Note: This page contains sample records for the topic gel electrophoresis 2-d from Science.gov.
While these samples are representative of the content of Science.gov,
they are not comprehensive nor are they the most current set.
We encourage you to perform a real-time search of Science.gov
to obtain the most current and comprehensive results.
Last update: August 15, 2014.
1

Silver staining of 2D electrophoresis gels.  

PubMed

Silver staining is used to detect proteins after electrophoretic separation on polyacrylamide gels. It -combines excellent sensitivity (in the low nanogram range) with the use of very simple and cheap equipment and chemicals. For its use in proteomics, two important additional features must be considered, compatibility with mass spectrometry and quantitative response. Both features are discussed in this chapter, and optimized silver staining protocols are proposed. PMID:22665294

Rabilloud, Thierry

2012-01-01

2

Detection of circular telomeric DNA without 2-D gel electrophoresis  

PubMed Central

The end of linear chromosomes form a lasso-like structure called the t-loop. Such t-loops resemble a DNA recombination intermediate, where the single-stranded 3? overhang is arrested in a stretch of duplex DNA. Presumably such a t-loop can also be deleted via a recombination process. This would result in the occurrence of circular extra-chromosomal telomeric DNA (t-circles), which are known to be abundantly present in immortal cells engaging the recombination-based alternative lengthening of telomeres pathway (ALT pathway). Little is known about the basic mechanism of telomeric recombination in these cells and what ultimately causes the generation of such t-circles. Current standard procedures for detecting these molecules involve 2-D gel electrophoresis or electron microscopy. However, both methods are labor-intense and sophisticated to perform. We would like to present a simpler, faster and equally sensitive method for detecting t-circles. Our approach is a telomere restriction fragment assay that involves the enzymatic preservation of circular DNA with Klenow enzyme followed by Bal31 degradation of the remaining linear DNA molecules. We show that with this approach t-circles can be detected in ALT cell lines, whereas no t-circles are present in telomerase-positive cell lines. We consider our approach being a valid method that can be used in experiments, in which t-circle generation is the experimental read out.

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

2011-01-01

3

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

Microsoft Academic Search

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

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

2004-01-01

4

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

PubMed Central

Background Two-dimensional gel electrophoresis (2DE) is a powerful technique for studying protein isoforms and their modifications. Existing commercial 2D image analysis tools rely on spot detection that limits analysis of complex protein profiles, e.g. spot appearance/disappearance or overlapping spots. Pixel-by-pixel correlation analysis, an analysis technique for identifying relations between protein patterns in gel images and external variables, can overcome such limitations in spot analysis. Results We have implemented the first publically available pixel-by-pixel correlation analysis tool, the software Gel2DE. 2D immunoblot time course analysis of p53 protein stabilization in response to ionizing irradiation shows that pixel-by-pixel analysis can yield an overall activation biosignature for p53, despite changing spots shape, size and position. Conclusions Pixel-by-pixel correlation of aligned 2D images permits analysis of complex protein patterns. We anticipate that the Gel2DE correlation software will be a useful tool for future bioinformatics discoveries through 2D gel electrophoresis.

2013-01-01

5

Segmentation of Protein Spots in 2D Gel Electrophoresis Images with Watersheds Using Hierarchical Threshold  

Microsoft Academic Search

\\u000a 2D Gel Electrophoresis (2DGE) image is the most widely used method for the isolation of the objective protein by comparative\\u000a analysis of the protein spot pattern in the gel plane. The process of protein analysis is the method, which compares the protein\\u000a pattern that is spread in the gel plane with the contrast group and finds interesting protein spot by

Youngho Kim; Jung-ja Kim; Yonggwan Won; In Yongho

2003-01-01

6

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

PubMed

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

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

2013-01-01

7

Gel Electrophoresis  

NSDL National Science Digital Library

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

Foundation, Wgbh E.

2007-04-19

8

2D-ToGo workflow: increasing feasibility and reproducibility of 2-dimensional gel electrophoresis.  

