electrophoresis. The rare-cutting endonucleases SfiI and XbaI generated 6 and 10 distinct genomic profiles, respectively, for the 22 strains analyzed, indicating that this technique may find application for epidemiologic studies. Summation of XbaI fragments from five E. coli O157:H7 strains estimated the genomic length at ca. 4.7 Mb. Images PMID:8215383

Harsono, K D; Kaspar, C W; Luchansky, J B



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)



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

Microsoft Academic Search

Diffusive transfer was examined as a blotting method to transfer proteins from polyacrylamide gels to membranes for ultraviolet matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The method is well-suited for transfers from isoelectric focusing (IEF) gels. Spectra have been obtained for 11 pmol of 66 kDa albumin loaded onto an IEF gel and subsequently blotted to polyethylene. Similarly, masses of

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



Proteomic analysis of tomato (Lycopersicum esculentum var. cerasifarm) expressing the HBsAg gene by 2-dimensional difference gel electrophoresis.  


In a previous study, an HBsAg gene-bearing transgenic tomato line was made available and it exhibited notable physiological alterations compared with the non-transgenic tomato (control). In particular, leaves of the transgenic plants were fleshy and dark. We hypothesized that a change in leaf proteins of the transgenic plants account for the observed phenotypes. In this study, total protein content in leaves of the transgenic plants was analyzed by 2-dimensional difference gel electrophoresis. A total number of 700 protein spots were detected on silver-stained gels, of which 368 protein spots were matched between the control and sample gels. Among these matched proteins, the expression levels of 122 proteins in the transgenic plants were upregulated while those of the rest were downregulated. In addition, 25 abundant proteins (value ratio?>?2.0) on silver-stained gels were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Sixteen differentially expressed proteins were identified, of which 13 were predicted to be involved in cell division, energy metabolism, protein synthesis and processing. The possible roles of these proteins in the transgenic tomato strain have been discussed. Taken together, our data indicate that significant alterations in protein expression occur in transgenic tomatoes bearing the HBsAg gene. Our findings will help broaden our knowledge of the mechanism by which exogenously expressed genes lead to phenotypic alterations in transgenic plants. PMID:24057504

Guo, Bin; He, Wei; Wu, Daochang; Che, Delu; Fan, Penghui; Xu, Lingling; Wei, Yahui



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



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



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


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



DNA polymorphisms in strains of Mycobacterium tuberculosis analyzed by pulsed-field gel electrophoresis: a tool for epidemiology.  

PubMed Central

Mycobacterium tuberculosis isolates were studied by comparing large restriction fragment (LRF) patterns produced by digestion of chromosomal DNA with infrequent-cutting endonucleases and pulsed-field gel electrophoresis. Four cultures of H37Rv and 36 clinical isolates of M. tuberculosis were compared by using DraI, AsnI, XbaI, and SpeI. DraI and AsnI allowed easy visual separation of 18 of 21 epidemiologically unrelated strains. XbaI and SpeI allowed discrimination of all 21 unrelated strains, including the 3 strains inseparable with DraI and AsnI, but comparison of LRF patterns was more tedious because of overlapping fragments. A total of 26 isolates belonging to 10 clusters of related isolates were compared by pulsed-field gel electrophoresis, with all related isolates giving identical LRF patterns. These included multiple isolates from the same patient or the same family. The same grouping of clustered isolates was obtained when BamHI DNA digests were hybridized with two probes from the insertion sequence IS6110. Long-term laboratory passage of H37Rv produced minimal detectable changes in LRF patterns. LRF patterns are useful tools for epidemiologic studies of tuberculosis without the need for radioactive or specific DNA probes. Images PMID:1352518

Zhang, Y; Mazurek, G H; Cave, M D; Eisenach, K D; Pang, Y; Murphy, D T; Wallace, R J



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



Protein Reverse Staining: High-Efficiency Microanalysis of Unmodified Proteins Detected on Electrophoresis Gels  

Microsoft Academic Search

A methodology is presented for efficiently gaining structural information from electrophoresed proteins after on-gel detection by imidazole-sodium dodecyl sulfate-zinc reverse staining. As a consequence of reverse staining, (a) protein bands arise transparent against a deep white-stained background, limits of detection being in the femtomole range; (b) there is no loss of image when the gel is kept in distilled water

C. Fernandezpatron; M. Calero; P. R. Collazo; J. R. Garcia; J. Madrazo; A. Musacchio; F. Soriano; R. Estrada; R. Frank; L. R. Castellanosserra; E. Mendez



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

Microsoft Academic Search

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

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



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



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


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

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



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.

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



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



Genotoxicity of select herbicides in Rana catesbeiana tadpoles using the alkaline single-cell gel DNA electrophoresis (comet) assay.  


Pesticides are broadly used for pest control in agriculture despite possible negative impacts they may pose to the environment. Thus, we examined the DNA damage caused by five herbicides commonly used in southern Ontario (Canada). Erythrocytes from Rana catesbeiana (bullfrog) tadpoles were evaluated for DNA damage following exposure to selected herbicides, using the alkaline single-cell gel DNA electrophoresis (SCG) or "comet" assay [Singh et al. (1988): Exp Cell Res 175:184-191; Ralph et al. (1996): Eviron Mol Mutagen 28:112-120]. This approach involves detection, under alkaline conditions, of DNA fragments that upon electrophoresis migrate from the nuclear care, resulting in a comet formation. The herbicides tested, along with their active ingredients, were AAtrex Nine-O (atrazine), Dual-960E (metalochlor), Roundup (glyphosate), Sencor-500F (metribuzin), and Amsol (2,4-D amine). Tadpoles were exposed in the laboratory for a 24-hr period to several concentrations of the herbicides dissolved in dechlorinated water. Methyl methanesulphonate was used as a positive control. The herbicides AAtrex Nine-O-, Dual-960E-, Roundup-, and Sencor-500F-treated tadpoles showed significant DNA damage when compared with unexposed control animals, whereas, Amsol-treated tadpoles did not. Unlike the other responding herbicides, Sencor-500F did not show a relationship between dosage and DNA damage. In summary, the results indicate that at least some of the herbicides currently used in southern Ontario are capable of inducing DNA damage in tadpoles. PMID:9142171

Clements, C; Ralph, S; Petras, M



Amino acid composition, molecular weight distribution and gel electrophoresis of walnut (Juglans regia L.) proteins and protein fractionations.  


As a by-product of oil production, walnut proteins are considered as an additional source of plant protein for human food. To make full use of the protein resource, a comprehensive understanding of composition and characteristics of walnut proteins are required. Walnut proteins have been fractionated and characterized in this study. Amino acid composition, molecular weight distribution and gel electrophoresis of walnut proteins and protein fractionations were analyzed. The proteins were sequentially separated into four fractions according to their solubility. Glutelin was the main component of the protein extract. The content of glutelin, albumin, globulin and prolamin was about 72.06%, 7.54%, 15.67% and 4.73% respectively. Glutelin, albumin and globulin have a balanced content of essential amino acids, except for methionine, with respect to the FAO pattern recommended for adults. SDS-PAGE patterns of albumin, globulin and glutelin showed several polypeptides with molecular weights 14.4 to 66.2 kDa. The pattern of walnut proteins in two-dimension electrophoresis (2-DE) showed that the isoelectric point was mainly in the range of 4.8-6.8. The results of size exclusion chromatogram indicated molecular weight of the major components of walnut proteins were between 3.54 and 81.76 kDa. PMID:24473146

Mao, Xiaoying; Hua, Yufei; Chen, Guogang



Novel moving reaction boundary-induced stacking and separation of human hemoglobins in slab polyacrylamide gel electrophoresis.  


We developed a novel polyacrylamide gel electrophoresis (PAGE) method to stack and separate human hemoglobins (Hbs) based on the concept of moving reaction boundary (MRB). This differs from the classic isotachophoresis (ITP)-based stacking PAGE in the aspect of buffer composition, including the electrode buffer (pH 8.62 Tris-Gly), sample buffer (pH 6.78 Tris-Gly), and separation buffer (pH 8.52 Tris-Gly). In the MRB-PAGE system, a transient MRB was formed between alkaline electrode buffer and acidic sample buffer, being designed to move toward the anode. Hbs carried partial positive charges in the sample buffer due to its pH below pI values of Hbs, resulting in electromigrating to the cathode. Hbs would carry negative charges quickly when migrated into the alkaline electrode buffer and be transported to the anode until meeting the sample buffer again. Thus, Hbs were stacked within a MRB until the transient MRB reached the separation buffer and then separated by zone electrophoresis with molecular sieve effect of the gel. The experimental results demonstrated that there were three clear and sharp protein zones of Hbs (HbA1c, HbA0, and HbA2) in MRB-PAGE, in contrast to only one protein zone (HbA0) in ITP-PAGE for large-volume loading (?15 ?l), indicating high stacking efficiency, separation resolution, and good sensitivity of MRB-PAGE. In addition, MRB-PAGE was performed in a conventional slab PAGE device, requiring no special device. Thus, it could be widely used in separation and analysis of diluted protein in a standard laboratory. PMID:23912834

Tang, Yun-Yun; Wang, Hou-Yu; Chen, Lu; Li, Si; Guo, Chen-Gang; Fan, Hui-Zhi; Cao, Cheng-Xi; Fan, Liu-Yin



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

Microsoft Academic Search

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

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



Blue native polyacrylamide gel electrophoresis and the monitoring of malate-and  

E-print Network

), phosphoenolpyruvate carbox- ykinase (PEPCK), citrate lyase (CL) and aspartate aminotransferase (AST). Malate dehydrogenase (MDH) was utilized as a coupling enzyme to detect either malate or oxaloacetate in the presence carbox- ykinase; CL; citrate lyase; MDH; malate dehydrogenase; 2D BN-PAGE; two-dimensional blue native

Appanna, Vasu


Improved Pulsed-Field Gel Electrophoresis for Typing Vancomycin-Resistant Enterococci  

Microsoft Academic Search

Vancomycin-resistant enterococci (VRE) have been isolated with increased frequency in all major medical centers in the United States, Canada, and Western Europe (10). Several typ- ing methods, such as phage typing (13), serotyping (15), bio- typing (4), biochemical fingerprinting (12) and, more recently, DNA restriction fragment analysis (8), total plasmid profile analysis (14), random amplified PCR (9), pulsed-field gel elec-




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



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



Esterases of the flounder ( Platichthys flesus , Pleuronectidae, Teleostei): Development of an identification protocol using starch gel electrophoresis and characterization of loci  

Microsoft Academic Search

Summary Fish esterases are among the most difficult enzymes to identify using starch gel electrophoresis because of the many loci that are simultaneously active, and especially because of duplication phenomena, satellite bands, and stain trails. In an attempt to simplify and clarify electropherograms, various staining and inhibitory methods were tested on esterases from the flounderPlatichthys flesus. A range of migration

P. Berrebi; P. Landaud; P. Borsa; J. F. Renno



A Method for Activity Staining after Native Polyacrylamide Gel Electrophoresis Using a Coupled Enzyme Assay and Fluorescence Detection: Application to the Analysis of Several Glycolytic Enzymes  

Microsoft Academic Search

We describe a method for the detection of isoforms of several glycolytic enzymes by activity staining after native PAGE. The staining is based on coupled enzyme assays carried out on the gel after electrophoresis and is linked to the disappearance of NADH, which is visualized by fluorescence. This method offers reliable and sensitive detection for phosphoenolpyruvate carboxylase, PPi-dependent phosphofructokinase, and

Jean Rivoal; Christopher R. Smith; Trevor F. Moraes; David H. Turpin; William C. Plaxton



16S Ribosomal DNA-Based Analysis of Bacterial Diversity in Purified Water Used in Pharmaceutical Manufacturing Processes by PCR and Denaturing Gradient Gel Electrophoresis  

Microsoft Academic Search

The bacterial community in partially purified water, which is prepared by ion exchange from tap water and is used in pharmaceutical manufacturing processes, was analyzed by denaturing gradient gel electrophoresis (DGGE). 16S ribosomal DNA fragments, including V6, -7, and -8 regions, were amplified with universal primers and analyzed by DGGE. The bacterial diversity in purified water determined by PCR-DGGE banding

Mako Kawai; Eiichi Matsutera; Hisashi Kanda; Nobuyasu Yamaguchi; Katsuji Tani; Masao Nasu



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.



Detection and Identification of Lactobacillus Species in Crops of Broilers of Different Ages by Using PCR-Denaturing Gradient Gel Electrophoresis and Amplified Ribosomal DNA Restriction Analysis  

Microsoft Academic Search

The microflora of the crop was investigated throughout the broiler production period (0 to 42 days) using PCR combined with denaturing gradient gel electrophoresis (PCR-DGGE) and selective bacteriological culture of lactobacilli followed by amplified ribosomal DNA restriction analysis (ARDRA). The birds were raised under conditions similar to those used in commercial broiler production. Lactobacilli predominated and attained populations of 108

Luo Guan; Karen E. Hagen; Gerald W. Tannock; Doug R. Korver; Gaylene M. Fasenko; Gwen E. Allison



Succession of bacterial and fungal communities during a traditional pot fermentation of rice vinegar assessed by PCR-mediated denaturing gradient gel electrophoresis  

Microsoft Academic Search

Denaturing gradient gel electrophoresis (DGGE) based on small subunit rRNA gene was applied to a traditional rice vinegar fermentation process in which the conversion of rice starch into acetic acid proceeded in a pot. The fungal DGGE profile indicated that the transition from Aspergillus oryzae to Saccharomyces sp. took place at the initial stage at which alcohol production was observed.

Shin Haruta; Shintaro Ueno; Isao Egawa; Kazunori Hashiguchi; Akira Fujii; Masanobu Nagano; Masaharu Ishii; Yasuo Igarashi



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



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

ERIC Educational Resources Information Center

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

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



Analytical and micropreparative peptide mapping by high performance liquid chromatography/electrospray mass spectrometry of proteins purified by gel electrophoresis.  

PubMed Central

We report the use of microbore reverse-phase high performance liquid chromatography connected on-line to an electrospray mass spectrometer for the separation/detection of peptides derived by proteolytic digestion of proteins separated by polyacrylamide gel electrophoresis. A small fraction (typically 10% of the total) of the peptides eluting from the column was diverted through a flow-splitting device into the ion source of the mass spectrometer, whereas the majority of the peptide samples was collected for further analyses. We demonstrate the feasibility of obtaining reproducible peptide maps from submicrogram amounts of protein applied to the gel and good correlation of the signal detected by the mass spectrometer with peptide detection by UV absorbance. Furthermore, independently verifiable peptide masses were determined from subpicomole amounts of peptides directed into the mass spectrometer. The method was used to analyze the 265-kDa and the 280-kDa isoforms of the enzyme acetyl-CoA carboxylase isolated from rat liver. The results provide compelling evidence that the two enzyme isoforms are translation products of different genes and suggest that these approaches may be of general utility in the definitive comparison of protein isoforms. We furthermore illustrate that knowledge of peptide masses as determined by this technique provides a major advantage for error-free data interpretation in chemical high-sensitivity peptide sequence analysis. PMID:8104612

Hess, D.; Covey, T. C.; Winz, R.; Brownsey, R. W.; Aebersold, R.




Microsoft Academic Search

A new Eucarya-specific 18S rDNA primer set was con- structed and tested using denaturing gradient gel electro- phoresis to analyze the genetic diversity of eukaryotic mi- croorganisms in aquatic environments. All eukaryal lines of descent exhibited four or fewer nucleotide mismatches in the forward primer sequence, except for the Microspora line of descent. The reverse primer annealed to a more

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


Revisit of imidazole-zinc reverse stain for protein polyacrylamide gel electrophoresis.  


Imidazole-zinc reverse stain (ZN stain) is known for its high sensitivity, ease of use, and cost-effective feature. ZN stain is compatible to many experiments of which those are proteomics-related in particular. Here, we describe the ZN staining procedures and the subsequent procedures incorporated in detail, along with the improvements of setup in aspects of visualization and documentation for post-processing ZN-stained gel images. PMID:22585514

Chen, Han-Min



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

Microsoft Academic Search

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

Chenping Xu; Yan Xu; Bingru Huang



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

PubMed Central

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 40C, 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



Capillary Electrophoresis of Proteins  

Microsoft Academic Search

1.1. Capillary Electrophoresis of Proteins Capillary electrophoresis (CE) is a separation technique that combines aspects of both gel electrophoresis and high performance liquid chromatography (HPLC). As is the case for gel electrophoresis, the separation in CE is based upon differential migration in an electrical field. Like HPLC, the detection of the migrating sample analytes may be monitored on-line or postcolumn\\/capillary

Mark Strege


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

PubMed Central

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

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



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


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

PubMed Central

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



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


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

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



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

PubMed Central

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




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

PubMed Central

Background Traditional medicines are among the oldest medicines and their extensive use in the recent years reflects the publics interest in alternatives to conventional medicine. Objectives The aim of this study was to investigate the genotoxicity of Dillsun herbal medicine in DNA damage of rat hepatocytes compared to sodium dichromate using a comet assay technique. Materials and Methods Male Wistar rats were caught and their liver was washed with a perfusion buffer, followed by another wash with collagenase buffer. Hepatocytes were isolated and transferred on to a petri dish which contained a washing buffer. Hepatocytes were then separated and the cells were filtered and centrifuged at 1500 rpm for 3 minutes. The hepatocytes were counted using neubauer slides and kept in a bioreactor for 30 minutes. Cells were then exposed to different doses of Dillsun such 0.2, 1, 2.5, 5 and 10 mg/mL. Sodium dichromate was the positive control and incubated buffer was used as a negative control. Cell suspensions were placed on slides pre-coated with low melting point agarose and were covered with agarose gel. Agarose gels were then lysed and electrophoresis was done, followed by neutralization and staining. Slides were analyzed by fluorescence microscopy. The size and extent of DNA damage visualized by this technique was evaluated by examining cells. Migration behavior was classified according to the Kobayashi pattern. Results The results indicated that with an increase of Dillsun dose, the mutagenicity index slightly increased but compared to the positive control, there were significant differences, which suggests that the crude extract of Dillsun in vitro did not have mutagenic effects. Conclusions In conclusion the results showed that Dillsun has no mutagenic effects when compared to the positive control. Although by increasing the Dillsun dose, DNA damage also increased but this increase was not significant. PMID:24624188

Kalantari, Heibatullah; Rezaei, Mohsen; Salehcheh, Maryam; Moosavi, Mehrnoosh; Varnaseri, Golnaz



A multifaceted analysis of viperid snake venoms by two-dimensional gel electrophoresis: an approach to understanding venom proteomics.  