PubMed

Two-dimensional gel electrophoresis (2-DE) is one of the most powerful methods for studying global protein profiles. However, due to the multiple manual steps involved in gel based processing it is challenging to achieve the necessary overall reproducibility for a reliable comparative analysis, especially between different laboratories. To improve the 2-DE technique for quantitative analyses we have set up a robust 2-DE workflow, called 2D-ToGo, which utilizes latest innovations concerning instrumentation, consumables and protocols. Quantitative data analyses indicate the high reproducibility between replicate gels processed at a single site (intra-laboratory variation: CV 20%). The data-sets of the inter-laboratory comparison revealed similar results displaying a variation of CV 23%. The technical improvements given by our 2-DE workflow have a positive impact on process robustness and most importantly, reproducibility. Accordingly, many of the well-known challenges for resolving and quantitating up to thousands of different protein components in a given biological sample are minimized. PMID:23679042

Posch, Anton; Franz, Thomas; Hartwig, Sonja; Knebel, Birgit; Al-Hasani, Hadi; Passlack, Waltraud; Kunz, Nancy; Hinze, Yvonne; Li, Xinping; Kotzka, Jorg; Lehr, Stefan

2013-07-01

9

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.

2012-01-20

10

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

NSDL National Science Digital Library

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

2008-10-06

11

Gel Electrophoresis  

NSDL National Science Digital Library

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

Yu, Julie

2007-01-01

12

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.

1998-06-10

13

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

NASA Astrophysics Data System (ADS)

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

Kaabouch, Naima; Schultz, Richard R.

2007-01-01

14

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.

Morisawa, Hiraku; Hirota, Mikako; Toda, Tosifusa

2006-01-01

15

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

PubMed

Recombinant monoclonal antibodies (rmAbs) are medicinal products obtained by rDNA technology. Consequently, like other biopharmaceuticals, they require the extensive and rigorous characterization of the quality attributes, such as identity, structural integrity, purity and stability. The aim of this work was to study the suitability of gel electrophoresis for the assessment of charge heterogeneity, post-translational modifications and the stability of the therapeutic, recombinant monoclonal antibody, trastuzumab. One-dimensional, SDS-PAGE, under reducing and non-reducing conditions, and two-dimensional gel electrophoresis were used for the determination of molecular mass (Mr), the isoelectric point (pI), charge-related isoform patterns and the stability of trastuzumab, subjected to stressed degradation and long-term conditions. For the assessment of the influence of glycosylation in the charge heterogeneity pattern of trastuzumab, an enzymatic deglycosylation study has been performed using N-glycosidase F and sialidase, whereas carboxypeptidase B was used for the lysine truncation study. Experimental data documented that 1D and 2D gel electrophoresis represent fast and easy methods to evaluate the quality of biological medicinal products. Important stability parameters, such as the protein aggregation, can be assessed, as well. PMID:24739811

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

2014-01-01

16

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

PubMed Central

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

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

2014-01-01

17

A 2D gel electrophoresis-based snapshot of the phosphoproteome in the cyanobacterium Synechocystis sp. strain PCC 6803.  

PubMed

Cyanobacteria are photoautotrophic prokaryotes that occur in highly variable environments. Protein phosphorylation is one of the most widespread means to adjust cell metabolism and gene expression to the demands of changing growth conditions. Using a 2D gel electrophoresis-based approach and a phosphoprotein-specific dye, we investigated the protein phosphorylation pattern in cells of the model cyanobacterium Synechocystis sp. strain PCC 6803. The comparison of gels stained for total and phosphorylated proteins revealed that approximately 5?% of the protein spots seemed to be phosphoproteins, from which 32 were identified using MALDI-TOF MS. For eight of them the phosphorylated amino acid residues were mapped by subsequent mass spectrometric investigations of isolated phosphopeptides. Among the phosphoproteins, we found regulatory proteins, mostly putative anti-sigma factor antagonists, and proteins involved in translation. Moreover, a number of enzymes catalysing steps in glycolysis or the Calvin-Benson cycle were found to be phosphorylated, implying that protein phosphorylation might represent an important mechanism for the regulation of the primary carbon metabolism in cyanobacterial cells. PMID:24275102

Mikkat, Stefan; Fulda, Sabine; Hagemann, Martin

2014-02-01

18

Hierarchical watershed transformation based on a-priori information for spot detection in 2D gel electrophoresis images  

NASA Astrophysics Data System (ADS)

For the spot detection in 2D electrophoresis images an approach which is based on the combination of the watershed transformation (WST) with a-priori knowledge is presented. To identify spot regions in the over segmented result of the WST two types of regions have to be found: Regions that correspond to a complete spot and regions that cover only a part of a spot. The first localization step, the gray value analysis, is based on the assumptions that spot regions have significantly higher gray values than the background and that they border on a background region. Since not all remaining regions are spot or partial spot regions, additionally a curvature analysis is done. Here the a-priori knowledge is used that regarding a gel image as a surface, the shape of a spot is obviously convex. Consequently, considering the second derivative all required spot and partial spot regions can be obtained by the regions of convex curvature. In a final merging step all partial spot regions covering one spot have to be combined to only one spot region. As merging criterion two spot characteristics are used. A spot should have an approximately elliptical shape and partial spot regions of one spot should have a local convex curvature in a small neighborhood along their boundary.