The complexity of Viperid venoms has long been appreciated by investigators in the fields of toxinology and medicine. However, it is only recently that the depth of that complexity has become somewhat quantitatively and qualitatively appreciated. With the resurgence of two-dimensional gel electrophoresis (2-DE) and the advances in mass spectrometry virtually all venom components can be visualized and identified given sufficient effort and resources. Here we present the use of 2-DE for examining venom complexity as well as demonstrating interesting approaches to selectively delineate subpopulations of venom proteins based on particular characteristics of the proteins such as antibody cross-reactivity or enzymatic activities. 2-DE comparisons between venoms from different species of the same genus (Bothrops) of snake clearly demonstrated both the similarity as well as the apparent diversity among these venoms. Using liquid chromatography/tandem mass spectrometry we were able to identify regions of the two-dimensional gels from each venom in which certain classes of proteins were found. 2-DE was also used to compare venoms from Crotalus atrox and Bothrops jararaca. For these venoms a variety of staining/detection protocols was utilized to compare and contrast the venoms. Specifically, we used various stains to visualize subpopulations of the venom proteomes of these snakes, including Coomassie, Silver, Sypro Ruby and Pro-Q-Emerald. Using specific antibodies in Western blot analyses of 2-DE of the venoms we have examined subpopulations of proteins in these venoms including the serine proteinase proteome, the metalloproteinase proteome, and the phospholipases A2 proteome. A functional assessment of the gelatinolytic activity of these venoms was also performed by zymography. These approaches have given rise to a more thorough understanding of venom complexity and the toxins comprising these venoms and provide insights to investigators who wish to focus on these venom subpopulations of proteins in future studies. PMID:15627971

Serrano, Solange M T; Shannon, John D; Wang, Deyu; Camargo, Antonio C M; Fox, Jay W



Brownian dynamics studies on DNA gel electrophoresis. I. Numerical method and ``periodic'' behavior of elongation-contraction motions  

NASA Astrophysics Data System (ADS)

The dynamics of a DNA molecule which is undergoing constant field gel electrophoresis (CFGE) is studied by a Brownian dynamics simulation method we have developed. In the method a DNA molecule is modeled as a chain of spherical electrolyte beads and the gel as a three-dimensional array of immobile beads. With the constraint for the separation of each pair of bonded beads to be less than a certain fixed value, as well as with the excluded volume effect, the simultaneous Langevin equations of motion for the beads are solved by means of the Lagrangian multiplier method. The resultant mobilities ? as a function of electric field coincide satisfactorily with the corresponding experimental results, once the time, the length, and the field of the simulation are properly scaled. In relatively strong fields "periodic" behavior is found in the chain dynamics and is examined through the time evolution of the radius of the longer principal axis, Rl(t). It is found that the mean width of a peak in Rl(t), or a period of one elongation-contraction process of the chain, is proportional to the number of beads in the chain, M, while the mean period between two such adjacent peaks is independent of M for large M. These results, combined with the observation that the chain moves to the field direction by the distance proportional to M in each elongation-contraction motion, yield the saturation of mobility for large M. This explains the reason that CFGE cannot separate DNA according to their size L(?M) for large L.

Azuma, Ryuzo; Takayama, Hajime



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



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

NASA Astrophysics Data System (ADS)

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

Larson, Ronald; Graham, Richard



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

PubMed Central

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

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



The osteopontin tissue level as a breast cancer biomarker in females after mastectomy measured by the capillary gel electrophoresis technique.  


A new diagnostic and prognostic biomarker may be of value in the diagnostic panel, especially among cancer diseases. The aim of the study was to evaluate the osteopontin (OPN) level measurable in the tumour tissue in females with diagnosed breast cancer after mastectomy, and to confirm its suitability to serve as a prognostic biomarker of the cancer.The OPN tissue levels and the classical risk factors were determined in twelve females. Tissue samples were collected and analysed by the capillary gel electrophoresis technique after previous appropriate preparation of the samples. A comparison between the OPN average values in the tissue of healthy versus cancer patients after mastectomy was performed using statistical univariate tests (ANOVA, t-test) and multivariate analysis (principal component analysis, PCA). The results demonstrate that the median values of the OPN in the tumour centre cancer tissue (10.940 ?g/g; within the range of 3.772-23.648) are significantly higher compared to healthy cells (5.173 ?g/g; within the range of 0.838- 17.583). Moreover, the increased tissue OPN level was correlated with the cancer stage.In this study osteopontin is presented as a possible candidate for a breast cancer biomarker. Further research is needed to obtain information on cancer signalling by means of the OPN threshold, and indication of its advanced stage. PMID:23244214

Konieczna, Lucyna; Skokowski, Jaroslaw; Oledzka, Ilona; Macur, Katarzyna; Belka, Mariusz; Baczek, Tomasz; Struck, Wiktoria; Markuszewski, Micha?



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


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



Molecular analysis of spoilage-related bacteria in pasteurized milk during refrigeration by PCR and denaturing gradient gel electrophoresis.  


Bacterial diversity in fluid milk products has been extensively studied in order to improve milk quality. Here, we illustrate the utility of viable counts and PCR-denaturing gradient gel electrophoresis (DGGE) for monitoring the microbial spoilage of pasteurized milk during shelf life. Five pasteurized milk samples stored at 4 degrees C were examined at 10 and 5 days before expiration and on the expiration day. With bacterial DNA extracted directly from the samples, PCR-DGGE analysis indicated that Pseudomonas became dominant in four samples. Meanwhile, the aerobic plate count of these four samples exceeded the regulatory limit of 20,000 CFU/ml at 5 days before expiration, and the rapid psychrotrophic count markedly surpassed the aerobic plate count on the expiration day. Streptococcus and Buttiauxella spp. were detected in several samples. Sequence analysis of DGGE fragments revealed high diversity among Pseudomonas spp. in the milk samples. P. putida and P. migulae grew to high numbers during refrigerated storage. Further identification of Pseudomonas at the species level was facilitated by PCR and multiplex PCR using species-specific primers; consequently, P. fluorescens and P. fragi were observed. These results highlight an important role of Pseudomonas in the shelf life of pasteurized milk. PMID:19343946

He, Hongfei; Dong, Jin; Lee, Chin Nyean; Li, Yong



Profiling of a microbial community under confined conditions in a fed-batch garbage decomposer by denaturing gradient gel electrophoresis.  


In order to determine the conditions for the maximum performance of a fed-batch composting (FBC) reactor, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was used to analyze the microbial communities established under the confined conditions of moisture content and environmental temperature. To evaluate the effects of microbial community structures on the performance of FBC reactors, degradation experiments using small-scale reactors and model waste were conducted under confined environmental conditions. A high degradation rate was observed under a wide range of MC conditions (30-60%) and at higher than usual temperatures (30-50 degrees C). The microbial communities that formed in the experimental FBC reactors were analyzed by DGGE of PCR-amplified 16S rRNA genes. The DGGE banding patterns at the same level as the degradation rates were similar even if the environmental conditions were different. Sequence analysis of the DGGE bands revealed the primary microbes which act in the reactor. PMID:17629695

Horisawa, Sakae; Sakuma, Yoh; Nakamura, Yasunori; Doi, Shuichi



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


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

Piwat, S; Teanpaisan, R



A pulsed-field gel electrophoresis (PFGE) map of twelve loci on chromosome 11q11-q13  

SciTech Connect

We report a pulsed-field gel electrophoresis map of 12 loci on proximal human chromosome 11q. Linkage studies have shown that this region of chromosome 11 contains the genes for familial atopic disease (APY) and multiple endocrine neoplasia type I (MEN1) (4). A physical map containing polymorphic loci will aid in the isolation of these disease genes. The map reported here has two noncontiguous groups of loci accounting for 8 of the 12 loci evaluated. One group spans a maximum distance of 1600 kb and included D11S146, BCL1, PRAD1, INT2, and HSTF1. The other group includes FTY1, C1NH, and COX8. TCN1, PGA, and PYGM did not yield any comigrating fragments and could not be physically linked on this PFGE map. These data enhance previously published physical maps of proximal 11q by refining the localization of and distances between markers in the BCL1 region. Additionally, new information about the locations and physical relationships between FTH1, C1NH, and COX 8 is presented. 14 refs., 3 figs.

Petty, E.M.; Bale, A.E. (Yale Univ. School of Medicine, New Haven, CT (United States)); Arnold, A. (Massachusetts General Hospital and Harvard Medical School, Boston (United States)); Marx, S.J. (National Inst. of Diabetes and Digestive and Kidney Diseases, Bethesda, MD (United States))



Succession of Bacterial Community Structure along the Changjiang River Determined by Denaturing Gradient Gel Electrophoresis and Clone Library Analysis  

PubMed Central

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

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



Survival of silage lactic acid bacteria in the goat gastrointestinal tract as determined by denaturing gradient gel electrophoresis.  


Aims:? To determine the survival rate of silage lactic acid bacteria (LAB) in the ruminant gastrointestinal tract. Methods and Results:? Wilted Italian ryegrass (Lolium multiflorum Lam.) silage (containing 19??10(6) ?CFU LAB?g(-1) ) was fed ad libitum to three goats equipped with rumen cannulae. Silage was given alone or with concentrates at a 1?:?1 ratio on a dry matter basis. Rumen fluid was then obtained 2, 4 and 8?h after the morning feeding. Denaturing gradient gel electrophoresis was performed to compare LAB communities in silage, rumen fluid and faeces. The LAB detected in the wilted silage included Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus murinus and Lactobacillus sakei. Bands indicative of Lact.?murinus were detected in either the rumen fluid or faeces, whereas the bands indicative of Lact.?plantarum, Lact.?brevis and Lact.?sakei were not. Although the rumen fluid LAB counts and volatile fatty acid concentrations were higher in goats fed silage plus concentrates compared with those fed silage alone, the LAB communities themselves remained unaffected. Sampling times and goat-to-goat variations did not affect the LAB communities found in the rumen fluid. Conclusion:? LAB communities found in the gut are not remarkably affected by the consumption of silage LAB, even when the silage is accompanied by concentrates that facilitate gut fermentation. Significance and Impact of the Study:? Although silage can improve probiotic function, it may be difficult for silage LAB to survive the digestive process in the ruminant gastrointestinal tract. PMID:22925065

Han, H; Takase, S; Nishino, N



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



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


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



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

PubMed Central

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial populations in DGGE profiles. To surmount this problem, we developed two new primer sets for specific amplification of bacterial 16S rDNA in wine fermentation samples without amplification of eukaryotic DNA. One primer set, termed WLAB1 and WLAB2, amplified lactic acid bacteria, while another, termed WBAC1 and WBAC2, amplified both lactic acid bacterial and acetic acid bacterial populations found in wine. Primer specificity and efficacy were examined with DNA isolated from numerous bacterial, yeast, and fungal species commonly found in wine and must samples. Importantly, both primer sets effectively distinguished bacterial species in wine containing mixtures of yeast and bacteria. PMID:14602643

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



Use of pulsed-field gel electrophoresis to investigate a pseudo-outbreak of Bacillus cereus in a pediatric unit.  

PubMed Central

Bacillus cereus is a well-known cause of food poisoning. It also causes rare systemic infections, usually in immunocompromised patients. Dissemination of this species in hospitals had been reported. Most of these episodes were pseudo-outbreaks and were usually secondary to equipment or environmental contamination. We report here on the use of pulsed-field gel electrophoresis (PFGE) to analyze a pseudo-outbreak of B. cereus in a pediatric unit. Different restriction endonucleases had been tested, and SmaI was found to give the best result for PFGE. Among the 26 clinical isolates of B. cereus and the type strain of the species, 15 distinct PFGE patterns were distinguished. PFGE after DNA macrorestriction with SmaI could clearly differentiate between the epidemiologically related isolates and the unrelated isolates. Because the same epidemic strain of B. cereus was isolated from the settle plates which were exposed near the outlet of the ventilation system, the source of this pseudo-outbreak was suspected to be the unit's air filtration system. This is one of the first reports of the application of PFGE to the study of B. cereus, and this method is useful for epidemiological investigation. PMID:9163476

Liu, P Y; Ke, S C; Chen, S L



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

PubMed Central

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

Piwat, S.; Teanpaisan, R.



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

SciTech Connect

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

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



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


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

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



Two-Dimensional Polyacrylamide Gel Electrophoresis Analysis of the Acid Tolerance Response in Listeria monocytogenes LO28  

PubMed Central

Listeria monocytogenes is capable of withstanding low pH after initial exposure to sublethal acidic conditions, a phenomenon termed the acid tolerance response (B. O'Driscoll, C. G. M. Gahan, and C. Hill, Appl. Environ. Microbiol. 62:1693-1698, 1996). Treatment of L. monocytogenes LO28 with chloramphenicol during acid adaptation abrogated the protective effect, suggesting that de novo protein synthesis is required for the acid tolerance response. Analysis of protein expression during acid adaptation by two-dimensional gel electrophoresis revealed changes in the levels of 53 proteins. Significant protein differences were also evident between nonadapted L. monocytogenes LO28 and a constitutively acid-tolerant mutant, ATM56. In addition, the analysis[S_TABC] revealed differences in protein expression between cells induced with a weak acid (lactic acid) and those induced with a strong acid (HCl). Comparison of both acid-adapted LO28 and ATM56 revealed that both are capable of maintaining their internal pH (pH(infi)) at higher levels than nonadapted control cells during severe acid stress. Collectively, the data demonstrate the profound alterations in protein synthesis which take place during acid adaptation in L. monocytogenes and ultimately lead to an increased ability to survive severe stress conditions. PMID:16535645

O'Driscoll, B.; Gahan, C.; Hill, C.



Characterisation of the bacterial community associated with early stages of Great Scallop (Pecten maximus), using denaturing gradient gel electrophoresis (DGGE).  


Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA was used to characterise and compare bacterial communities associated with scallop larvae (Pecten maximus), in different production units in a shellfish hatchery. Water and larvae samples were collected from three different aquaculture systems; stagnant, flow-through and a flow- through system with seawater treated with ozone. Samples were also collected from different algal cultures, inlet tanks and water pipes leading to the different aquaculture systems. Clear differences were seen between the bacterial community associated with the larvae and in the water from the different aquaculture systems. However, there were high similarities in the community composition between different water samples and between larvae samples collected at different time periods, indicating a high stability in the bacterial communities. Fifty three percent of the sequences from these samples were similar to 16S rRNA gene sequences of members of the gamma-subclass of the Proteobacteria. The different algal cultures had different bacterial communities, however 73 percent of the sequences were similar to 16S rRNA gene sequences of members of the alpha-subclass of the Proteobacteria. Differences in the DGGE profiles were also seen between the samples taken from the inlet tanks and water pipes, indicating a change in the bacterial community composition as the water passed through the pipes. To our knowledge this is the first study investigating bacterial communities associated with Great Scallop larvae in different aquaculture systems including noncultured components. PMID:12866858

Sandaa, Ruth-Anne; Magnesen, Thorolf; Torkildsen, Lise; Bergh, Oivind



Simulating Electrophoresis.  

ERIC Educational Resources Information Center

Describes a DNA fingerprinting simulation that uses vegetable food coloring and plastic food containers instead of DNA and expensive gel electrophoresis chambers. Allows students to decipher unknown combinations of dyes in a method similar to that used to decipher samples of DNA in DNA fingerprint techniques. (JRH)

Moertel, Cheryl; Frutiger, Bruce



Protein profiling of human pancreatic islets by two-dimensional gel electrophoresis and mass spectrometry.  


Completion of the human genome sequence has provided scientists with powerful resources with which to explore the molecular events associated with disease states such as diabetes. Understanding the relative levels of expression of gene products, especially of proteins, and their post-translational modifications will be critical. However, though the pancreatic islets play a key role in glucose homeostasis, global protein expression data in human are decidedly lacking. We here report the two-dimensional protein map and database of human pancreatic islets. A high level of reproducibility was obtained among the gels and a total of 744 protein spots were detected. We have successfully identified 130 spots corresponding to 66 different protein entries and generated a reference map of human islets. The functionally characterized proteins include enzymes, chaperones, cellular structural proteins, cellular defense proteins, signaling molecules, and transport proteins. A number of proteins identified in this study (e.g., annexin A2, elongation factor 1-alpha 2, histone H2B.a/g/k, heat shock protein 90 beta, heat shock 27 kDa protein, cyclophilin B, peroxiredoxin 4, cytokeratins 7, 18, and 19) have not been previously described in the database of mouse pancreatic islets. In addition, altered expression of several proteins, like GRP78, GRP94, PDI, calreticulin, annexin, cytokeratins, profilin, heat shock proteins, and ORP150 have been associated with the development of diabetes. The data presented in this study provides a first-draft reference map of the human islet proteome, that will pave the way for further proteome analysis of pancreatic islets in both healthy and diabetic individuals, generating insights into the pathophysiology of this condition. PMID:15952740

Ahmed, Meftun; Forsberg, Jens; Bergsten, Peter



Agarose gel electrophoresis of joint fluid using Hyrys-Hydrasys SEBIA system as a new prognostic tool for periprosthetic osteolysisin revision arthroplasty  

PubMed Central

Rationale. Prevention of wear-mediated osteolysis, the most common complication in total joint arthroplasty, is a great challenge for orthopedic surgery. Despite the diversity of current biomarkers of periprosthetic osteolysis (products of wear, bone turnover and inflammatory biomarkers), the major interferences and the great amount of sample necessary for analysis limit their use in clinical practice. Objective. The aim of this paper is to present three new electrophoretic methods using Hyrys-Hydrasys SEBIA system that have been used for the first time in Electrophoresis Laboratory of our hospital in the analysis of joint fluid for the prevention of periprosthetic osteolysis in revision arthroplasty. Methods and results. Analytical aspects of agarose gel electrophoresis of joint fluid proteins and lipoproteins as well as SDS-agarose gel electrophoresis of joint fluid proteins, their performances and clinical value are presented. The decreased level of albumin and increased level of alpha1 and alpha2 globulins were frequent changes detected on SEBIA electropherograms and good indicator for the presence of an inflammatory reaction generated by particle debris. In addition, a slightly increase of LDL mobility could provide good information about a high oxidative stress. Moreover, the Ig G assessed by using SDS-agarose gel electrophoresis could be a potential biomarker for an immunological reaction towards orthopedic implants. Discussion. Electrophoresis of joint fluid using Hyrys-Hydrasys SEBIA France system is a new analytical technique able to remove the most of current biomarkers disadvantages due to the determination of particular proteins (acute phase proteins, albumin, lipoproteins, and immunoglobulins) by using minimal amounts of joint fluid with minor interferences, minimal cost and rapid results. Abbreviations CTX, crosslinked C-telopeptide; IL- interleukins; Ig G, immunoglobulin G; LDL, low density lipoprotein; NTX, crosslinked N-telopeptide; PICP, procollagen I C terminal extension peptide; SDS, sodium dodecyl sulphate PMID:24146682

Chiva, A



Comparison of automated repetitive-sequencebased polymerase chain reaction and spa typing versus pulsed-field gel electrophoresis for molecular typing of methicillin-resistant Staphylococcus aureus  

Microsoft Academic Search

Automated repetitive polymerase chain reaction (PCR) (DiversiLab, bioMrieux, St. Laurent, Quebec, Canada) and single locus sequence typing of the Staphylococcus protein A (spa) gene with spa-type assignment by StaphType RIDOM software were compared to pulsed-field gel electrophoresis (PFGE) as the gold standard method for methicillin-resistant Staphylococcus aureus (MRSA) typing. Fifty-four MRSA isolates were typed by all methods: 10 of known

Deirdre L. Church; Barbara L. Chow; Tracie Lloyd; Daniel B. Gregson



Comparison of Protein A Gene Sequencing with Pulsed-Field Gel Electrophoresis and Epidemiologic Data for Molecular Typing of Methicillin-Resistant Staphylococcus aureus  

Microsoft Academic Search

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




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



Vertical distribution of bacterial community structure in the sediments of two eutrophic lakes revealed by denaturing gradient gel electrophoresis (DGGE) and multivariate analysis techniques  

Microsoft Academic Search

Vertical distribution of bacterial community structure was investigated in the sediments of two eutrophic lakes of China,\\u000a Lake Taihu and Lake Xuanwu. Profiles of bacterial communities were generated using a molecular fingerprinting technique, denaturing\\u000a gradient gel electrophoresis (DGGE) followed by DNA sequence analysis, and the results were interpreted with multivariate\\u000a statistical analysis. To assess changes in the genetic diversity of

Jin Zeng; Liuyan Yang; Jiayun Li; Yi Liang; Lin Xiao; Lijuan Jiang; Dayong Zhao



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



Probing for drug-induced multiplex signal transduction pathways using high resolution two-dimensional gel electrophoresis: application to ?-adrenoceptor stimulation in the rat C6 glioma cell  

Microsoft Academic Search

Whole-cell [32P]-protein phosphorylation assays and two-dimensional gel electrophoresis (2-DGE) were applied to the analysis of the ?-adrenoceptor (?AR)-linked signal transduction pathway. Rat C6 glioma cells were stimulated with isoproterenol and the protein lysates were resolved by 2-DGE. Two dimensional [32P]-phosphoprotein `maps' were generated depicting the modulation of intracellular proteins after isoproterenol stimulation versus unstimulated cells. A total of 274 distinct

Stephen M Storm; Xavier Z Khawaja



Moving pictures and pulsed-field gel electrophoresis show only linear mitochondrial DNA molecules from yeasts with linear-mapping and circular-mapping mitochondrial genomes  

Microsoft Academic Search

The mobility of mitochondrial DNA (mtDNA) in pulsed-field gel electrophoresis (PFGE) and its appearance in moving pictures\\u000a from fluorescence microscopy were used to investigate the mitochondrial genome structure for five Pichia and Williopsis strains of yeast. An apocytochrome b-gene hybridization probe identified only linear mtDNA molecules for each strain when\\u000a total cellular DNA was fractionated by PFGE. Most of the

Michael A. Jacobs; Shannon R. Payne; Arnold J. Bendich



A Single-Sample Method for Determination of Carbohydrate and Protein Contents Glycoprotein Bands Separated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis  

Microsoft Academic Search

A method is described for determination of carbohydrate and protein contents of glycoproteins separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and then electroblotted onto polyvinylidene difluoride (PVDF) membranes. Blots were stained, and appropriate pieces of PVDF membranes were excised, destained, and subjected to sequential hydrolysis with 0.2 M trifluoroacetic acid (TFA) for 1 h at 80C, then with 2 M

Ewa Zdebska; Jerzy Ko?cielak



Application of polymerase chain reaction-denaturing gradient gel electrophoresis for comparison of direct and indirect extraction methods of soil DNA used for microbial community fingerprinting  

Microsoft Academic Search

We used polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to compare bacterial community patterns\\u000a obtained with target DNA extracted from a soil by direct and indirect methods. For this purpose, two direct extraction methods,\\u000a i.e. cell lysis by bead beating and cell disruption by grinding in liquid N, and two indirect methods, i.e. cell extraction\\u000a followed by DNA extraction, and

J. Kozdrj; J. D. van Elsas



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




Pulsed-Field Gel Electrophoresis Typing of Oxacillin-Resistant Staphylococcus aureus Isolates from the United States: Establishing a National Database  

Microsoft Academic Search

Received 2 June 2003\\/Returned for modification 10 July 2003\\/Accepted 22 August 2003 Oxacillin-resistant Staphylococcus aureus (ORSA) is a virulent pathogen responsible for both health care- associated and community onset disease. We used SmaI-digested genomic DNA separated by pulsed-field gel electrophoresis (PFGE) to characterize 957 S. aureus isolates and establish a database of PFGE patterns. In addition to PFGE patterns of

Linda K. McDougal; Christine D. Steward; George E. Killgore; Jasmine M. Chaitram; Sigrid K. McAllister; Fred C. Tenover



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




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.



Genetic Analysis of Enteropathogenic and Enterohemorrhagic Escherichia coli Serogroup O103 Strains by Molecular Typing of Virulence and Housekeeping Genes and Pulsed-Field Gel Electrophoresis  

Microsoft Academic Search

We investigated the genetic relationships of 54 Escherichia coli O103 strains from humans, animals, and meat by molecular typing of housekeeping and virulence genes and by pulsed-field gel electrophoresis (PFGE). Multilocus sequence typing (MLST) of seven housekeeping genes revealed seven profiles, I through VII. MLST profiles I plus III cover 45 Shiga toxin-producing E. coli (STEC) O103:H2 strains from Australia,

Lothar Beutin; Stefan Kaulfuss; Sylvia Herold; Eric Oswald; Herbert Schmidt


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


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



Characterization of Finnish Borrelia burgdorferi sensu lato isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and with monoclonal antibodies.  

PubMed Central

Thirty-seven Borrelia burgdorferi strains, isolated in 1992 from Ixodes ricinus in Finland, were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting and indirect immunofluorescence assay (IFA) with five to nine monoclonal antibodies (MAbs). By SDS-PAGE results and reactivities to MAbs H3TS, J 8.3, I 17.3, and D6, the 37 isolates were assigned to the species B. burgdorferi sensu stricto (n = 7), Borrelia afzelii (n = 17), or Borrelia garinii (n = 13). Twenty more isolates examined only by IFA and with part of the MAbs were distributed as follows: 9 B. burgdorferi sensu stricto and 11 other species. Among 16 of 37 isolates displaying a SDS-PAGE patterns considered typical of that of B. garinii, 3 were negative by the test with MAb D6; the rest were positive. The three MAb D6-negative isolates reacted with MAb J 8.3 but not with MAb I 17.3. It is suggested that these isolates of a previously undescribed type represent atypical B. afzelii strains deficient in the expression of OspB proteins. The misleading species designation by the SDS-PAGE result is described. The IFA results were generally consistent with those obtained by immunoblotting. The exception was for 3 of 29 isolates that were positive with MAb H5332 by immunoblotting but that were IFA negative. In the present material of 57 strains, all 16 B. burgdorferi sensu stricto isolates originated from the Aland Islands. B. afzelii and B. garinii were isolated from all three regions where ticks were collected. The distributive difference seems to offer a basis for comparative clinico-epidemiological studies of Lyme borreliosis. PMID:7559935

Tuomi, J; Rantamaki, L K; Tanskanen, R; Junttila, J



Association of Streptomyces community composition determined by PCR-denaturing gradient gel electrophoresis with indoor mold status.  


Both Streptomyces species and mold species have previously been isolated from moisture-damaged building materials; however, an association between these two groups of microorganisms in indoor environments is not clear. In this study, we used a culture-independent method, PCR-denaturing gradient gel electrophoresis (PCR-DGGE), to investigate the composition of the Streptomyces community in house dust. Twenty-three dust samples each from two sets of homes categorized as high-mold and low-mold based on mold-specific quantitative PCR analysis were used in the study. Taxonomic identification of prominent bands was performed by cloning and sequencing. Associations between DGGE amplicon band intensities and home mold status were assessed using univariate analyses as well as multivariate recursive partitioning (decision trees) to test the predictive value of combinations of bands intensities. In the final classification tree, a combination of two bands was significantly associated with mold status of the home (p?=?0.001). The sequence corresponding to one of the bands in the final decision tree matched a group of Streptomyces species that included Streptomyces coelicolor and Streptomyces sampsonii, both of which have been isolated from moisture-damaged buildings previously. The closest match for the majority of sequences corresponding to a second band consisted of a group of Streptomyces species that included Streptomyces hygroscopicus, an important producer of antibiotics and immunosuppressors. Taken together, the study showed that DGGE can be a useful tool for identifying bacterial species that may be more prevalent in mold-damaged buildings. PMID:25331035

Johansson, Elisabet; Reponen, Tiina; Meller, Jarek; Vesper, Stephen; Yadav, Jagjit



High-Resolution Differentiation of Cyanobacteria by Using rRNA-Internal Transcribed Spacer Denaturing Gradient Gel Electrophoresis  

PubMed Central

For many ecological studies of cyanobacteria, it is essential that closely related species or strains can be discriminated. Since this is often not possible by using morphological features, cyanobacteria are frequently studied by using DNA-based methods. A powerful method for analysis of the diversity and dynamics of microbial populations and for checking the purity and affiliation of cultivated strains is denaturing gradient gel electrophoresis (DGGE). We realized high-resolution discrimination of a variety of cyanobacteria by means of DGGE analysis of sections of the internal transcribed spacer between the 16S and 23S rRNA genes (rRNA-ITS). A forward primer specific for cyanobacteria, targeted at the 3? end of the 16S rRNA gene, was designed. The combination of this primer and three different reverse primers targeted to the rRNA-ITS or to the 23S rRNA gene yielded PCR products of different sizes from cultures of all 16 cyanobacterial genera that were tested but not from other bacteria. DGGE profiles produced from the shortest section of rRNA-ITS consisted of one band for all but one cyanobacterial genera, and those generated from longer stretches of rRNA-ITS yielded DGGE profiles containing one to four bands. The suitability of DGGE for detecting intrageneric and intraspecific variation was tested by using strains of the genus Microcystis. Many strains could be discriminated by means of rRNA-ITS DGGE, and the resolution of this method was strikingly higher than that obtained with previously described methods. The applicability of the developed DGGE assays for analysis of cyanobacteria in field samples was demonstrated by using samples from freshwater lakes. The advantages and disadvantages associated with the use of each developed primer set are discussed. PMID:14602623

Janse, Ingmar; Meima, Marion; Kardinaal, W. Edwin A.; Zwart, Gabriel



Evaluation of the taxonomic utility of six-enzyme pulsed-field gel electrophoresis in reconstructing Salmonella subspecies phylogeny.  


Pulsed-field gel electrophoresis (PFGE) remains an important tool in the molecular epidemiological evaluation of strains emerging from disease outbreak clusters. Recent studies of Escherichia coli O157:H7 and Salmonella Enteritidis have noted marked improvements in the discriminatory power of PFGE when combining band profiles from up to six restriction enzyme datasets into a single concatenated analysis. This approach has provided more accurate assignments of genetic relationships among closely related strains and allowed effective phylogenetic inference of host and geographical reservoirs. Although this approach enhances epidemiological congruence among closely related strains, it remains unclear to what extent six-enzyme PFGE pattern similarity reiterates evolutionary relatedness among more distantly related Salmonella strains (i.e., serovar or subspecies levels). Here, taxonomic accuracy of six-enzyme PFGE is tested phylogenetically across two distinct Salmonella enterica populations-Salmonella reference collection B (SARB), representing the breadth of taxonomic diversity of S. enterica subspecies I only, and Salmonella reference collection C (SARC), comprising the seven disparate subspecies of S. enterica plus S. bongori. Cladistic analysis of SAR strains revealed substantial polyphyly between the two strain collections such that numerous SARB strains clustered more closely with diverged SARC subspecies rather than with other members of subspecies I. Also, in several cases, SARC sibling strains from the same subspecies were evolutionary obscured-broken into distant locales on the most parsimonious six-enzyme trees. Genetic diversity among SARB and SARC strains was comparable at 45% and 47%, respectively, while congruence testing revealed discordance among individual enzyme datasets. While six-enzyme PFGE is effective in ascertaining accurate genetic relationships for more closely related strains (e.g., strains within the same serovar), reconstitution of evolutionarily meaningful strain groupings may be elusive for Salmonella at the serovar level and above. Thus, caution is warranted when applying PFGE with a limited number of enzymes as the primary phylogenetic marker in these instances. PMID:20959148

Trujillo, Socrates; Keys, Christine E; Brown, Eric W



[Typing of Shigella sonnei strains isolated in some provinces of Turkey using antimicrobial resistance and pulsed field gel electrophoresis methods].  


Shigella species may lead to large epidemics owing to their low infective doses and frequent transmission from person to person with high secondary attack rates. Shigella sonnei is one of the most prevalent causative agent of infectious gastroenteritis in developing and developed countries and it is the most frequently reported Shigella serotype from Turkey in recent years. The aim of this study was to determine the types of S. sonnei strains isolated in different provinces of Turkey [in Marmara earthquake regions (Izmit, n=5; Adapazari, n=6; Yalova, n=2) in 1999 and in Ankara (n=17) in 1997, 2000 and 2001] according to their antimicrobial resistance and pulsed field gel electrophoresis (PFGE) patterns. All isolates were found sensitive to gentamicin, ceftriaxone, imipenem, nalidixic acid and ciprofloxacin. Twenty three (76.6%) of isolates were found resistant to streptomycin, 21 (70%) to trimethoprim-sulfamethoxazole, 20 (66.6%) to tetracycline, 6 (20%) to ampicillin, 3 (10%) to ampicillin/sulbactam and 1 (3.3%) to chloramphenicol. Three (%10) isolates were detected as intermediate susceptible to tetracycline and cefoperazone, while four isolates (13.3%) were susceptible to all antimicrobial agents tested. A total of nine different patterns were obtained according to antimicrobial resistance patterns. PFGE was performed by Xbal restriction enzyme. Isolates were grouped into A (n=24) and B (n=6) main PFGE types and into 13 closely or possibly related types. A total of 15 different PFGE patterns were identified among the isolates. It was determined that isolates from the same clone disseminated in Ankara during the years 2000-2001. Overall, different clones of S. sonnei strains were in dissemination in the provinces included. This study indicated that different S. sonnei clones were in circulation in Turkey and these results constitute the basic molecular preliminary data for the National Enteric Pathogens Laboratory Network in Turkey. PMID:19149077

Akali, Alper; Levent, Belkis; Akba?, Efsun; Esen, Berrin



Plasmid Profile and Pulsed-Field 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



Qualitative and quantitative proteomics by two-dimensional gel electrophoresis, peptide mass fingerprint and a chemically-coded affinity tag (CCAT).  


The chemically-coded affinity tag (CCAT) method combines standard electrophoresis protocols with MALDI-TOF-MS analysis to identify and quantify protein abundances in complex samples in one step. This method is designed to fit into the workflow of SDS-PAGE or two-dimensional electrophoresis (2-DE) only requiring basic proteome laboratory equipment. Prior to electrophoresis two protein samples are separately labelled with a heavy or a light version of the CCAT reagent via reduced cysteines in the proteins. Equal amounts are then combined and electrophoretically separated. Proteins can then be excised from the gel to obtain their peptide mass fingerprint by mass spectrometry. This fingerprint enabled not only identification, but also quantification by comparing relative peak intensities of CCAT-labelled peptides. In this article, we display how the CCAT method can be used to analyse two protein samples in one gel and that the peak intensities of labelled peptides reflect the abundance of a protein in it. PMID:14651868

Watt, Steven Alexander; Patschkowski, Thomas; Kalinowski, Jrn; Niehaus, Karsten



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



Proteomic analysis of surface proteins of Trichinella spiralis muscle larvae by two-dimensional gel electrophoresis and mass spectrometry  

PubMed Central

Background Trichinella spiralis is a zoonotic tissue-dwelling parasitic nematode that infects humans and other mammals. Its surface proteins are recognized as antigenic in many infected hosts, being directly exposed to the hosts immune system and are the main target antigens that induce the immune responses. The larval surface proteins may also interact with intestinal epithelial cells and may play an important role in the invasion and development process of T. spiralis. The purpose of this study was to analyze and characterize the surface proteins of T. spiralis muscle larvae by two-dimensional gel electrophoresis (2-DE) and mass spectrometry. Methods The surface proteins of T. spiralis muscle larvae were stripped from the cuticle of live larvae by the cetyltrimethylammonium bromide (CTAB) and sodium deoxycholate. The surface protein stripping was examined by an immunofluorescent test (IFT). The surface proteins were analyzed by SDS-PAGE and Western blotting, and then identified by 2-DE and MALDI-TOF/TOF mass spectrometry analysis. Results The IFT results showed that the surface proteins-stripped larvae were not recognized by sera of mice immunized with surface antigens. Western blotting showed 7 of 12 protein bands of the surface proteins were recognized by mouse infection sera at 18 dpi and at 42 dpi. The 2-DE results showed that a total of approximately 33 proteins spots were detected with molecular weights varying from 10 to 66kDa and isoelectric point (pI) from 4 to 7. Twenty-seven of 33 protein spots were identified and characterized to correlate with 15 different proteins. Out of the 14 proteins identified as T. spiralis proteins, 5 proteins (partial P49 antigen, deoxyribonuclease II family protein, two serine proteases, and serine proteinase) had catalytic and hydrolase activity. All of these 5 proteins were also associated with metabolic processes and 2 of the five proteins were associated with cellular processes. Conclusions In this study, T. spiralis muscle larval surface proteins have been identified, which will provide useful information to elucidate the host-parasite interaction, identify the invasion-related proteins, early diagnostic antigens and the targets for a vaccine. PMID:24330777



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

E-print Network

of Bio-Engineering; c Mechanical Engineering, d University of Maryland, College Park, MD; e Pathogenetics was also verified via an electrophoresis gel. Twelve amplifications were tested (Table Sup-1.6 mm as a proof-of-concept for the 2D- PCR approach. This initial effort demonstrated a substantial

Shapiro, Benjamin


oriGNAI3: a narrow zone of preferential replication initiation in mammalian cells identified by 2D gel and competitive PCR replicon mapping techniques.  

PubMed Central

The nature of mammalian origins of DNA replication remains controversial and this is primarily because two-dimensional gel replicon mapping techniques have identified broad zones of replication initiation whereas several other techniques, such as quantitative PCR, have disclosed more discrete sites of initiation at the same chromosomal loci. In this report we analyze the replication of an amplified genomic region encompassing the 3'-end of the GNAI3 gene, the entire GNAT2 gene and the intergenic region between them in exponentially growing Chinese hamster fibroblasts. These cells express GNAI3 but not GNAT2 . The replication pattern was first analyzed by two-dimensional neutral-alkaline gel electrophoresis. Surprisingly, the results revealed a small preferential zone of replication initiation, of at most 1.7 kb, located in a limited part of the GNAI3 - GNAT2 intergenic region. Mapping of this initiation zone was then confirmed by quantitative PCR. The agreement between the two techniques exploited here strengthens the hypothesis that preferred sites of replication initiation do exist in mammalian genomes. PMID:9580680

Toledo, F; Baron, B; Fernandez, M A; Lachages, A M; Mayau, V; Buttin, G; Debatisse, M



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


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



Salmonella Cerro isolated over the past twenty years from various sources in the US represent a single predominant Pulsed-Field Gel Electrophoresis type  

PubMed Central

Salmonella Cerro prevalence in US dairy cattle has increased significantly during the past decade. Comparison of 237 Salmonella isolates collected from various human and animal sources between 1986 and 2009 using pulsed- field gel electrophoresis, antimicrobial resistance typing, and spvA screening, showed very limited genetic diversity, indicating clonality of this serotype. Improved subtyping methods are clearly needed to analyze the potential emergence of this serotype. Our results thus emphasize the critical importance of population-based pathogen surveillance for the detection and characterization of potentially emerging pathogens, and caution to critically evaluate the adequacy of diagnostic tests for a given study population and diagnostic application. PMID:21349663

Hoelzer, K.; Cummings, K.J.; Wright, E.M.; Rodriguez-Rivera, L.D.; Roof, S.E.; Moreno Switt, A.I.; Dumas, N; Root, T.; Schoonmaker-Bopp, D.J.; Grohn, Y.T.; Siler, J.D.; Warnick, L.D.; Hancock, D.D.; Davis, M.A.; Wiedmann, M.



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



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.



Introducing basic molecular biology to Turkish rural and urban primary school children via hands-on PCR and gel electrophoresis activities.  


This study includes the results of a 2-day education project titled "Molecular Biology Laboratory Summer School, MoBiLYO." The project was held at a University Research Center by scientists from Department of Pharmacology and graduate students. The project was composed of introductory lectures, model construction, DNA isolation, polymerase chain reaction (PCR), and gel electrophoresis. The participants were 13-year-old eighth-graders attending primary schools affiliated with Ministry of National Education in urban and rural areas of Izmir, Turkey. The purpose of this study was to introduce basic molecular biology concepts through individually performed experiments such as PCR and gel electrophoresis integrated with creative drama. The students were assessed at the beginning and the end of each project day via mini-tests, experimental and presentation skills evaluation forms. Data showed that students' knowledge about DNA structure and basic molecular biology techniques significantly increased. On the basis of experimental and presentational skills, there was no significant difference between kids from urban and rural schools or between public and boarding public schools, whereas the average score of girls was significantly higher than that of boys. In conclusion, individually performed experiments integrated with creative drama significantly increased students' perception of complex experimental procedures on basic molecular biology concepts. Data suggests that integration of these concepts into the science and technology curriculum of Turkish primary education may support the recruitment of future scientists who can handle rapidly developing genomic techniques that will affect our everyday life. PMID:24474053

Selli, Cigdem; Y?ld?r?m, Gokce; Kaymak, Aysegul; Karacicek, Bilge; Ogut, Deniz; Gungor, Turkan; Erem, Erdem; Ege, Mehmet; Bmen, Nilay; Tosun, Metiner



Rapid and efficient isotachophoretic preconcentration in free solution coupled with gel electrophoresis separation on a microchip using a negative pressure sampling technique.  