Wegner, Susan; Pleissner, Klaus-Peter; Oswald, Helmut; Fleck, Eckart

1999-05-01

19

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

PubMed

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

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

2007-12-01

20

Proteome analysis of round-headed and normal spermatozoa by 2-D fluorescence difference gel electrophoresis and mass spectrometry.  

PubMed

Globozoospermia is a severe form of teratozoospermia characterized by round-headed spermatozoa with an absent acrosome, an aberrant nuclear membrane and midpiece defects. Globozoospermia is diagnosed by the presence of 100% round-headed spermatozoa on semen analysis, and patients with this condition are absolutely infertile. The objective of this study was to investigate the differences in protein expression between human round-headed and normal spermatozoa. Two-dimensional (2-D) fluorescence difference gel electrophoresis (DIGE) coupled with mass spectrometry (MS) was used in this study. Over 61 protein spots were analysed in each paired normal/round-headed comparison, using DIGE technology along with an internal standard. In total, 35 protein spots identified by tandem mass spectrometry (MS/MS) exhibited significant changes (paired t-test, P < 0.05) in the expression level between normal and round-headed spermatozoa. A total of nine proteins were found to be upregulated and 26 proteins were found to be downregulated in round-headed spermatozoa compared with normal spermatozoa. The differentially expressed proteins that we identified may have important roles in a variety of cellular processes and structures, including spermatogenesis, cell skeleton, metabolism and spermatozoa motility. PMID:19823175

Liao, Ting-Ting; Xiang, Zhen; Zhu, Wen-Bing; Fan, Li-Qing

2009-11-01

21

Proteome analysis of round-headed and normal spermatozoa by 2-D fluorescence difference gel electrophoresis and mass spectrometry  

PubMed Central

Globozoospermia is a severe form of teratozoospermia characterized by round-headed spermatozoa with an absent acrosome, an aberrant nuclear membrane and midpiece defects. Globozoospermia is diagnosed by the presence of 100% round-headed spermatozoa on semen analysis, and patients with this condition are absolutely infertile. The objective of this study was to investigate the differences in protein expression between human round-headed and normal spermatozoa. Two-dimensional (2-D) fluorescence difference gel electrophoresis (DIGE) coupled with mass spectrometry (MS) was used in this study. Over 61 protein spots were analysed in each paired normal/round-headed comparison, using DIGE technology along with an internal standard. In total, 35 protein spots identified by tandem mass spectrometry (MS/MS) exhibited significant changes (paired t-test, P < 0.05) in the expression level between normal and round-headed spermatozoa. A total of nine proteins were found to be upregulated and 26 proteins were found to be downregulated in round-headed spermatozoa compared with normal spermatozoa. The differentially expressed proteins that we identified may have important roles in a variety of cellular processes and structures, including spermatogenesis, cell skeleton, metabolism and spermatozoa motility.

Liao, Ting-Ting; Xiang, Zhen; Zhu, Wen-Bing; Fan, Li-Qing

2009-01-01

22

Electrophoresis and Gel Analysis  

NSDL National Science Digital Library

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

Foundation, Wgbh E.

2011-09-22

23

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

PubMed

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

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

2009-01-01

24

Gel Electrophoresis of Dyes  

NSDL National Science Digital Library

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

Stephens, Janice; Leach, Jan

2011-01-01

25

Applicability of 2D gel electrophoresis and liquid chromatography in proteomic analysis of urine using mass spectrometry MALDI-TOF.  

PubMed

Proteomics including the studies of the structure, function and dependences between proteins is more and more extensively applied in human medicine and veterinary medicine. The analysis of protein profiles of tissues and body fluid from healthy and ill individuals allows to identify diagnostic, prognostic and predictive markers in various pathological states in people and animals. This paper presents preparation of urine samples for analysis in the mass spectrometer MALDI-TOF (Ultraflextreme, Bruker, Bremen, Germany) by means of two methods: liquid chromatography based on the system Nano-LC (PROTEINER FC II, Bruker Daltonics, Bremen Germany). and two-direction electrophoresis 2DE (GE Healthcare, United Kingdom). Both methods enable separation of the mixture under consideration into individual fractions of high purity indispensable for obtaining readable mass spectra. The purpose of this paper is to determine applicability of these methods in analysis of protein composition of urine samples. PMID:24195300

Banach, T; Adaszek, ?; Wy?upek, D; Winiarczyk, M; Winiarczyk, S

2013-01-01

26

Profiling of host cell proteins by two-dimensional difference gel electrophoresis (2D-DIGE): Implications for downstream process development.  