We present a novel isotachophoresis-gel electrophoresis (ITP-GE) microchip system designed for rapid and efficient isotachophoretic preconcentration coupled with gel electrophoresis separation by using a negative pressure sampling technique. The overall ITP-GE procedure involves only three steps: sample loading, ITP preconcentration and GE separation and was controlled by a simple and compact negative pressure sampling device, which is composed of a vacuum vessel, a three-way electromagnetic valve and a single high voltage power supply. During the sample loading stage, a negative pressure was applied via a three-way electromagnetic valve in headspace of the two sealed sample waste reservoirs (SWs). A sandwiched sample zone between a leading and a terminating electrolyte zone was formed in the channel intersection in less than 1s. Once the three-way electromagnetic valve was switched to connect SWs to ambient atmosphere to release vacuum in SWs, ITP preconcentration in free solution and GE separation in the 4% hydroxyethylcellulose (HEC) sieving material were consequently activated under the electric potentials applied. The performance of present approach was evaluated by using DNA fragments as model analytes. Compared to conventional cross microchip GE using electrokinetic pinched injection, an average signal enhancement of 185-fold was obtained with satisfactory resolution. The results demonstrated the ITP-GE approach possessing an exciting potential of high sensitivity and short sampling time with significant simplification in operation and instrumentation. PMID:19328490

Qi, Li-Ya; Yin, Xue-Feng; Liu, Jin-Hua



Proteome analysis of rice tissues by two-dimensional electrophoresis: an approach to the investigation of gibberellin regulated proteins  

Microsoft Academic Search

Protein databases constructed using high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) were used to explore the proteome expressed in various rice tissues. Proteins from leaf sheath, root, and cultured suspension cells were systematically analyzed using 2D-PAGE, mass spectrometry and Edman sequencing, followed by database searching. In all, 79 of the 431 spots detected by 2D-PAGE in the leaf sheath, 73 of

N. Tanaka; H. Konishi; M. M. K. Khan; S. Komatsu



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 104 ELECTROPHORESIS Silver Stain Markers Designed for molecular weight determinations on silver stained gels, Silver Stain SDS

Lebendiker, Mario


A shortcut organic dye-based staining method for the detection of DNA both in agarose and polyacrylamide gel electrophoresis.  


In this study, we describe a brief, sensitive and safe organic dye-based staining method for the visualization of DNA both in agarose and polyacrylamide gels by using Victoria Pure Blue BO (VPBBO). Down to 0.8-1.6 ng of ? DNA/HindIII markers in agarose gels and 0.4-0.8 ng of pUC18 DNA/Mspl markers in polyacrylamide gels can be successfully detected within 15 and 10 min by the new developed technique, respectively. Moreover, the mechanism of the VPBBO staining was investigated and further confirmed by electrospray ionization mass spectrometry (ESI-MS) and molecular docking. The results indicated that the interaction between VPBBO and DNA is mainly due to groove binding. PMID:23296513

Cong, Weitao; Chen, Mao; Zhu, Zhongxin; Liu, Zhiguo; Nan, Jia; Ye, Weijian; Ni, Maowei; Zhao, Ting; Jin, Litai



Comparison of capillary electrophoresis sequencing with the new CEQ 2000 DNA Analysis System to conventional gel based systems for HIV drug resistance analysis 1 1 The two first authors contributed equally to this study  

Microsoft Academic Search

To date the majority of sequencing technologies have been based on use of gel plates. In this study sequencing by capillary electrophoresis for HIV-1 genotyping on the CEQ 2000 sequencer (Beckman Coulter Inc.) has been investigated and compared to an in house protocol on the Prism-377 sequencer (Applied Biosystems) and to the HIV-1 TruGene kit (Visible Genetics Inc.), two gel

Patrick Merel; Isabelle Pellegrin; Isabele Garrigue; Anne Caumont; Marie-Hlne Schrive; Valrie Birac; Pascal Bonot; Herv Fleury



Denaturing gradient gel electrophoresis analysis of bacterial community profiles in the rhizosphere of cry1AC-carrying Brassica rapa subsp. pekinensis.  


The effect of genetically modified (GM) Brassica rapa subsp. pekinensis (Chinese cabbage) expressing Bt toxin gene (cry1AC) to the rhizosphere bacterial community was examined using the denaturing gradient gel electrophoresis (DGGE) fingerprinting method. From the visual comparison of the DGGE profiles, there were no significant differences between the profiles of Bt and control rhizosphere in both Suwon and Yesan samples. From the sequence analysis of the individual bands, Sphingomonas sp. of Alphaproteobacteria and several actinobacterial members were identified as the main bacterial taxa in both Suwon and Yesan samples. In the multiple correspondence analysis, no clear separation between Bt and control rhizosphere was seen in both Suwon and Yesan datasets. The profiles of bulk soils were separated from those of rhizosphere. The DGGE fingerprinting analyses indicated that Bt crops did not significantly alter the genetic composition of rhizosphere bacterial communities. PMID:18337686

Jung, Sera; Park, Semi; Kim, Daeha; Kim, Seung Bum



Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of gentically modified crops. 3. Assessing unintended effects.  


The current procedures to assess the safety of food and feed derived from modern biotechnology include the investigation of possible unintended effects. To improve the probability of detecting unintended effects, profiling techniques such as proteomics are currently tested as complementary analytical tools to the existing safety assessment. An optimized two-dimensional gel electrophoresis (2DE) method was used as a proteomics approach to investigate insertional and pleiotropic effects on the proteome due to genetic engineering. Twelve transgenic Arabidopsis thaliana lines were analyzed by 2DE, and their seed proteomes were compared to that of their parental line as well as to 12 Arabidopsis ecotype lines. The genetic modification of the Arabidopsis lines, using three different genes and three different promoters, did not cause unintended changes to the analyzed seed proteome. Differences in spot quantity between transgenic and nontransgenic lines fell in the range of values found in the 12 Arabidopsis ecotype lines or were related to the introduced gene. PMID:16536592

Ruebelt, Martin C; Lipp, Markus; Reynolds, Tracey L; Schmuke, Jon J; Astwood, James D; DellaPenna, Dean; Engel, Karl-Heinz; Jany, Klaus-Dieter



Identification of Potential Diagnostic and Vaccine Candidates of Helicobacter pylori by Two-Dimensional Gel Electrophoresis, Sequence Analysis, and Serum Profiling  

PubMed Central

There is great interest in characterizing the proteins of the gastric pathogen Helicobacter pylori, especially those to which humans respond immunologically, because of the potential importance of such proteins in diagnosis and vaccine development. Two-dimensional gel electrophoresis was used to separate and identify potential antigens of H. pylori ATCC 43504. Over 30 proteins were reactive in Western blots with pooled sera from 14 infected patients. These proteins were analyzed by N-terminal sequence analysis. Fourteen proteins were determined to be distinct from any proteins previously described from H. pylori; the others were previously isolated and characterized proteins. Analysis of eight distinct H. pylori strains showed that most of these antigens were produced by all of the strains. We propose that collection of new antigens such as those recognized here will be useful in serologic tests for detecting and monitoring H. pylori infection and may also serve as potential targets for antimicrobial agent or vaccine development. PMID:9665963

McAtee, C. Patrick; Lim, Moon Young; Fung, Kevin; Velligan, Mark; Fry, Kirk; Chow, Theresa; Berg, Douglas E.



The alkaline single-cell gel electrophoresis/comet assay: a way to study DNA repair in radicle cells of germinating Vicia faba.  


Dry seeds are known to accumulate DNA damage with time of storage. Repair of DNA lesions during germination of Vicia faba seeds was followed in the radicles using the alkaline single-cell gel electrophoresis/comet assay. In this assay nuclei were liberated, mixed with agarose and spread out over a microscope slide. After lysis of the nuclear membrane and unwinding of the DNA duplex, DNA was stretched during electrophoresis, giving a comet-like migration pattern. The more DNA was damaged, the higher its mobility. DNA repair took place rapidly the first hours of imbibition and more slowly until ca 33 h after onset of germination. A small amount of heavily damaged cells remained present. Labelling with BrdU provided the possibility to localize repair patches and replicated sites in the comet migration pattern. At 15 h of germination, incorporation of BrdU in radicle DNA was situated at random over the entire comet. At 33 h, DNA repair was more or less accomplished and BrdU was mainly localized in the 'heads' of most comets. PMID:11321247

Koppen, G; Verschaeve, L



Effect of bromodeoxyuridine on radiation-induced DNA damage and repair based on DNA fragment size using pulsed-field gel electrophoresis  

SciTech Connect

We have used biphasic linear ramping pulsed-field gel electrophoresis (PFGE) to understand the effect of incorporation of bromodeoxyuridine (BrdUrd) on radiation-induced DNA damage and repair. This technique permits a determination of the fragment size distribution produced immediately after irradiation as well as during the repair period. We found that incorporation of BrdUrd increased the induction and decreased the repair of radiation damage. The fragment size distribution was consistent with a random breakage model. When we found that significantly more damage was detected after irradiation of deproteinized DNA compared to intact cells, we studied the effects of BrdUrd incorporation on the radiation response of cells or DNA at various phases of preparation for electrophoresis: cells adherent to the culture dish (A), trypsinized cells (B), agarose-embedded cells (C) and deproteinized DNA (D). Although there was a general tendency to detect more damage when irradiation was performed later in the preparation process, steps B and C were the only successive steps which were significantly different. These findings demonstrate that incorporation of BrdUrd randomly increases the induction of radiation damage and decreases its repair at the level of 200 kbp to 5 Mbp fragments. Furthermore, they confirm that the amount of damage detected depends upon the conditions of the cells or DNA at the time of irradiation. 34 refs., 5 figs., 2 tabs.

Lawrence, T.S.; Davis, M.A.; Normolle, D.P. [Univ. of Michigan, Ann Arbor, MI (United States)



[Solid-phase microextraction coupled with capillary electrophoresis for doping analysis of propranolol enantiomers in urine using a sol-gel derived calix [4] arene fiber].  


A new type fiber coated with diglycidyloxy calix [4] arene/hydroxy-terminated silicone oil (diglycidyloxy-C [4] arene/OH-TSO) made by sol-gel method was prepared for capillary electrophoresis (CE) sample pretreatment. By using headspace solid-phase microextraction (HS-SPME) combined with a novel back-extraction facility coupled off-line to capillary zone electrophoresis (CZE), the determination of propranolol enantiomers in urine was achieved with combination of ultrasonic back-extraction and field amplified sample injection (FASI) technologies. Extraction and back-extraction parameters were optimized. The clean-up effect and preconcentration effect were realized without derivatization during the SPME process in terms of this strongly polar and thermally stable compound. Preconcentration of the sample by calix [4] arene fiber increased the sensitivity, yielding a limit of detection (LOD) of 0.01 mg/L by CZE-diode array detection (DAD). Method repeatability (relative standard deviations (RSD) < 6.5%) and fiber reusability (> 150 extraction procedures) were observed over a wide linear range of propranolol (0.05 - 10 mg/L) in urine samples. Compared with commercial SPME stationary phases, the new coating showed higher extraction efficiency and this SPME-CZE-DAD procedures could meet the demand of minimum required performance limits (MRPL) set by the World Anti-Doping Agency (WADA) for the detection of propranolol in urine samples. PMID:16827299

Zhou, Xingwang; Li, Xiujuan; Zeng, Zhaorui



Standardization and international multicenter validation of a PulseNet pulsed-field gel electrophoresis protocol for subtyping Shigella flexneri isolates.  


Shigella flexneri is one of the agents most frequently linked to diarrheal illness in developing countries and often causes outbreaks in settings with poor hygiene or sanitary conditions. Travel is one of the means by which S. flexneri can be imported into developed countries, where this pathogen is not commonly seen. A robust and discriminatory subtyping method is needed for the surveillance of S. flexneri locally and regionally, and to aid in the detection and investigation of outbreaks. The PulseNet International network utilizes standardized pulsed-field gel electrophoresis (PFGE) protocols to carry out laboratory-based surveillance of foodborne pathogens in combination with epidemiologic data. A multicenter validation was carried out in nine PulseNet laboratories located in North and South America, Europe, and Asia, and it demonstrated that a new protocol is highly robust and reproducible for subtyping of S. flexneri. This protocol, already approved for PulseNet laboratories, applies NotI and XbaI as primary and secondary restriction enzymes, respectively, under electrophoresis conditions of initial switch time of 5 s to final switch time of 35 s, at 6 volts/cm. PMID:22506731

Pichel, Mariana; Brengi, Silvina P; Cooper, Kara L F; Ribot, Efrain M; Al-Busaidy, Suleiman; Araya, Pamela; Fernndez, Jorge; Vaz, Tania Ibelli; Kam, Kai Man; Morcos, Myriam; Nielsen, Eva M; Nadon, Celine; Pimentel, Guillermo; Prez-Gutirrez, Enrique; Gerner-Smidt, Peter; Binsztein, Norma



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


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



Polyclonal infections due to Mycobacterium avium complex in patients with AIDS detected by pulsed-field gel electrophoresis of sequential clinical isolates.  

PubMed Central

Invasive infection with organisms of the Mycobacterium avium complex (MAC) is common among patients with advanced human immunodeficiency virus infection. In previous studies, we analyzed multiple individual colonies of MAC isolated from specimens obtained at the same time and observed that 14 to 20% of patients are simultaneously infected with more than one strain. In this study, we examined sequential isolates from 12 patients with AIDS who had two or more MAC isolates available from clinical specimens collected more than 1 week apart; the intervals between the first and last specimens ranged from 8 to 192 (median, 46) days. For each isolate, restriction digests of genomic DNA were analyzed by pulsed-field gel electrophoresis; DNA was prepared by using a protocol, described here in detail, which had been optimized for conditions of bacterial growth and lysis. The pulsed-field gel electrophoresis analysis identified four patients (33%) infected with two different MAC strains. Both M. avium and M. intracellulare were cultured from blood specimens from two patients. In each of the four patients, the second strain was identified from a culture taken within 14 days of the initial study isolate, and in three of these patients, the first strain was detected again in a subsequent culture. These observations suggest that the presence of two different strains among isolates from sequential cultures may reflect ongoing polyclonal infection. We conclude that polyclonal infection with MAC is common among patients with AIDS. The identification of such infections may be critical in the development of effective treatments. Images PMID:7929773

Slutsky, A M; Arbeit, R D; Barber, T W; Rich, J; von Reyn, C F; Pieciak, W; Barlow, M A; Maslow, J N



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




Exposures of Sus scrofa to a TASER() conducted electrical weapon: no effects on 2-dimensional gel electrophoresis patterns of plasma proteins.  


In an earlier study, we found significant changes in red-blood-cell, leukocyte, and platelet counts, and in red-blood-cell membrane proteins, following exposures of anesthetized pigs to a conducted electrical weapon. In the current study, we examined potential changes in plasma proteins [analyzed via two-dimensional gel electrophoresis (2-DGE)] following two 30s exposures of anesthetized pigs (Sus scrofa) to a TASER () C2 conducted electrical weapon. Patterns of proteins, separated by 2-DGE, were consistent and reproducible between animals and between times of sampling. We determined that the blood plasma collection, handling, storage, and processing techniques we used are suitable for swine blood. There were no statistically significant changes in plasma proteins following the conducted-electrical-weapon exposures. Overall gel patterns of fibrinogen were similar to results of other studies of both pigs and humans (in control settings, not exposed to conducted electrical weapons). The lack of significant changes in plasma proteins may be added to the body of evidence regarding relative safety of TASER C2 device exposures. PMID:25319243

Jauchem, James R; Cerna, Cesario Z; Lim, Tiffany Y; Seaman, Ronald L



Comparison of capillary electrophoresis sequencing with the new CEQ 2000 DNA Analysis System to conventional gel based systems for HIV drug resistance analysis.  


To date the majority of sequencing technologies have been based on use of gel plates. In this study sequencing by capillary electrophoresis for HIV-1 genotyping on the CEQ 2000 sequencer (Beckman Coulter Inc.) has been investigated and compared to an 'in house' protocol on the Prism-377 sequencer (Applied Biosystems) and to the HIV-1 TruGene kit (Visible Genetics Inc.), two gel plate-based systems. Plasma from 20 HAART-treated patients with virological failure were analyzed for protease (PR) and reverse transcriptase (RT) genes. A total of 520 RT codons (26/patient) and 360 PR codons (18/patient) related to antiretroviral drug resistance were evaluated. The overall agreement between CEQ 2000 and Prism-377 results was 100% for the RT and PR primary and secondary mutations. The overall agreement between CEQ 2000 and TruGene was 100% for primary and > or =97% for secondary mutations. Discrepant results would have never led to errors in genotype interpretation. Performances for a 24 patients/week/one technician genotyping throughput were analyzed. For Prism-377, TruGene and CEQ 2000, manual processing required 5, 4 and 2,5 days, sequence data analysis needed additional 3, 1 and 2 days and cost/patient was approximately 49, 214 and 39 $, respectively. The CEQ 2000 sequencer offers a reliable alternative for fast and cost effective HIV drug resistance analysis. PMID:11543879

Merel, P; Pellegrin, I; Garrigue, I; Caumont, A; Schrive, M H; Birac, V; Bonot, P; Fleury, H



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



Identification of increased amounts of eppin protein complex components in sperm cells of diabetic and obese individuals by difference gel electrophoresis.  


Metabolic disorders like diabetes mellitus and obesity may compromise the fertility of men and women. To unveil disease-associated proteomic changes potentially affecting male fertility, the proteomes of sperm cells from type-1 diabetic, type-2 diabetic, non-diabetic obese and clinically healthy individuals were comparatively analyzed by difference gel electrophoresis. The adaptation of a general protein extraction procedure to the solubilization of proteins from sperm cells allowed for the resolution of 3187 fluorescent spots in the difference gel electrophoresis image of the master gel, which contained the entirety of solubilized sperm proteins. Comparison of the pathological and reference proteomes by applying an average abundance ratio setting of 1.6 and a p ? 0.05 criterion resulted in the identification of 79 fluorescent spots containing proteins that were present at significantly changed levels in the sperm cells. Biometric evaluation of the fluorescence data followed by mass spectrometric protein identification revealed altered levels of 12, 71, and 13 protein species in the proteomes of the type-1 diabetic, type-2 diabetic, and non-diabetic obese patients, respectively, with considerably enhanced amounts of the same set of one molecular form of semenogelin-1, one form of clusterin, and two forms of lactotransferrin in each group of pathologic samples. Remarkably, ?-galactosidase-1-like protein was the only protein that was detected at decreased levels in all three pathologic situations. The former three proteins are part of the eppin (epididymal proteinase inhibitor) protein complex, which is thought to fulfill fertilization-related functions, such as ejaculate sperm protection, motility regulation and gain of competence for acrosome reaction, whereas the putative role of the latter protein to function as a glycosyl hydrolase during sperm maturation remains to be explored at the protein/enzyme level. The strikingly similar differences detected in the three groups of pathological sperm proteomes reflect a disease-associated enhanced formation of predominantly proteolytically modified forms of three eppin protein complex components, possibly as a response to enduring hyperglycemia and enhanced oxidative stress. PMID:21525168

Paasch, Uwe; Heidenreich, Falk; Pursche, Theresia; Kuhlisch, Eberhard; Kettner, Karina; Grunewald, Sonja; Kratzsch, Jrgen; Dittmar, Gunnar; Glander, Hans-Jrgen; Hoflack, Bernard; Kriegel, Thomas M



DNA electrophoresis in a monodisperse porous medium  

Microsoft Academic Search

Electrophoresis of DNA migrating in an ordered matrix is studied and compared with classical agarose gel electrophoresis. A well-defined migration medium is obtained by crystallization of monodisperse silica spheres. Electrophoretic mobility of DNA is measured with fluorescence recovery after photobleaching experiments. The main result is that, as it was the case for gel electrophoresis, diffusion and electrophoretic mobility of DNA

L. Meistermann; B. Tinland



A charge-coupled-device camera image analysis system for quantifying DNA distributions in agarose gels after pulsed-field gel electrophoresis  

SciTech Connect

A charge-coupled-device camera system was coupled to a personal computer and, with uniformity in illumination and detection (within 4-8%) along each lane, was used for quantifying the distribution of DNA molecules that migrate from the PFGE well (plug) into the lane at distances varying from 1 to 50 mm (with 0.5 mm/pixel). By using a specially designed transmission filter for transmitting 470-725 nm fluorescence from ethidium bromide-stained DNA while eliminating most of the fluorescence (<400 nm) from the agarose gel, and by using neutral density filters to prevent saturation of the camera, the fluorescence intensity is linearly related to the amount of DNA varying from {approximately} 0.03 {mu}g in a 3-mm-diameter cylindrical plug 5 mm long (equal to background) to {approximately} 4 {mu}g (where ethidium bromide staining saturates). The percentage DNA released from the plug and distribution in the lane (with 1-2 mm resolution) obtained by quantifying DNA fluorescence were not significantly different from the same data obtained by analysis of radioactivity of the same DNA labeled with [{sup 3}H]dThd. However, scattering of fluorescence from one lane into an adjacent lane 3 mm away and as far as 10 mm from the plug into the lane presented a problem. This problem was overcome by using a form with slots to cover every other lane when the images were obtained and either (1) cutting the lane from the plug and moving it 15 mm away or (2) imaging the intact gel and applying a correction for {approximately} 7% of the fluorescence from the plug tailing out {approximately} 10 mm beyond the first 1 mm in the lane. In addition, the following were required: (1) carefully controlled staining and destaining procedures, and (2) a low background that is obtained as an average uniform background in each lane 5 mm beyond where DNA migration stops. 31 refs., 7 figs.

Dewey, W.C.; Thompson, L.L.; Trinh, M.L. [Univ. of California, San Francisco, CA (United States); Latz, D.L. [Univ. of California, San Francisco, CA (United States)]|[Univ. of Heidelberg (Germany); Ward, J.F. [Uni8v. of California, San Diego, La Jolla, CA (United States)



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


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



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


An inverse-shifting polymerase chain reaction (IS-PCR) combined with short-end capillary gel electrophoresis (CGE) was developed for genotyping of intron 22 inversion Type 1 (Inv22-1) and Type 2 (Inv22-2) of hemophilia A (HA). Severe HA cases are affected by intron 22 inversion around 45-50%. Inv22-1 has higher frequency than Inv22-2. The aim of this study is to distinguish them by genotyping. In order to improve Inv22 genotyping efficiency, five primers were designed and applied to differentiate the wild type, Inv22-1, Inv22-2 and carrier. Three amplicons of 405, 457 and 512bp were recognized for wild type; 333, 457 and 584bp for Inv22-1; 385, 405 and 584bp for Inv22-2. The Inv22-1 carrier has 5 amplicons including 333, 405, 457, 512, 584bp and Inv22-2 carrier is differentiated by 385, 405, 457, 512 and 584bp. The amplicons between Inv22-1 and Inv22-2 carriers are only different in 333bp for Inv22-1 carrier and 385bp for Inv22-2 carrier. Capillary gel electrophoresis (CGE) was used for separation within 5min. The separation voltage was set at 8kV (cathode at detector), and the temperature was kept at 25C. The sieving matrix was 89mM Tris, 89mM boric acid, 2mM EDTA containing 0.4% (w/v) HPMC and 1?M of YO-PRO()-1 Iodide. Total of 50 HA patients (including 35 non-Inv22, 14 Inv22-1, and one Inv22-2 patients) and 7 HA carriers were diagnosed in the application. Seven random samples (5 patients and 2 carriers) were subjected to comparison and gave identical results of DNA sequencing and this modified IS-PCR. PMID:25159417

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



Comparison of first dimension IPG and NEPHGE techniques in two-dimensional gel electrophoresis experiment with cytosolic unfolded protein response in Saccharomyces cerevisiae  

PubMed Central

Background Two-dimensional gel electrophoresis (2DE) is one of the most popular methods in proteomics. Currently, most 2DE experiments are performed using immobilized pH gradient (IPG) in the first dimension; however, some laboratories still use carrier ampholytes-based isoelectric focusing technique. The aim of this study was to directly compare IPG-based and non-equilibrium pH gradient electrophoresis (NEPHGE)-based 2DE techniques by using the same samples and identical second dimension procedures. We have used commercially available Invitrogen ZOOM IPGRunner and WITAvision systems for IPG and NEPHGE, respectively. The effectiveness of IPG-based and NEPHGE-based 2DE methods was compared by analysing differential protein expression during cytosolic unfolded protein response (UPR-Cyto) in Saccharomyces cerevisiae. Results Protein loss during 2DE procedure was higher in IPG-based method, especially for basic (pI?>?7) proteins. Overall reproducibility of spots was slightly better in NEPHGE-based method; however, there was a marked difference when evaluating basic and acidic protein spots. Using Coomassie staining, about half of detected basic protein spots were not reproducible by IPG-based 2DE, whereas NEPHGE-based method showed excellent reproducibility in the basic gel zone. The reproducibility of acidic proteins was similar in both methods. Absolute and relative volume variability of separate protein spots was comparable in both 2DE techniques. Regarding proteomic analysis of UPR-Cyto, the results exemplified parameters of general comparison of the methods. New highly basic protein Sis1p, overexpressed during UPR-Cyto stress, was identified by NEPHGE-based 2DE method, whereas IPG-based method showed unreliable results in the basic pI range and did not provide any new information on basic UPR-Cyto proteins. In the acidic range, the main UPR-Cyto proteins were detected and quantified by both methods. The drawback of NEPHGE-based 2DE method is its failure to detect some highly acidic proteins. The advantage of NEPHGE is higher protein capacity with good reproducibility and quality of spots at high protein load. Conclusions Comparison of broad range (pH310) gradient-based 2DE methods suggests that NEPHGE-based method is preferable over IPG (Invitrogen) 2DE method for the analysis of basic proteins. Nevertheless, the narrow range (pH47) IPG technique is a method of choice for the analysis of acidic proteins. PMID:23889826



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


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



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


The current study was conducted to determine the usefulness of 2 molecular techniques, automated repetitive extragenic palindromic-PCR (REP-PCR) and denaturing gradient gel electrophoresis (DGGE), to identify Salmonella serotypes of poultry origin. Salmonella continues to be a foodborne pathogen of principal concern in the United States. The interspersed conserved repetitive sequence of the bacterial genome and the 16-23S rDNA intergenic spacer region were amplified for REP-PCR and DGGE, respectively. Fifty-four Salmonella isolates from 2 turkey processing plants (A and B) were used for this comparison. Serotypes consisted of Brandenburg, Derby, Hadar, and Typhimurium, with n=6, 21, 12, and 15, respectively. The REP-PCR was fully automated, whereas DGGE was run on an acrylamide gel and the image was captured digitally. Both dendrograms were created using the unweighted pair group method with arithmetic average. There were more variations in percentage similarity in DGGE when compared with REP-PCR. The banding patterns were more distinct and uniform in the REP-PCR group than with DGGE. The results from the REP-PCR were generated within 1 h, whereas the DGGE required approximately 1 d to run. These data suggest that DGGE and REP-PCR are useful tools for identifying Salmonella serotypes isolated from poultry production or processing environments. In addition, REP-PCR is more rapid, may have a higher discriminatory power, but may be less cost-effective than DGGE. However, more research may be needed to validate this argument. Both DGGE and REP-PCR displayed high sensitivity in discriminating among Salmonella serotypes and either method could be considered as an alternative to more expensive and time-consuming conventional antibody-based serotyping methodologies. PMID:20460676

Anderson, P N; Hume, M E; Byrd, J A; Hernandez, C; Stevens, S M; Stringfellow, K; Caldwell, D J



Application of nested PCR-DGGE (denaturing gradient gel electrophoresis) for the analysis of ciliate communities in soils.  


Ciliates play important roles as prey and predators in ecosystems. Changes in the ciliate community can affect the composition and population of microfauna and microflora in ecosystems. To investigate the structure of ciliate communities, we developed a nested PCR-DGGE method, which combines a universal eukaryotic-specific primer set in the first PCR step with a ciliate-specific primer set in the second PCR step, to amplify 18S rRNA genes from ciliates. The 300 bp DGGE fragments generated more bands on the gel than the 600 bp DGGE fragments. Prior to bead beating, DNA extraction of ciliates from soil samples was optimized with a combination of freeze-thaw cycles and ultrasonication. We applied this nested PCR-DGGE method to agricultural soils amended with 0, 120, 300, and 600 t ha? year? of livestock slurry. The results from the DGGE profiles and principal component analysis (PCA) revealed that the supplement of slurry to soils influenced the ciliate communities. From phylogenetic analysis, 108 DGGE bands were assigned to six classes, which included Spirotrichea and Colpodea, of the subphylum Intramacronucleata, and one class of the subphylum Postciliodesmatophora. These results indicated that a wide variety of taxonomic groups were detected by DGGE profiling. Thus, the nested PCR-DGGE method described here could clearly differentiate between ciliate communities within soil samples and allowed for the phylogenetic identification of these ciliates at the class level. PMID:22791045

Shimano, Satoshi; Sambe, Mitsuo; Kasahara, Yasuhiro



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, Maria; Ceron, Jose J.; de Torre, Carlos; Escribano, Damian; Gutierrez, Ana M.; Tecles, Fernando



Porcine salivary analysis by 2-dimensional gel electrophoresis in 3 models of acute stress: a pilot study.  


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



Prevalence of Clostridium botulinum in Finnish Trout Farms: Pulsed-Field Gel Electrophoresis Typing Reveals Extensive Genetic Diversity among Type E Isolates  

PubMed Central

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, Sebastian; Bjorkroth, Johanna; Hyytia, Eija; Korkeala, Hannu



Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella Species in Human Feces by Using Group-Specific PCR Primers and Denaturing Gradient Gel Electrophoresis  

PubMed Central

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments generated by PCR with 16S ribosomal DNA-targeted group-specific primers was used to detect lactic acid bacteria (LAB) of the genera Lactobacillus, Pediococcus, Leuconostoc, and Weissella in human feces. Analysis of fecal samples of four subjects revealed individual profiles of DNA fragments originating not only from species that have been described as intestinal inhabitants but also from characteristically food-associated bacteria such as Lactobacillus sakei, Lactobacillus curvatus, Leuconostoc mesenteroides, and Pediococcus pentosaceus. Comparison of PCR-DGGE results with those of bacteriological culture showed that the food-associated species could not be cultured from the fecal samples by plating on Rogosa agar. On the other hand, all of the LAB species cultured from feces were detected in the DGGE profile. We also detected changes in the types of LAB present in human feces during consumption of a milk product containing the probiotic strain Lactobacillus rhamnosus DR20. The analysis of fecal samples from two subjects taken before, during, and after administration of the probiotic revealed that L. rhamnosus was detectable by PCR-DGGE during the test period in the feces of both subjects, whereas it was detectable by culture in only one of the subjects. PMID:11375166

Walter, Jens; Hertel, Christian; Tannock, Gerald W.; Lis, Claudia M.; Munro, Karen; Hammes, Walter P.



Multiplex polymerase chain reaction-capillary gel electrophoresis: a promising tool for GMO screening--assay for simultaneous detection of five genetically modified cotton events and species.  


A multiplex polymerase chain reaction assay coupled to capillary gel electrophoresis for amplicon identification by size and color (multiplex PCR-CGE-SC) was developed for simultaneous detection of cotton species and 5 events of genetically modified (GM) cotton. Validated real-time-PCR reactions targeting Bollgard, Bollgard II, Roundup Ready, 3006-210-23, and 281-24-236 junction sequences, and the cotton reference gene acp1 were adapted to detect more than half of the European Union-approved individual or stacked GM cotton events in one reaction. The assay was fully specific (<1.7% of false classification rate), with limit of detection values of 0.1% for each event, which were also achieved with simulated mixtures at different relative percentages of targets. The assay was further combined with a second multiplex PCR-CGE-SC assay to allow simultaneous detection of 6 cotton and 5 maize targets (two endogenous genes and 9 GM events) in two multiplex PCRs and a single CGE, making the approach more economic. Besides allowing simultaneous detection of many targets with adequate specificity and sensitivity, the multiplex PCR-CGE-SC approach has high throughput and automation capabilities, while keeping a very simple protocol, e.g., amplification and labeling in one step. Thus, it is an easy and inexpensive tool for initial screening, to be complemented with quantitative assays if necessary. PMID:19610365

Nadal, Anna; Esteve, Teresa; Pla, Maria



Soil clone library analyses to evaluate specificity and selectivity of PCR primers targeting fungal 18S rDNA for denaturing-gradient gel electrophoresis (DGGE).  


We evaluated the fungal specificity and detection bias of four fungal 18S rRNA gene (18S rDNA) primer sets for denaturing-gradient gel electrophoresis (DGGE). We constructed and compared clone libraries amplified from upland and paddy field soils with each primer set (1, NS1/GCFung; 2, FF390/FR1-GC; 3, NS1/FR1-GC; and 4, NS1/EF3 for the first PCR and NS1/FR1-GC for the second PCR). Primer set 4 (for nested PCR) showed the highest specificity for fungi but biased specific sequences. Sets 1, 2, and 3 (for single PCR) amplified non-fungal eukaryotic sequences (from 7 to 16% for upland soil and from 20 to 31% for paddy field soil) and produced libraries with similar distributions of fungal 18S rDNA sequences at both the phylum and the class level. Set 2 tended to amplify more diverse fungal sequences, maintaining higher specificity for fungi. In addition, clone analyses revealed differences among primer sets in the frequency of chimeras. In upland field soil, the libraries amplified with primer sets 3 and 4, which targeted long fragments, contained many chimeric 18S rDNA sequences (18% and 48%, respectively), while the libraries obtained with sets 1 and 2, which targeted short fragments, contained fewer chimeras (5% and 10%, respectively). PMID:21576883

Takada Hoshino, Yuko; Morimoto, Sho



Parallel Characterization of Anaerobic Toluene- and Ethylbenzene-Degrading Microbial Consortia by PCR-Denaturing Gradient Gel Electrophoresis, RNA-DNA Membrane Hybridization, and DNA Microarray Technology  

PubMed Central

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis. PMID:12088997

Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Said; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.



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.



Bacterial community composition during two consecutive NE Monsoon periods in the Arabian Sea studied by denaturing gradient gel electrophoresis (DGGE) of rRNA genes  

NASA Astrophysics Data System (ADS)

Horizontal and vertical variations in bacterial community composition were examined in samples collected during two Joint Global Ocean Flux Study (JGOFS) Arabian Sea cruises in 1995. The cruises, 11 months apart, took place during two consecutive NE Monsoon periods (January and December). Bacteria were harvested by filtration from samples collected in the mixed layer, mid-water, and deep sea at stations across the study area. Total bacterial community genomic DNA was analyzed by PCR amplification of 16S rRNA gene fragments, followed by denaturing gradient gel electrophoresis (DGGE). In total, 20 DGGE bands reflecting unique or varying phylotypes were excised, cloned and sequenced. Amplicons were dominated by bacterial groups commonly found in oceanic waters (e.g., the SAR11 cluster of ?-Proteobacteria and cyanobacteria), but surprisingly none of the sequenced amplicons were related to ?-Proteobacteria or to members of the Cytophaga-Flavobacter-Bacteroides phylum. Amplicons related to magnetotactic bacteria were found for the first time in pelagic oceanic waters. The DGGE banding patterns revealed a dominance of ?15 distinguishable amplicons in all samples. In the mixed layer the bacterial community was dominated by the same ?15 phylotypes at all stations, but unique phylotypes were found with increasing depth. Except for cyanobacteria, comparison of the bacterial community composition in surface waters from January and December 1995 showed only minor differences, despite significant differences in environmental parameters. These data suggest a horizontal homogeneity and some degree of seasonal predictability of bacterial community composition in the Arabian Sea.

Riemann, Lasse; Steward, Grieg F.; Fandino, Laura B.; Campbell, Lisa; Landry, Michael R.; Azam, Farooq



Source tracking of Escherichia coli by 16S-23S intergenic spacer region denaturing gradient gel electrophoresis (DGGE) of the rrnB ribosomal operon.  


This research validates a novel approach for source tracking based on denaturing gradient gel electrophoresis (DGGE) analysis of DNA extracted from Escherichia coli isolates. Escherichia coli from different animal sources and from river samples upstream from, at, and downstream of a combined sewer overflow were subjected to DGGE to determine sequence variations within the 16S-23S intergenic spacer region (ISR) of the rrnB ribosomal operon. The ISR was analyzed to determine if E. coli isolates from various animal sources could be differentiated from each other. DNA isolated from the E. coli animal sources was PCR amplified to isolate the rrnB operon. To prevent amplification of all 7 E. coli ribosomal operons by PCR amplification using universal primers, sequence-specific primers were utilized for the rrnB operon. Another primer set was then used to prepare samples of the 16S-23S ISR for DGGE. Comparison of PCR-DGGE results between human and animal sources revealed differences in the distribution and frequency of the DGGE bands produced. Human and Canada Goose isolates had the most unique distribution patterns and the highest percent of unique isolates and were grouped separately from all other animal sources. Method validation suggests that there are enough host specificity and genetic differences for use in the field. Field results at and around a combined sewer overflow indicate that this method can be used for microbial source tracking. PMID:18026210

D'Elia, Thomas V; Cooper, Chester R; Johnston, Carl G



Separation of native allophycocyanin and R-phycocyanin from marine red macroalga Polysiphonia urceolata by the polyacrylamide gel electrophoresis performed in novel buffer systems.  


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



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.



Culture-based and denaturing gradient gel electrophoresis analysis of the bacterial community structure from the intestinal tracts of earthworms(Eisenia fetida).  


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



Comparison of multiple-locus variable-number tandem repeat analysis, pulsed-field gel electrophoresis, and phage typing for subtype analysis of Salmonella enterica serotype Enteritidis.  


Strain subtyping is an important tool for detection of outbreaks caused by Salmonella enterica serotype Enteritidis. Current subtyping methods, however, yield less than optimal subtype discrimination. In this study, we describe the development and evaluation of a multiple-locus variable-number tandem repeat analysis (MLVA) method for subtyping Salmonella serotype Enteritidis. The discrimination ability and epidemiological concordance of MLVA were compared with those of pulsed-field gel electrophoresis (PFGE) and phage typing. MLVA provided greater discrimination among non-epidemiologically linked isolates than did PFGE or phage typing. Epidemiologic concordance was evaluated by typing 40 isolates from four food-borne disease outbreaks. MLVA, PFGE, and, to a lesser extent, phage typing exhibited consistent subtypes within an outbreak. MLVA was better able to differentiate isolates between the individual outbreaks than either PFGE or phage typing. The reproducibility of MLVA was evaluated by subtyping sequential isolates from an infected individual and by testing isolates following multiple passages and freeze-thaw cycles. PFGE and MLVA patterns were reproducible for isolates that were frozen and passaged multiple times. However, 2 of 12 sequential isolates obtained from an individual over the course of 36 days had an MLVA type that differed at one locus and one isolate had a different phage type. Overall, MLVA typing of Salmonella serotype Enteritidis had enhanced resolution, good reproducibility, and good epidemiological concordance. These results indicate that MLVA may be a useful tool for detection and investigation of outbreaks caused by Salmonella serotype Enteritidis. PMID:17151203

Boxrud, D; Pederson-Gulrud, K; Wotton, J; Medus, C; Lyszkowicz, E; Besser, J; Bartkus, J M



Comparison of Multiple-Locus Variable-Number Tandem Repeat Analysis, Pulsed-Field Gel Electrophoresis, and Phage Typing for Subtype Analysis of Salmonella enterica Serotype Enteritidis?  