PubMed

Host cell proteins (HCPs) constitute a major group of impurities for biologic drugs produced using cell culture technology. HCPs are required to be closely monitored and adequately removed in the downstream process. However, HCPs are a complex mixture of proteins with significantly diverse molecular and immunological properties. An overall understanding of the composition of HCPs and changes in their molecular properties upon changes in upstream and harvest process conditions can greatly facilitate downstream process design. This article describes the use of a comparative proteomic profiling method viz. two-dimensional difference gel electrophoresis (2D-DIGE) to examine HCP composition in the harvest stream of CHO cell culture. The effect of upstream process parameters such as cell culture media, bioreactor control strategy, feeding strategy, and cell culture duration/cell viability on HCP profile was examined using this technique. Among all the parameters studied, cell viability generated the most significant changes on the HCP profile. 2D-DIGE was also used to compare the HCP differences between monoclonal antibody producing and null cell cultures. The HCP species in production cell culture was found to be well represented in null cell culture, which confirms the suitability of using the null cell culture for immunoassay reagent generation. 2D-DIGE is complimentary to the commonly used HCP immunoassay. It provides a direct comparison of the changes in HCP composition under different conditions and can reveal properties (pI, MW) of individual species, whereas the immunoassay sensitively quantifies total HCP amount in a given sample. PMID:19739084

Jin, Mi; Szapiel, Nicolas; Zhang, Jennifer; Hickey, John; Ghose, Sanchayita

2010-02-01

27

Two-dimensional difference gel electrophoresis.  

PubMed

Two-dimensional difference gel electrophoresis (2D DIGE) is a modified form of 2D electrophoresis (2DE) that allows one to compare two or three protein samples simultaneously on the same gel. The proteins in each sample are covalently tagged with different color fluorescent dyes that are designed to have no effect on the relative migration of proteins during electrophoresis. Proteins that are common to the samples appear as "spots" with a fixed ratio of fluorescent signals, whereas proteins that differ between the samples have different fluorescence ratios. With the appropriate imaging system, difference gel electrophoresis (DIGE) is capable of reliably detecting as little as 0.2 fmol of protein, and protein differences down to ±15%, over a ?20,000-fold protein concentration range. DIGE combined with digital image analysis therefore greatly improves the statistical assessment of proteome variation. Here we describe a protocol for conducting DIGE experiments, which takes 2-3 days to complete. PMID:22585495

Minden, Jonathan S

2012-01-01

28

Copolymers For Capillary Gel Electrophoresis  

DOEpatents

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

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

2005-08-09

29

Introduction to Agarose Gel Electrophoresis  

NSDL National Science Digital Library

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

Bioengineering, Climb: C.

30

Comparison of proteins expressed by Pseudomonas aeruginosa strains representing initial and chronic isolates from a cystic fibrosis patient: an analysis by 2-D gel electrophoresis and capillary column liquid chromatography-tandem mass spectrometry.  

PubMed

Isolates of Pseudomonas aeruginosa from chronic lung infections in cystic fibrosis (CF) patients have phenotypes distinct from those initially infecting CF patients, as well as from other clinical or environmental isolates. To gain a better understanding of the differences in these isolates, protein expression was followed using two-dimensional (2-D) gel electrophoresis and protein identification by peptide sequencing using micro-capillary column liquid chromatography-tandem mass spectrometry (microLC/MS/MS). The isolates selected for this analysis were from the sputum of a CF patient: strain 383 had a nonmucoid phenotype typical of isolates from the environment, and strain 2192, obtained from the same patient, had a mucoid phenotype typical of isolates from chronic CF lung infections. Strains 383 and 2192 were confirmed to be genetically identical by restriction endonuclease analysis, random amplified polymorphic DNA-PCR, and pulsed-field gel electrophoresis. Conditions of protein extraction were optimized for consistent high-resolution separation of several hundred proteins from these clinical isolates as detected by Coomassie staining of 2-D gels. Fourteen proteins were selected for analysis; this group included those whose expression was common between both strains as well as unique for each strain. The proteins were identified by microLC/MS/MS of the peptides produced by an in-gel tryptic digestion and compared to translated data from the Pseudomonas Genome Project; optimization of this technique has allowed for the comparison of proteins expressed by strains 383 and 2192. PMID:11021925