PubMed Central

Strain subtyping is an important tool for detection of outbreaks caused by Salmonella enterica serotype Enteritidis. Current subtyping methods, however, yield less than optimal subtype discrimination. In this study, we describe the development and evaluation of a multiple-locus variable-number tandem repeat analysis (MLVA) method for subtyping Salmonella serotype Enteritidis. The discrimination ability and epidemiological concordance of MLVA were compared with those of pulsed-field gel electrophoresis (PFGE) and phage typing. MLVA provided greater discrimination among non-epidemiologically linked isolates than did PFGE or phage typing. Epidemiologic concordance was evaluated by typing 40 isolates from four food-borne disease outbreaks. MLVA, PFGE, and, to a lesser extent, phage typing exhibited consistent subtypes within an outbreak. MLVA was better able to differentiate isolates between the individual outbreaks than either PFGE or phage typing. The reproducibility of MLVA was evaluated by subtyping sequential isolates from an infected individual and by testing isolates following multiple passages and freeze-thaw cycles. PFGE and MLVA patterns were reproducible for isolates that were frozen and passaged multiple times. However, 2 of 12 sequential isolates obtained from an individual over the course of 36 days had an MLVA type that differed at one locus and one isolate had a different phage type. Overall, MLVA typing of Salmonella serotype Enteritidis had enhanced resolution, good reproducibility, and good epidemiological concordance. These results indicate that MLVA may be a useful tool for detection and investigation of outbreaks caused by Salmonella serotype Enteritidis. PMID:17151203

Boxrud, D.; Pederson-Gulrud, K.; Wotton, J.; Medus, C.; Lyszkowicz, E.; Besser, J.; Bartkus, J. M.



Comparative horizontal starch gel isoenzyme electrophoresis of Lymnaea (Bullastra) cumingiana (Pulmonata: Lymnaeidae) and related taxa in the Indo-Pacific region.  


Foot muscle tissue extracts from six lymnaeid species of the Indo-Pacific region [Lymnaea (Bullastra) cumingiana and L. (Radix) quadrasi from the Philippines, L. (R.) rubiginosa from Indonesia and Thailand, and L. (R.) viridis from Guam and Hong Kong] were subjected to horizontal starch gel isoenzyme electrophoresis and assayed for seven isoenzymes (AcP, AlP, CA, EST, LAP, CAT and GOT) to elucidate their taxonomic relationships. L. cumingiana exhibited banding patterns for EST, LAP and CAT uniquely different from the rest, thus supporting the hypothesis that it is a distinct species. Zymogram patterns for AlP, CA, EST and LAP attest to the close affinity between L. quadrasi and L. rubiginosa (Indonesia and Thailand). Minor differences suggest a closer relationship between the two geographical strains of L. rubiginosa than with L. quadrasi, lending support to the hypothesis that L. quadrasi is inseparable as a race or variety from the typical L. swinhoei Adams, which in turn is but a race of L. auricularia, which also encompasses L. rubiginosa. The two geographical strains of L. viridis from Guam and Hong Kong showed the greatest consistency with regards to similarity and congruence in banding patterns. Non-specific esterases (EST) were the most useful in distinguishing the six species from each other. PMID:7825010

Monzon, R B; Thammapalerd, N; Kitikoon, V; Temcharoen, P; Sornmani, S; Viyanant, V



Use of 16S Ribosomal DNA PCR and Denaturing Gradient Gel Electrophoresis for Analysis of the Microfloras of Healing and Nonhealing Chronic Venous Leg Ulcers  

PubMed Central

The bacterial microfloras of 8 healing and 10 nonhealing chronic venous leg ulcers were compared by using a combination of cultural analysis and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene products. Cultural analysis of the microflora revealed that the majority of both wound types carried the aerobes Staphylococcus and Pseudomonas spp. (89 and 80%, respectively). Sequencing of 16S ribosomal DNAs selected on the basis of DGGE profiling allowed the identification of strains not detected by cultural means. Of considerable interest was the finding that more than 40% of the sequences represented organisms not cultured from the wound from which they were amplified. DGGE profiles also revealed that all of the wounds possessed one apparently common band, identified by sequencing as Pseudomonas sp. The intensity of this PCR signal suggested that the bacterial load of nonhealing wounds was much higher for pseudomonads compared to healing wounds and that it may have been significantly underestimated by cultural analysis. Hence, the present study shows that DGGE could give valuable additional information about chronic wound microflora that is not apparent from cultural analysis alone. PMID:15297496

Davies, Charlotte E.; Hill, Katja E.; Wilson, Melanie J.; Stephens, Phil; Hill, C. Michael; Harding, Keith G.; Thomas, David W.



Denaturing gradient gel electrophoresis for nonlethal detection of Aeromonas salmonicida in salmonid mucus and its potential for other bacterial fish pathogens.  


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



Determination of microbial diversity in Daqu, a fermentation starter culture of Maotai liquor, using nested PCR-denaturing gradient gel electrophoresis.  


This study endeavored to investigate the diversity of microbes present during the shaping, ripening and drying of Daqu, a fermentation starter culture and substrata complex of Maotai alcoholic spirit. A nested PCR-denaturing gradient gel electrophoresis technique was utilized with different combinations of primers. The results showed the presence of bacteria, yeasts and molds. The microflora, which originate from wheat, were readily detectable during every stage of the fermentation process. However, the microbial structure had clear differences in the shaping, ripening and drying processes. In the shaping stage, there was a high level of diversity of the LAB (lactic acid bacteria) and fungi in the shaped samples. In the ripening stage, however, a reduction of diversity of fungi with a high level of diversity of the Bacilli was observed in the ripened samples. In the drying stage, the diversity of Bacilli and fungi, especially acid-producing bacteria, reduced dramatically. Interestingly, uncultured Lactococcus sp., Microbacterium testaceum, Cochliobolus sp., and Thermoascus crustaceus were the first to be detected in the fermentation starters used in liquor production. This study revealed the microbial diversity and distributions during the shaping, ripening and drying of Daqu-making, facilitating evaluation of the hygienic conditions and aiding in the design of specific starter and/or adjunct cultures. PMID:22806111

Xiu, Liu; Kunliang, Guo; Hongxun, Zhang



Improved Identification of Rapidly Growing Mycobacteria by a 16S-23S 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.



Succession of bacterial and fungal communities during a traditional pot fermentation of rice vinegar assessed by PCR-mediated denaturing gradient gel electrophoresis.  


Denaturing gradient gel electrophoresis (DGGE) based on small subunit rRNA gene was applied to a traditional rice vinegar fermentation process in which the conversion of rice starch into acetic acid proceeded in a pot. The fungal DGGE profile indicated that the transition from Aspergillus oryzae to Saccharomyces sp. took place at the initial stage at which alcohol production was observed. The early stage was characterized by the coexistence of Saccharomyces sp. and lactic acid bacteria. Almost all of the bacterial DGGE bands related to lactic acid bacteria were replaced by bands derived from Lactobacillus acetotolerance and Acetobacter pasteurianus at the stage at which acetic acid started to accumulate. The microbial succession, tested in three different pots, was found to be essentially identical. Among the bacteria isolated at the early stage, some species differed from those detected by DGGE. This is the first report to reveal the microbial community succession that occurs during a unique vinegar fermentation process, as determined by a culture-independent method. PMID:16499984

Haruta, Shin; Ueno, Shintaro; Egawa, Isao; Hashiguchi, Kazunori; Fujii, Akira; Nagano, Masanobu; Ishii, Masaharu; Igarashi, Yasuo



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


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



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



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



The key role of pulsed-field gel electrophoresis in investigation of a large multiserotype and multistate food-borne outbreak of Salmonella infections centered in Pennsylvania.  


Five different serotypes of Salmonella enterica were implicated in a large outbreak linked to fresh Roma tomatoes served at gas station deli counters in Pennsylvania and nearby states during July 2004: S. enterica serotypes Javiana, Anatum, Thompson, Typhimurium, and Muenchen. One of these serotypes, Anatum, was isolated from both tomatoes and patients. Pulsed-field gel electrophoresis (PFGE) played a key role in identifying the outbreak-associated isolates and distinguishing them from unrelated sporadic isolates. It also demonstrated that the genetic fingerprints of serotype Anatum isolates derived from patients were indistinguishable from those derived from tomatoes. Rapid communication of PFGE fingerprints with other public health laboratories through the Centers for Disease Control and Prevention's PulseNet USA national molecular surveillance network for bacterial food-borne pathogens facilitated the tracking of this outbreak in other states. The work described in this report emphasizes the laboratory's role in core public health functions and services, thereby providing a highly visible example of public health in action. PMID:16954249

Sandt, Carol H; Krouse, Donna A; Cook, Charles R; Hackman, Amy L; Chmielecki, Wayne A; Warren, Nancy G



Laboratory investigation of a multistate food-borne outbreak of Escherichia coli O157:H7 by using pulsed-field gel electrophoresis and phage typing.  


Two hundred thirty-three isolates of Escherichia coli O157:H7 were analyzed by both pulsed-field gel electrophoresis (PFGE) and bacteriophage typing. All 26 isolates from persons whose illness was associated with a recent multistate outbreak of E. coli O157:H7 infections linked to the consumption of undercooked hamburgers and all 27 isolates from incriminated lots of hamburger meat had the same phage type and the same PFGE pattern. Twenty-five of 74 E. coli O157:H7 isolates from Washington State and 10 of 27 isolates from other states obtained during the 6 months before the outbreak had the same phage type as the outbreak strain, but only 1 isolate had the same PFGE pattern. PFGE thus appeared to be a more sensitive method than bacteriophage typing for distinguishing outbreak and non-outbreak-related strains. The PFGE patterns of seven preoutbreak sporadic isolates and five sporadic isolates from the outbreak period differed from that of the outbreak strain by a single band, making it difficult to identify these isolates as outbreak or non-outbreak related. Phage typing and PFGE with additional enzymes were helpful in resolving this problem. While not as sensitive as PFGE, phage typing was helpful in interpreting PFGE data and could have been used as a simple, rapid screen to eliminate the need for performing PFGE on unrelated isolates. PMID:7883892

Barrett, T J; Lior, H; Green, J H; Khakhria, R; Wells, J G; Bell, B P; Greene, K D; Lewis, J; Griffin, P M



A study on the vertical profile of bacterial DNA structure in the Puruogangri (Tibetan Plateau) ice core using denaturing gradient gel electrophoresis  

NASA Astrophysics Data System (ADS)

The bacterial DNA structures at different depths in the Puruogangri (Tibetan Plateau) ice core (83.45 m) were investigated by the denaturing gradient gel electrophoresis (DGGE) DNA fingerprinting technique. DGGE profiles indicated that the bacterial species diversity in glacial ice is high, and indigenous species represented by common bands in all samples may grow on the glacial surface. Bacterial diversity, as estimated by Shannon indices (mean 2.91; SD 0.25; n = 13), was comparable to that of soil habitats and had a positive correlation with Ca2+ concentration (R = 0.71; P < 0.01), a good proxy of dust. This suggested that the soil ecosystem was the main source of bacteria in this glacier. The low similarity indices (0-43%) were found between the ice-core samples, which corresponded to the episodic deposition under defined climatic conditions and low activity of microorganisms in glacial ice. The profiles of bacterial species composition in glacial ice may be a bioindicator of climatic changes or dating.

Zhang, Xinfang; Yao, Tandong; An, Lizhe; Tian, Lide; Xu, Shijian


Comparative Evaluation of an Automated Repetitive-Sequence-Based PCR Instrument versus Pulsed-Field Gel Electrophoresis in the Setting of a Serratia marcescens Nosocomial Infection Outbreak?  

PubMed Central

A semiautomated, repetitive-sequence-based PCR (rep-PCR) instrument (DiversiLab system) was evaluated in comparison with pulsed-field gel electrophoresis (PFGE) to investigate an outbreak of Serratia marcescens infections in a neonatal intensive care unit (NICU). A selection of 36 epidemiologically related and 8 epidemiologically unrelated isolates was analyzed. Among the epidemiologically related isolates, PFGE identified five genetically unrelated patterns. Thirty-two isolates from patients and wet nurses showed the same PFGE profile (pattern A). Genetically unrelated PFGE patterns were found in one patient (pattern B), in two wet nurses (patterns C and D), and in an environmental isolate from the NICU (pattern G). Rep-PCR identified seven different patterns, three of which included the 32 isolates of PFGE type A. One or two band differences in isolates of these three types allowed isolates to be categorized as similar and included in a unique cluster. Isolates of different PFGE types were also of unrelated rep-PCR types. All of the epidemiologically unrelated isolates were of different PFGE and rep-PCR types. The level of discrimination exhibited by rep-PCR with the DiversiLab system allowed us to conclude that this method was able to identify genetic similarity in a spatio-temporal cluster of S. marcescens isolates. PMID:20237095

Ligozzi, Marco; Fontana, Roberta; Aldegheri, Marco; Scalet, Giovanna; Lo Cascio, Giuliana



Approach to Analyze the Diversity of Myxobacteria in Soil by Semi-Nested PCR-Denaturing Gradient Gel Electrophoresis (DGGE) Based on Taxon-Specific Gene  

PubMed Central

The genotypic diversity of insoluble macromolecules degraded myxobacteria, provided an opportunity to discover new bacterial resources and find new ecological functions. In this study, we developed a semi-nested-PCR-denaturing gradient gel electrophoresis (DGGE) strategy to determine the presence and genotypic diversity of myxobacteria in soil. After two rounds of PCR with myxobacteria-specific primers, an 194 bp fragment of mglA, a key gene involved in gliding motility, suitable for DGGE was obtained. A large number of bands were observed in DGGE patterns, indicating diverse myxobacteria inhabiting in soils. Furthermore, sequencing and BLAST revealed that most of the bands belonged to the myxobacteria-group, and only three of the twenty-eight bands belonged to other group, i.e., Deinococcus maricopensis. The results verified that myxobacterial strains with discrepant sequence compositions of gene mglA could be discriminated by DGGE with myxobacteria-specific primers. Collectively, the developed semi-nested-PCR-DGGE strategy is a useful tool for studying the diversity of myxobacteria. PMID:25280065

Li, Baiyuan; Yao, Qing; Zhu, Honghui



Evidence for recombination between N- and B-tropic murine leukemia viruses: analysis of three virion proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.  

PubMed Central

We have sodium dodecyl sulfate-polyacrylamide gel electrophoresis to analyze the virion proteins of an N- and a B-tropic C-type virus derived from the BALB/c mouse and 21 putative recombinants, designated XLP-N viruses, obtained from seven crosses between these N- and B-tropic viruses. All the XLP-N viruses are N-tropic but posses the XC plaque morphology of their B-tropic virus parent. Three virion proteins, p15, p30, and gp70, of the parental viruses each differ in electrophoretic mobility. Two recombinants were found that possess a p15 that comigrates with p15 of the B virus; 19 possess a p15 that comigrates with N virus p15. Sixteen recombinants possess a gp70 that migrates like the gp70 of the B virus: four have gp70 with an electrophoretic mobility like that of the N virus gp70. All 21 recombinants possess a p30 that comigrates with p30 of their N virus parent. Given the origin and phenotype of XLP-N viruses, these results would seem to provide good evidence that these viruses are recombinants. Images PMID:197267

Schindler, J; Hynes, R; Hopkins, N



Molecular characterization of mild-to-moderate hemophilia A: Detection of the mutation in 25 of 29 patients by denaturing gradient gel electrophoresis  

SciTech Connect

To date it has been difficult to characterize completely a genetic disorder, such as hemophilia A, in which the involved gene is large and unrelated affected individuals have different mutations, most of which are point mutations. Toward this end, the authors analyzed the DNA of 29 patients with mild-to-moderate hemophilia A in which the causative mutation is likely to be a missense mutation. Using computer analysis, they determined the melting properties of factor VIII gene sequences to design primer sets for PCR amplification and subsequent denaturing gradient gel electrophoresis (DGGE). A total of 45 primer sets was chosen to amplify 99% of the coding region of the gene and 41 of 50 splice junctions. To facilitate detection of point mutations, they mixed DNA from two male patients, and both homoduplexes and heteroduplexes were analyzed. With these 45 primer sets, 26 DNAs containing previously identified point mutations were easily distinguishable from normal. After analyzing the 29 patient with unknown mutations, they identified the disease-producing mutation in 25 (86%). Two polymorphisms and two rare normal variants were also found. Therefore, DGGE after computer analysis is a powerful method for nearly complete characterization of disease-producing mutations and polymorphisms in large genes such as that for factor VIII.

Higuchi, Miyoko; Antonarakis, S.E.; Kasch, L. Economou-Petersen, E.; Kazazian, H.H. Jr. (Johns Hopkins Univ., Baltimore, MD (United States)); Oldenburg, J.; Olek, K. (Univ. of Bonn (West Germany)); Arai, Morio; Inaba, Hiroshi (Tokyo Medical College (Japan))



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.



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


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



Carnosol, rosemary ingredient, induces apoptosis in adult T-cell leukemia/lymphoma cells via glutathione depletion: proteomic approach using fluorescent two-dimensional differential gel electrophoresis.  


Adult T-cell leukemia/lymphoma (ATL) is a fatal malignancy caused by infection with human T-lymphotropic virus type-1 and there is no accepted curative therapy for ATL. We searched for biological active substances for the prevention and treatment of ATL from several species of herbs. The ATL cell growth-inhibitory activity and apoptosis assay showed that carnosol, which is an ingredient contained in rosemary (Rosmarinus officinalis), induced apoptosis in ATL cells. Next, to investigate the apoptosis-inducing mechanism of carnosol, we applied proteomic analysis using fluorescent two-dimensional differential gel electrophoresis and mass spectrometry. The proteomic analysis showed that the expression of reductases, enzymes in glycolytic pathway, and enzymes in pentose phosphate pathway was increased in carnosol-treated cells, compared with untreated cells. These results suggested that carnosol affected the redox status in the cells. Further, the quantitative analysis of glutathione, which plays the central role for the maintenance of intracellular redox status, indicated that carnosol caused the decrease of glutathione in the cells. Further, N-acetyl-L-cystein, which is precursor of glutathione, canceled the efficiency of carnosol. From these results, it was suggested that the apoptosis-inducing activity of carnosol in ATL cells was caused by the depletion of glutathione. PMID:24323765

Ishida, Yo-ichi; Yamasaki, Masao; Yukizaki, Chizuko; Nishiyama, Kazuo; Tsubouchi, Hirohito; Okayama, Akihiko; Kataoka, Hiroaki



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



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.



Cost-Effectiveness and Efficacy of spa, SCCmec, and PVL Genotyping of Methicillin-Resistant Staphylococcus aureus as Compared to Pulsed-Field Gel Electrophoresis  

PubMed Central

Pulsed-field gel electrophoresis (PFGE) is a valuable molecular typing assay used for methicillin-resistant Staphylococcus aureus (MRSA) surveillance and genotyping. However, there are several limitations associated with PFGE. In Alberta, Canada, the significant increase in the number of MRSA isolates submitted to the Provincial Laboratory for Public Health (ProvLab) for PFGE typing led to the need for an alternative genotyping method. In this study, we describe the transition from PFGE to Staphylococcus protein A (spa), Staphylococcal cassette chromosome (SCCmec), and Panton-Valentine leukocidin (PVL) typing. A total of 1915 clinical MRSA isolates collected from 2005 to 2009 were used to develop and validate an algorithm for assigning PFGE epidemic types using spa, SCCmec, and PVL typing and the resulting data was used to populate a new Alberta MRSA typing database. An additional 12620 clinical MRSA isolates collected from 2010 to 2012 as part of ongoing routine molecular testing at ProvLab were characterized using the new typing algorithm and the Alberta MRSA typing database. Switching to spa, SCCmec, and PVL from PFGE typing substantially reduced hands-on and turn-around times while maintaining historical PFGE epidemic type designations. This led to an approximate $77,000 reduction in costs from 2010 to 2012. PFGE typing is still required for a small subset of MRSA isolates that have spa types that are rare, novel, or associated with more than one PFGE epidemic type. PMID:24244440

Li, Vincent; Chui, Linda; Louie, Lisa; Simor, Andrew; Golding, George R.; Louie, Marie



Characterization of the platelet membrane glycoprotein abnormalities in Bernard-Soulier syndrome and comparison with normal by surface-labeling techniques and high-resolution two-dimensional gel electrophoresis.  

PubMed Central

The platelets from three patients with Bernard-Soulier syndrome have been analyzed by surface-labeling coupled with two-dimensional gel electrophoresis and compared with normals. As well as the previously described absence or deficiency in glycoprotein (GP) Ib(alpha) it could be shown that GP Ib beta and an additional low molecular weight glycoprotein GP17 were not detectable using carbohydrate-labeling methods or deficient to the same extent as the GPIb alpha subunit. In addition, the thrombin cleavable glycoprotein could not be detected using carbohydrate-labeling methods in two patients and was deficient in a third. This finding was confirmed in a fourth patient by one-dimensional gel electrophoresis. Thus, the changes in the membrane of Bernard-Soulier platelets are more complex than previously thought. Images PMID:6284798

Clemetson, K J; McGregor, J L; James, E; Dechavanne, M; Lscher, E F



Contour-clamped homogeneous electric field electrophoresis of Staphylococcus aureus  

Microsoft Academic Search

Contour-clamped homogeneous electric field (CHEF) electrophoresis is a technique of pulsed-field gel electrophoresis that enables the resolution of large fragments of DNA that cannot be resolved by conventional gel electrophoresis. The procedure involves the application of controlled electric fields that change direction at a predetermined angle to samples of DNA that have been embedded in an agarose gel matrix and

Warren B Grubb; Frances G O'Brien; Edet E Udo



Comparison of Multiple-Locus Variable-Number Tandem-Repeat Analysis with Pulsed-Field Gel Electrophoresis, spa Typing, and Multilocus Sequence Typing for Clonal Characterization of Staphylococcus aureus Isolates  

Microsoft Academic Search

Multiple-locus variable-number tandem-repeat analysis (MLVA), a new PCR-based method of typing Staph- ylococcus aureus, was compared to pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST) on a group of 59 S. aureus (mostly methicillin-resistant) clinical isolates. The aim of the study was to establish possible criteria of clustering MLVA patterns and to check concordance levels between the

Natalia Malachowa; Artur Sabat; Marek Gniadkowski; Jolanta Krzyszton-Russjan; Joanna Empel; Jacek Miedzobrodzki; Klaudia Kosowska-Shick; Peter C. Appelbaum; Waleria Hryniewicz



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




Comparative evaluation of an automated ribotyping system versus pulsed-field gel electrophoresis for epidemiological typing of clinical isolates of Escherichia coli and Pseudomonas aeruginosa from patients with recurrent gram-negative bacteremia  

Microsoft Academic Search

Ribotyping and macrorestriction analysis of chromosomal DNA using pulsed-field gel electrophoresis (PFGE) are among the more useful molecular epidemiologic typing methods. Because these techniques are labor intensive, automation of one or more steps may allow clinical laboratories to apply molecular typing methods. We compared the recently developed automated ribotyping system, the RiboPrinter Microbial Characterization System (DuPont), with PFGE as a

M. A. Pfaller; C. Wendt; R. J. Hollis; R. P. Wenzel; S. J. Fritschel; J. J. Neubauer; L. A. Herwaldt



Elucidating the Key Member of a Linuron-Mineralizing Bacterial Community by PCR and Reverse Transcription-PCR Denaturing Gradient Gel Electrophoresis 16S rRNA Gene Fingerprinting and Cultivation  

PubMed Central

A bacterial community from Danish agricultural soil was enriched with linuron [N-(3,4-dichlorophenyl)-N?-methoxy-N?-methylurea] as the sole carbon and nitrogen source. The community mineralized [ring-U-14C]linuron completely to 14CO2 and 14C-biomass. Denaturing gradient gel electrophoresis analysis and cultivation revealed that a Variovorax sp. was responsible for the mineralization activity. PMID:16000836

S?rensen, Sebastian R.; Rasmussen, Jim; Jacobsen, Carsten S.; Jacobsen, Ole S.; Juhler, Rene K.; Aamand, Jens



Use of restriction fragment length polymorphisms resolved by pulsed-field gel electrophoresis for subspecies identification of mycobacteria in the Mycobacterium avium complex and for isolation of DNA probes.  

PubMed Central

Mycobacterial strains from the Mycobacterium avium complex were compared with each other and with Mycobacterium phlei isolates by restriction endonuclease digestion of chromosomal DNA with SspI and analysis by pulsed-field gel electrophoresis. Characteristic profiles were observed for known typed strains, and five groups were identified. Primary bovine isolates identified as Mycobacterium paratuberculosis by classical methods were shown to fall into both the M. paratuberculosis- and M. avium-like groups. M. paratuberculosis 18 was in the latter category. Two Mycobacterium intracellulare strains of different Schaefer serotypes had different digestion profiles. In addition, this system was exploited for the preparation of DNA probes by the isolation, digestion, and subcloning of DNA fragments separated by pulsed-field gel electrophoresis. Probe JC12 hybridized only to M. avium complex strains, but not to M. phlei, showing characteristic hybridization profiles for each of the groups previously identified by pulsed-field gel electrophoresis. The approach taken in the study lends itself to the comparative analysis of members of the M. avium complex and to the isolation and characterization of DNA probes with specificity for these mycobacteria. Images PMID:1352787

Coffin, J W; Condon, C; Compston, C A; Potter, K N; Lamontagne, L R; Shafiq, J; Kunimoto, D Y



DNA double-strand breaks in mammalian cells exposed to gamma-rays and very heavy ions. Fragment-size distributions determined by pulsed-field gel electrophoresis.  


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



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



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



Diversity of pulsed-field gel electrophoresis pulsotypes, serovars, and antibiotic resistance among Salmonella isolates from wild amphibians and reptiles in the California Central Coast.  


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, with 27 isolates resistant to more than one antibiotic, including a subspecies IIIb isolate from snake having resistance to five different antibiotics. Twenty-three isolates were resistant to more than one class of antibiotic, and six isolates were resistant to three classes. While these subspecies of IIIa and IIIb cause fewer instances of human illness, they may serve as reservoirs of antibiotic resistance, determinants in the environment, and be sources of contamination of leafy greens associated with product recalls. PMID:23577627

Gorski, Lisa; Jay-Russell, Michele T; Liang, Anita S; Walker, Samarpita; Bengson, Yingjia; Govoni, Jessica; Mandrell, Robert E



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



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



Field-inversion gel electrophoresis analysis of the induction and rejoining of DNA double-strand breaks in cells embedded in agarose.  


Among the techniques available for the measurement of the induction and rejoining of DNA double-strand breaks (DSBs), pulsed-field gel electrophoresis appears to have the greatest potential to improve the sensitivity limits to study these lesions in the dose range closest to that used in cell survival experiments. Encapsulating the cells in agarose during the experimental procedure allows the accurate and reproducible measurement of rejoining kinetics with a very minimal time delay immediately after irradiation. The method allows direct comparison of the amount of initial DNA damage sustained with repair kinetics in experiments designed to elucidate the mechanisms underlying differences in radiosensitivity between cell lines, together with analysis of the effect of different radiation qualities. The sensitivity limits of the method are 1 Gy for the double-strand break induction experiments and 10 Gy for rejoining experiments. Under selected conditions, no significant degradation of DNA had been observed in rodent cell lines during repair incubation up to 17 h in either irradiated cells or unirradiated controls (background levels for neutron experiments, 2.2 +/- 0.3% at Time 0 compared to 2.3 +/- 0.5% after 17 h of incubation; background levels for X-ray experiments, 2.3 +/- 0.6% at Time 0 and 3.7 +/- 1.1% after 17 h of incubation). In preliminary experiments with the A549 human oat cell carcinoma cell line, DNA DSB background levels remained constant in unirradiated controls up to 4 h in the range reported for the rodent cell line. PMID:8386387

Kysela, B P; Michael, B D; Arrand, J E



Numerical Analysis of Grassland Bacterial Community Structure under Different Land Management Regimens by Using 16S Ribosomal DNA Sequence Data and Denaturing Gradient Gel Electrophoresis Banding Patterns  

PubMed Central

Bacterial diversity in unimproved and improved grassland soils was assessed by PCR amplification of bacterial 16S ribosomal DNA (rDNA) from directly extracted soil DNA, followed by sequencing of ?45 16S rDNA clones from each of three unimproved and three improved grassland samples (A. E. McCaig, L. A. Glover, and J. I. Prosser, Appl. Environ. Microbiol. 65:17211730, 1999) or by denaturing gradient gel electrophoresis (DGGE) of total amplification products. Semi-improved grassland soils were analyzed only by DGGE. No differences between communities were detected by calculation of diversity indices and similarity coefficients for clone data (possibly due to poor coverage). Differences were not observed between the diversities of individual unimproved and improved grassland DGGE profiles, although considerable spatial variation was observed among triplicate samples. Semi-improved grassland samples, however, were less diverse than the other grassland samples and had much lower within-group variation. DGGE banding profiles obtained from triplicate samples pooled prior to analysis indicated that there was less evenness in improved soils, suggesting that selection for specific bacterial groups occurred. Analysis of DGGE profiles by canonical variate analysis but not by principal-coordinate analysis, using unweighted data (considering only the presence and absence of bands) and weighted data (considering the relative intensity of each band), demonstrated that there were clear differences between grasslands, and the results were not affected by weighting of data. This study demonstrated that quantitative analysis of data obtained by community profiling methods, such as DGGE, can reveal differences between complex microbial communities. PMID:11571155

McCaig, Allison E.; Glover, L. Anne; Prosser, James I.



Diversity of lactic acid bacteria from modified atmosphere packaged sliced cooked meat products at sell-by date assessed by PCR-denaturing gradient gel electrophoresis.  


The predominant lactic acid bacteria (LAB) microbiota associated with three types of modified atmosphere packaged (MAP) sliced cooked meat products (i.e. ham, turkey and chicken) was analyzed at sell-by date using a combination of culturing and molecular population fingerprinting. Likewise routine analyses during industrial MAP production, meat samples were plated on the general heterotrophic Plate Count Agar (PCA) and on the LAB-specific de Man, Rogosa, Sharpe (MRS) agar under different temperature and atmosphere conditions. Subsequently, community DNA extracts were prepared from culturable bacterial fractions harvested from both media and used for PCR targeting the V3 hyper-variable region of the 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE) of PCR amplicons (PCR-DGGE). Irrespective of aerobic or anaerobic incubation conditions, V3-16S rDNA DGGE fingerprints of culturable fractions from PCA and MRS medium displayed a high level of similarity indicating that LAB constituted the most dominant group in the culturable bacterial community. Comparison of DGGE profiles of fractions grown at 20, 28 or 37 degrees C indicated that part of the culturable community consisted of psychrotrophs. Four DGGE bands were common among cooked ham, turkey and chicken products, suggesting that these represent the microbiota circulating in the plant where all three MAP product types were sliced and packaged. Based on band sequencing and band position analysis using LAB reference strains, these four bands could be assigned to Lactobacillus sakei and/or the closely related Lactobacillus fuchuensis, Lactobacillus curvatus, Carnobacterium divergens and Leuconostoc carnosum. In conclusion, the PCR-DGGE approach described in this study allows to discriminate, identify and monitor core and occasional LAB microbiota of MAP sliced cooked meat products and provides valuable complementary information to the current plating procedures routinely used in industrial plants. PMID:19913685

Audenaert, Kris; D'Haene, Klaas; Messens, Kathy; Ruyssen, Tony; Vandamme, Peter; Huys, Geert



Application of Pulsed-Field Gel Electrophoresis and Binary Typing as Tools in Veterinary Clinical Microbiology and Molecular Epidemiologic Analysis of Bovine and Human Staphylococcus aureus Isolates  

PubMed Central

Thirty-eight bovine mammary Staphylococcus aureus isolates from diverse clinical, temporal, and geographical origins were genotyped by pulsed-field gel electrophoresis (PFGE) after SmaI digestion of prokaryotic DNA and by means of binary typing using 15 strain-specific DNA probes. Seven pulsed-field types and four subtypes were identified, as were 16 binary types. Concordant delineation of genetic relatedness was documented by both techniques, yet based on practical and epidemiological considerations, binary typing was the preferable method. Genotypes of bovine isolates were compared to 55 previously characterized human S. aureus isolates through cluster analysis of binary types. Genetic clusters containing strains of both human and bovine origin were found, but bacterial genotypes were predominantly associated with a single host species. Binary typing proved an excellent tool for comparison of S. aureus strains, including methicillin-resistant S. aureus, derived from different host species and from different databases. For 28 bovine S. aureus isolates, detailed clinical observations in vivo were compared to strain typing results in vitro. Associations were found between distinct genotypes and severity of disease, suggesting strain-specific bacterial virulence. Circumstantial evidence furthermore supports strain-specific routes of bacterial dissemination. We conclude that PFGE and binary typing can be successfully applied for genetic analysis of S. aureus isolates from bovine mammary secretions. Binary typing in particular is a robust and simple method and promises to become a powerful tool for strain characterization, for resolution of clonal relationships of bacteria within and between host species, and for identification of sources and transmission routes of bovine S. aureus. PMID:10790124

Zadoks, Ruth; van Leeuwen, Willem; Barkema, Herman; Sampimon, Otlis; Verbrugh, Henri; Schukken, Ynte Hein; van Belkum, Alex



Recovery of Bordetella holmesii from Patients with Pertussis-Like Symptoms: Use of Pulsed-Field Gel Electrophoresis To Characterize Circulating Strains  

PubMed Central

A 4-year retrospective study showing that we isolated Bordetella holmesii, but not Bordetella pertussis, from patients with pertussis-like symptoms was performed. From 1995 through 1998, we isolated B. holmesii from 32 nasopharyngeal specimens that had been submitted from patients suspected of having pertussis. Previously, B. holmesii had been associated mainly with septicemia and was not thought to be associated with respiratory illness. A study was undertaken to describe the characteristics of the B. holmesii isolates recovered and why we were successful in detecting the organism in nasopharyngeal specimens. B. holmesii isolates were characterized for drug sensitivities and for genetic relatedness by pulsed-field gel electrophoresis (PFGE). These isolates, an additional strain of B. holmesii isolated from a blood culture and previously confirmed by the Centers for Disease Control and Prevention, Atlanta, Ga., and 14 other clinical isolates of Bordetella spp., including 4 of B. bronchiseptica, 5 of B. parapertussis, and 5 of B. pertussis, were studied. They were all separately inoculated on three Bordet Gengou (BG) selective media containing either 0.625 ?g of oxacillin per ml, 40 ?g of cephalexin per ml, or 2.5 ?g of methicillin per ml, on BG agar with no antibiotic (control), and on charcoal agar (CA) with and without 40 ?g of cephalexin per ml. We found that cephalexin, the antibiotic commonly incorporated in both CA and BG agar for the recovery of Bordetella spp., is inhibitory to the growth of B. holmesii. In addition, the genotypic analysis of the 32 B. holmesii isolates by PFGE following restriction with XbaI and SpeI identified the dominant strains circulating during the study period. PMID:10834997

Mazengia, Eyob; Silva, Ellen A.; Peppe, Joseph A.; Timperi, Ralph; George, Harvey



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




Comparison of Two Multilocus Variable-Number Tandem-Repeat Methods and Pulsed-Field Gel Electrophoresis for Differentiating Highly Clonal Methicillin-Resistant Staphylococcus aureus Isolates?  

PubMed Central

In the United Kingdom, EMRSA-15 and EMRSA-16 account for the majority (?90%) of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) infections. Currently, the standard typing technique, pulsed-field gel electrophoresis (PFGE), is laborious and insufficient for discriminating between closely related subtypes of EMRSA-15 and -16. The objective of the present study was to compare the usefulness of multilocus variable-number tandem-repeat fingerprinting (MLVF) and multilocus variable-number tandem-repeat analysis (MLVA) with PFGE for subtyping these highly clonal MRSA lineages. A panel of 85 MRSA isolates (41 EMRSA-15, 20 EMRSA-16, and 24 MRSA isolates with diverse PFGE patterns) was investigated. In addition, a further 29 EMRSA-15s with identical PFGE patterns from two geographically linked but epidemiologically distinct outbreaks and several sporadic cases were analyzed. PFGE, MLVF, and MLVA resolved 66 (Simpson's index of diversity [SID] = 0.984), 51 (SID = 0.95), and 42 (SID = 0.881) types, respectively, among the 85 MRSA isolates. MLVF was more discriminatory than MLVA for EMRSA-15 and -16 strains, but both methods had comparable discriminatory powers for distinguishing isolates in the group containing diverse PFGE types. MLVF was comparable to PFGE for resolving the EMRSA-15s but had a lower discriminatory power for the EMRSA-16s. MLVF and MLVA resolved the 29 isolates with identical PFGE patterns into seven and six subtypes, respectively. Importantly, both assays indicated that the two geographically related outbreaks were caused by distinct subtypes of EMRSA-15. Taken together, the data suggest that both methods are suitable for identifying and tracking specific subtypes of otherwise-indistinguishable MRSA. However, due to its greater discriminatory power, MLVF would be the most suitable alternative to PFGE for hospital outbreak investigations. PMID:20702668

Holmes, A.; Edwards, G. F.; Girvan, E. K.; Hannant, W.; Danial, J.; Fitzgerald, J. R.; Templeton, K. E.



Application of Denaturing Gradient Gel Electrophoresis (DGGE) To Study the Diversity of Marine Picoeukaryotic Assemblages and Comparison of DGGE with Other Molecular Techniques  

PubMed Central

We used denaturing gradient gel electrophoresis (DGGE) to study the diversity of picoeukaryotes in natural marine assemblages. Two eukaryote-specific primer sets targeting different regions of the 18S rRNA gene were tested. Both primer sets gave a single band when used with algal cultures and complex fingerprints when used with natural assemblages. The reproducibility of the fingerprints was estimated by quantifying the intensities of the same bands obtained in independent PCR and DGGE analyses, and the standard error of these estimates was less than 2% on average. DGGE fingerprints were then used to compare the picoeukaryotic diversity in samples obtained at different depths and on different dates from a station in the southwest Mediterranean Sea. Both primer sets revealed significant differences along the vertical profile, whereas temporal differences at the same depths were less marked. The phylogenetic composition of picoeukaryotes from one surface sample was investigated by excising and sequencing DGGE bands. The results were compared with an analysis of a clone library and a terminal restriction fragment length polymorphism fingerprint obtained from the same sample. The three PCR-based methods, performed with three different primer sets, revealed very similar assemblage compositions; the same main phylogenetic groups were present at similar relative levels. Thus, the prasinophyte group appeared to be the most abundant group in the surface Mediterranean samples as determined by our molecular analyses. DGGE bands corresponding to prasinophytes were always found in surface samples but were not present in deep samples. Other groups detected were prymnesiophytes, novel stramenopiles (distantly related to hyphochytrids or labyrinthulids), cryptophytes, dinophytes, and pelagophytes. In conclusion, the DGGE method described here provided a reasonably detailed view of marine picoeukaryotic assemblages and allowed tentative phylogenetic identification of the dominant members. PMID:11425706

Dez, Beatriz; Pedrs-Ali, Carlos; Marsh, Terence L.; Massana, Ramon



Comparison of multilocus sequence typing, pulsed-field gel electrophoresis, and antimicrobial susceptibility typing for characterization of Salmonella enterica serotype Newport isolates.  


In the United States, multidrug-resistant phenotypes of Salmonella enterica serotype Newport (commonly referred to as MDR-AmpC) have emerged in animals and humans and have become a major public health problem. Although pulsed-field gel electrophoresis (PFGE) is the current "gold standard" typing method for Salmonella, multilocus sequence typing (MLST) may be more relevant to investigations exploring evolutionary and population biology relationships. In this study, 81 Salmonella enterica serotype Newport isolates from humans, food animals, and retail foods were examined for antimicrobial susceptibility and characterized using PFGE and MLST of seven genes, aroC, dnaN, hemD, hisD, purE, sucA, and thrA. Forty-nine percent of the isolates were resistant to nine or more of the tested antimicrobials. Salmonella isolates displayed resistance most often to sulfamethoxazole (57%), streptomycin (56%), tetracycline (56%), ampicillin (52%), and ceftiofur (49%) and, to a lesser extent, to kanamycin (19%), trimethoprim-sulfamethoxazole (17%), and gentamicin (11%). A total of 43 PFGE patterns were generated using XbaI, indicating a genetically diverse population. The largest PFGE cluster contained isolates from clinically ill swine, cattle, and humans. MLST resulted in 12 sequence types (STs), with one type encompassing 62% of the strains. Ten new sequence types and one novel allele type were identified. Furthermore, MLST typing showed that strains closely related by PFGE clustered in major STs, whereas more distantly related strains were separated into two clusters by PFGE. The results of this study demonstrated that the MLST scheme employed here clustered S. enterica serovar Newport isolates in distinct molecular populations, and strain discrimination was enhanced by combining PFGE, antimicrobial susceptibility, and MLST results. PMID:16825363

Harbottle, H; White, D G; McDermott, P F; Walker, R D; Zhao, S



Proteomic differential display analysis for TS-1-resistant and -sensitive pancreatic cancer cells using two-dimensional gel electrophoresis and mass spectrometry.  


TS-1 is an oral anticancer agent containing two biochemical modulators for 5-fluorouracil (5-FU) and tegafur (FT), a metabolically activated prodrug of 5-FU. TS-1 has been recognized as an effective anticancer drug using standard therapies for patients with advanced pancreatic cancer along with gemcitabine. However, a high level of inherent and acquired tumor resistance to TS-1 induces difficulty in the treatment. To identify proteins linked to the TS-1-resistance of pancreatic cancer, we profiled protein expression levels in samples of TS-1-resistant and -sensitive pancreatic cancer cell lines by using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The cytotoxicity of a 5-FU/5-chloro-2,4-dihydroxypyridine (CDHP) combination towards pancreatic cancer cell lines was evaluated by MTS assay. Panc-1, BxPC-3, MiaPaCa-2 and PK59 showed high sensitivity to the 5-FU/CDHP combination (TS-1-sensitive), whereas PK45p and KLM-1 were much less sensitive (TS-1-resistant). Proteomic analysis showed that eleven spots, including T-complex protein 1 subunit beta, ribonuclease inhibitor, elongation factor 1-delta, peroxiredoxin-2 and superoxide dismutase (Cu-Zn), appeared to be down-regulated, and 29 spots, including hypoxia up-regulated protein 1, lamin-A/C, endoplasmin, fascin and annexin A1, appeared to be up-regulated in TS-1-resistant cells compared with -sensitive cells. These results suggest that the identified proteins showing different expression between TS-1-sensitive and -resistant pancreatic cancer cells possibly relate to TS-1-sensitivity. These findings could be useful to overcome the TS-1-resistance of pancreatic cancer cells. PMID:21737628

Yoshida, Kanako; Kuramitsu, Yasuhiro; Murakami, Kohei; Ryozawa, Shomei; Taba, Kumiko; Kaino, Seiji; Zhang, Xiulian; Sakaida, Isao; Nakamura, Kazuyuki



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



Qualitative and quantitative proteomics by two-dimensional gel electrophoresis, peptide mass fingerprint and a chemically-coded affinity tag (CCAT)  

Microsoft Academic Search

The chemically-coded affinity tag (CCAT) method combines standard electrophoresis protocols with MALDI-TOF-MS analysis to identify and quantify protein abundances in complex samples in one step. This method is designed to fit into the workflow of SDS-PAGE or two-dimensional electrophoresis (2-DE) only requiring basic proteome laboratory equipment. Prior to electrophoresis two protein samples are separately labelled with a heavy or a

Steven Alexander Watt; Thomas Patschkowski; Jrn Kalinowski; Karsten Niehaus



Getting the Most out of Electrophoresis Units  

ERIC Educational Resources Information Center

At Oklahoma City Community College, they have developed gel electrophoresis activities that support active learning of many scientific concepts, including: pH, electrolysis, oxidation reduction, electrical currents, potentials, conductivity, molarity, gel electrophoresis, DNA and protein separation, and DNA fingerprinting. This article presents

Mulvihill, Charlotte



Identification of and Spatio-Temporal Differences between Microbial Assemblages from Two Neighboring Sulfurous Lakes: Comparison by Microscopy and Denaturing Gradient Gel Electrophoresis  

PubMed Central

The microbial assemblages of Lake Cis and Lake Vilar (Banyoles, northeast Spain) were analyzed in space and time by microscopy and by performing PCR-denaturing gradient gel electrophoresis (DGGE) and sequence 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 during a phytoplankton bloom) were analyzed. Although the lakes have the same climatic conditions and the same water source, the limnological parameters were different, as were most of the morphologically distinguishable photosynthetic bacteria enumerated by microscopy. The phylogenetic affiliations of the predominant DGGE bands were inferred by performing a comparative 16S rRNA sequence analysis. Sequences obtained from Lake Cis samples were related to gram-positive bacteria and to members of the division Proteobacteria. Sequences obtained from Lake Vilar samples were related to members of the Cytophaga-Flavobacterium-Bacteroides phylum and to cyanobacteria. Thus, we found that like the previously reported differences between morphologically distinct inhabitants of the two lakes, there were also differences among the community members whose morphologies did not differ conspicuously. The changes in the species composition from winter to spring were also marked. The two lakes both contained sequences belonging to phototrophic green sulfur bacteria, which is consistent with microscopic observations, but these sequences were different from the sequences of cultured strains previously isolated from the lakes. Euryarchaeal sequences (i.e., methanogen- and thermoplasma-related sequences) also were present in both lakes. These euryarchaeal group sequences dominated the archaeal sequences in Lake Cis but not in Lake Vilar. In Lake Vilar, a new planktonic population related to the crenarchaeota produced the dominant archaeal band. The phylogenetic analysis indicated that new bacterial and archaeal lineages were present and that the microbial diversity of these assemblages was greater than previously known. We evaluated the correspondence between the abundances of several morphotypes and DGGE bands by comparing microscopy and sequencing results. Our data provide evidence that the sequences obtained from the DGGE fingerprints correspond to the microorganisms that are actually present at higher concentrations in the natural system. PMID:10653710

Casamayor, Emilio O.; Schafer, Hendrik; Baneras, Lluis; Pedros-Alio, Carlos; Muyzer, Gerard



Interactions of atrazine and 2,4-D with human serum albumin studied by gel and capillary electrophoresis, and FTIR spectroscopy.  


The herbicides 6-chloro-N-ethyl-N'-(1-methylethyl)-1,3,5-triazine-2,4-diamine (atrazine) and 2,4-dichlorophenoxyacetic acid (2,4-D) are widely used in agricultural practice to fight dicotyledon weeds mainly in maize, cereals, and lucerne. As a result, these compounds are found not only in the plants, soil, and water, but also in the cultivated ground in the following years as well as in agricultural products such as fruits, milk, butter, and sugar beet. The toxicological effects of herbicides occur in vivo, when transported to the target organ through the bloodstream. It has been suggested that human serum albumin (HSA) serves as a carrier protein to transport 2,4-D to molecular targets. This study was designed to examine the interaction of atrazine and 2,4-D with HSA in aqueous solution at physiological pH with herbicide concentrations of 0.0001-1 mM, and final protein concentration of 1% w/v. Gel and capillary electrophoresis, UV-visible and Fourier transform infrared spectroscopic methods were used to determine the drug binding mode, the drug binding constant, and the protein secondary structure in aqueous solution. Structural analysis showed that different types of herbicide-HSA complexes are formed with stoichiometric ratios (drug/protein) of 3:1 and 11:1 for atrazine and 4.5:1 and 10:1 for 2,4-D complexes. Atrazine showed a weak binding affinity (K=3.50 x 10(4) M(-1)), whereas two bindings (K(1)=2.50 x 10(4) M(-1) and K(2)=8.0 x 10(3) M(-1)) were observed for 2,4-D complexes. The herbicide binding results in major protein secondary structural changes from that of the alpha-helix 55% to 45--39% and beta-sheet 22% to 24--32%, beta-anti 12% to 10--22% and turn 11% to 12--15%, in the drug-HSA complexes. The observed spectral changes indicate a partial unfolding of the protein structure, in the presence of herbicides in aqueous solution. PMID:11451446

Purcell, M; Neault, J F; Malonga, H; Arakawa, H; Carpentier, R; Tajmir-Riahi, H A



Pulsed-field gel electrophoresis profile homogeneity of Mycobacterium avium subsp. paratuberculosis isolates from cattle and heterogeneity of those from sheep and goats  

PubMed Central

Background Mycobacterium avium subsp. paratuberculosis (Map) causes paratuberculosis in animals and is suspected of causing Crohn's Disease in humans. Characterization of strains led to classify paratuberculosis isolates in two main types, cattle type strains, found affecting all host species, and sheep type strains, reported affecting mainly sheep. In order to get a better understanding of the epidemiology of paratuberculosis a large set of Map isolates obtained from different species over the last 25 years have been characterized. Five-hundred and twenty isolates from different hosts (cattle, sheep, goats, bison, deer and wild boar) and origins had been cultured and typed by IS1311 restriction-endonuclease-analysis. Two-hundred and sixty-nine isolates were further characterized by pulsed-field gel electrophoresis (PFGE) using SnaBI and SpeI endonucleases. Differences in strain isolation upon various media conditions were also studied. Results All bovines, 4 and 26% of Spanish sheep and goats, respectively, and the deer and wild boar studied, carried IS1311-Cattle type strains. IS1311-Sheep type encompassed 96% and 74% of Spanish sheep and goats, and all three Portuguese sheep. Thirty-seven distinct multiplex PFGE profiles were found, giving 32 novel profiles. Profiles 2-1 and 1-1 accounted for the 85% of cattle isolates. Ten distinct profiles were detected in Spanish sheep, none of them with an incidence higher than 25%. Profile 16-11 (43%) and another three profiles were identified in Spanish caprine cultures. The hierarchical analysis, clustered all profiles found in cattle, "wild" hosts and some small ruminants within the same group. The other group included 11 profiles only found in Spanish sheep and goats, including Spanish pigmented profiles. Differences in growth requirements associated with isolate genotype were observed. Conclusion Cattle in Spain are infected with cattle type strains, while sheep and goats are mainly infected with sheep type strains. Although 7H9 broth based culture media seem to broadly cover the growth requirements of most Map strains, the use of various solid media is recommended to reduce any recovery biases. High genetic homogeneity of isolates from cattle, and heterogeneity of those from sheep and goats have been detected. PMID:17352818

Sevilla, Iker; Garrido, Joseba M; Geijo, Marivi; Juste, Ramon A



Analysis of the dynamics of fungal communities in soil via fungal-specific PCR of soil DNA followed by denaturing gradient gel electrophoresis.  


A molecular method for profiling of fungal communities in soil was applied in experiments in soil microcosms, with two objectives, (1) to assess the persistence of two selected fungal species in soil, and (2) to analyze the response of the natural fungal community to a spill of sulphurous petrol in the same soil. To achieve the aims, two soil DNA extraction methods, one originally designed for the direct extraction of bacterial community DNA and the other one aimed to obtain fungal DNA, were tested for their efficiency in recovering DNA of fungal origin from soil. Both methods allowed for the efficient extraction of DNA from introduced Trichoderma harzianum spores as well as Arthrobotrys oligospora mycelial fragments, at comparable rates. Several PCR amplification systems based on primers specific for fungal 18S ribosomal RNA genes were tested to design strategies for the assessment of fungal communities in soil. The PCR systems produced amplicons of expected size with DNA of most fungi studied, which included members of the Ascomycetes, Basidiomycetes, Zygomycetes and Chytridiomycetes. On the other hand, the 18S rRNA genes of Oomycetes (including key plant pathogens) were poorly amplified. Plant (Solanum tuberosum), nematode (Meloidogyne sp.) and bacterial DNA was not amplified. For studies of soil fungal communities, a nested PCR approach was selected, in which the first PCR provided the required specificity for fungi, whereas the second (nested) PCR served to produce amplicons separable on denaturing gradient gels. Denaturing gradient gel electrophoresis (DGGE) allowed the resolution of mixtures of PCR products of several different fungi, as well as products resulting from mixed-template amplifications, into distinct banding patterns. The persistence of fungal species in soil was assessed using T. harzianum spores and A. oligospora hyphal fragments added to silt loam soil microcosms. Using PCR-DGGE, these fungi were detectable for about 14 days and 2 months, respectively. Both singly-inoculated soils and soils that had received mixed inoculants revealed, next to bands resulting from indigenous fungi, the expected bands in the DGGE profiles. The A. oligospora specific amplicon, by virtue of its unique migration in the denaturing gradient, was well detectable, whereas the T. harzianum specific product comigrated with products from indigenous fungi. PCR-DGGE analysis of DNA obtained from the silt loam soil treated with dibenzothiophene-containing petrol showed the progressive selection of specific fungal bands over time, whereas this selection was not observed in untreated soil microcosms. Cloning of individual molecules from the selected bands and analysis of their sequences revealed a complex of targets which clustered with the 18S rDNA sequences of the closely-related species Nectria haematococca, N. ochroleuca and Fusarium solani. Fungal isolates obtained from the treated soil on PDA plates were identified as Trichoderma sp., whereas those on Comada agar fell into the Cylindrocarpon group (anamorph of Nectria spp). PMID:11121612

van Elsas, J D; Duarte, G F; Keijzer-Wolters, A; Smit, E



Study of disposable microdevices for DNA electrophoresis  

E-print Network

A study was undertaken to determine if a microfluidic chip, made of economical plastic materials, is feasible. The chip was designed to perform gel electrophoresis, specifically of DNA fragments for either sequencing or ...

Timp, Winston (Winston G.)



Serogrouping and pulsed-field gel electrophoresis analysis of Listeria monocytogenes isolates from cases of human infection in Hungary 20042012 molecular typing of Listeria monocytogenes in Hungary.  


Eighty isolates of Listeria monocytogenes cultured from human infections in Hungary between 2004 and 2012 were serotyped by the PCR technique of Doumithet al. [9] and characterised by pulsed-field gel electrophoresis (PFGE). Most of the isolates belonged to two serogroups: 53 isolates (66.3%) to serovar group 4b,4d,4e and 21isolates (25.8%) to serogroup 1/2a,3a. Although many pulsotypes were identified a particular pulsotype proved highly excelling comprising of 31 isolates after digestion by both ApaI and AscI restriction enzymes. All strains from this pulsotype belonged toserovar group 4b,4d,4e. Interestingly 24% of isolates from invasive samples(cerebrospinal fluid, blood) belonged to two distinct pulsotypes in the less common serovar group 1/2a,3a. Several small clusters of cases caused by isolates with identical pulsotypes were identified. PMID:25046881

Pszti, Judit; Kirly, Jzsefn; Fzi, Mikls



One-dimensional and two-dimensional immobilized metal affinity electrophoresis.  


Immobilized metal affinity electrophoresis (IMAEP) is a straightforward method in which metal ions are embedded in a polyacrylamide gel strip with a negligible electrophoretic migration. Due to the preferential binding between metal ions and the phosphate group, this method uses immobilized metal ions like iron, manganese, aluminum, or titanium to capture phosphoproteins from a mixture of phosphoprotein and nonphosphoproteins. IMAEP has also been incorporated into a traditional two-dimensional (2D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) system (isoelectric focusing-PAGE) to increase its resolving power. In 2D IMAEP, the metal ions in polyacrylamide gel strip are overlaid on top of the second dimensional polyacrylamide gel to stop electrophoretic migration of phosphoproteins. Data shows that there is no detrimental effect of SDS in IMAEP on the extraction of phosphoproteins from a mixture of proteins. In addition, SDS exposes phosphate groups by unfolding the phosphoproteins to facilitate metal ion-phosphate binding while supplying the protein with negative charges. PMID:22585494

Lee, Bao-Shiang; Lasanthi, G D; Jayathilaka, P; Huang, Jin-Sheng; Gupta, Shalini



Analysis of electrophoresis performance  

NASA Technical Reports Server (NTRS)

The SAMPLE computer code models electrophoresis separation in a wide range of conditions. Results are included for steady three dimensional continuous flow electrophoresis (CFE), time dependent gel and acetate film experiments in one or two dimensions and isoelectric focusing in one dimension. The code evolves N two dimensional radical concentration distributions in time, or distance down a CFE chamber. For each time or distance increment, there are six stages, successively obtaining the pH distribution, the corresponding degrees of ionization for each radical, the conductivity, the electric field and current distribution, and the flux components in each direction for each separate radical. The final stage is to update the radical concentrations. The model formulation for ion motion in an electric field ignores activity effects, and is valid only for low concentrations; for larger concentrations the conductivity is, therefore, also invalid.

Roberts, G. O.



Relative Quantitative Comparisons of the Extracellular Protein Profiles of Staphylococcus aureus UAMS-1 and Its sarA, agr, and sarA agr Regulatory Mutants Using One-Dimensional Polyacrylamide Gel Electrophoresis and Nanocapillary Liquid Chromatography Coupled with Tandem Mass Spectrometry  

Microsoft Academic Search

One-dimensional polyacrylamide gel electrophoresis followed by nanocapillary liquid chromatography cou- pled with mass spectrometry was used to analyze proteins isolated from Staphylococcus aureus UAMS-1 after 3, 6, 12, and 24 h of in vitro growth. Protein abundance was determined using a quantitative value termed normalized peptide number, and overall, proteins known to be associated with the cell wall were more

Richard C. Jones; Joanna Deck; Ricky D. Edmondson; Mark E. Hart



Development and Validation of a Stability-Indicating Capillary Electrophoresis Method for the Determination of Zolpidem Tartrate in Tablet Dosage Form with Positive Confirmation using 2D- and 3D-DAD Fingerprints  

PubMed Central

The aim of the current study was to develop a simple, precise, and accurate capillary zone electrophoresis method for the determination of zolpidem tartrate in tablet dosage form. Separation was conducted in normal polarity mode at 25C, 22 kV, using hydrodynamic injection for 10 s. Separation was achieved using a background electrolyte of 20 mM disodium hydrogen phosphate adjusted with phosphoric acid (85%), pH at 5.50, and detection at 254 nm. Using the above optimized conditions, complete determination took place in less than 3 min using amiloride HCl as the internal standard. The method was linear over the range of 31000 ?g mL?1 with a correlation coefficient of 0.9999. Forced degradation studies were conducted by introducing a sample of zolpidem tartrate standard and pharmaceutical sample solutions to different forced degradation conditions, being neutral (water), basic (0.1 M NaOH), acidic (0.1 M HCl), oxidative (10% H2O2), temperature (60C in oven for 3 days), and photolytic (exposure to UV light at 254 nm for 2 h). Degradation products resulting from the stress studies did not interfere with the detection of zolpidem tartrate and the assay can be considered stability-indicating. PMID:24959406

Al Azzam, Khaldun M.; Yit, Lee Kam; Saad, Bahruddin; Shaibah, Hassan