Hanna, S L; Sherman, N E; Kinter, M T; Goldberg, J B

2000-10-01

31

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

PubMed

The in vitro stationary phase proteome of the human pathogen Shigella dysenteriae serotype 1 (SD1) was quantitatively analyzed in Coomassie Blue G250 (CBB)-stained 2D gels. More than four hundred and fifty proteins, of which 271 were associated with distinct gel spots, were identified. In parallel, we employed 2D-LC-MS/MS followed by the label-free computationally modified spectral counting method APEX for absolute protein expression measurements. Of the 4502 genome-predicted SD1 proteins, 1148 proteins were identified with a false positive discovery rate of 5% and quantitated using 2D-LC-MS/MS and APEX. The dynamic range of the APEX method was approximately one order of magnitude higher than that of CBB-stained spot intensity quantitation. A squared Pearson correlation analysis revealed a reasonably good correlation (R2 = 0.67) for protein quantities surveyed by both methods. The correlation was decreased for protein subsets with specific physicochemical properties, such as low Mr values and high hydropathy scores. Stoichiometric ratios of subunits of protein complexes characterized in E. coli were compared with APEX quantitative ratios of orthologous SD1 protein complexes. A high correlation was observed for subunits of soluble cellular protein complexes in several cases, demonstrating versatile applications of the APEX method in quantitative proteomics. PMID:19563668

Kuntumalla, Srilatha; Braisted, John C; Huang, Shih-Ting; Parmar, Prashanth P; Clark, David J; Alami, Hamid; Zhang, Quanshun; Donohue-Rolfe, Arthur; Tzipori, Saul; Fleischmann, Robert D; Peterson, Scott N; Pieper, Rembert

2009-01-01

32

Unified Theory for Gel Electrophoresis and Gel Filtration  

Microsoft Academic Search

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

David Rodbard; Andreas Chrambach

1970-01-01

33

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

PubMed

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

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

2014-10-01

34

Denaturing gradient gel electrophoresis (DGGE).  

PubMed

Denaturing Gradient Gel Electrophoresis (DGGE) is a technique used to separate short- to medium-length DNA fragments based on their melting characteristics. It has been used frequently for identifying single-nucleotide polymorphisms without the need for DNA sequencing and as a molecular fingerprinting method for complex ecosystem communities, in particular in conjunction with amplification of microbial 16S rRNA genes. Here, the principles of DGGE, based on partial DNA strand separation at a given position in a gradient of chemical denaturant, are described, and an example protocol, optimized for fingerprinting of 200-300 bp fragments of bacterial 16S rRNA genes, is given. PMID:23913290

Strathdee, Fiona; Free, Andrew

2013-01-01

35

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...

36

Conducting polymer electrodes for gel electrophoresis.  

PubMed

In nearly all cases, electrophoresis in gels is driven via the electrolysis of water at the electrodes, where the process consumes water and produces electrochemical by-products. We have previously demonstrated that ?-conjugated polymers such as poly(3,4-ethylenedioxythiophene) (PEDOT) can be placed between traditional metal electrodes and an electrolyte to mitigate electrolysis in liquid (capillary electroosmosis/electrophoresis) systems. In this report, we extend our previous result to gel electrophoresis, and show that electrodes containing PEDOT can be used with a commercial polyacrylamide gel electrophoresis system with minimal impact to the resulting gel image or the ionic transport measured during a separation. PMID:24586761

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

2014-01-01

37

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.

1992-07-21

38

Proteomic analysis by two-dimensional differential in gel electrophoresis (2D DIGE) of the early response of Pisum sativum to Orobanche crenata.  

PubMed

Crenate broomrape (Orobanche crenata) is considered to be the major constraint for legume crops in Mediterranean countries. Strategies of control have been developed, but only marginal successes have been achieved. For the efficient control of the parasite, a better understanding of its interaction and associated resistance mechanisms at the molecular level is required. The pea response to this parasitic plant and the molecular basis of the resistance was studied using a proteomic approach based on 2D DIGE and MALDI-MSMS analysis. For this purpose, two genotypes showing different levels of resistance to O. crenata, as well as three time points (21, 25, and 30 d after inoculation) have been compared. Multivariate statistical analysis identified 43 differential protein spots under the experimental conditions (genotypes/treatments), 22 of which were identified using a combination of peptide mass fingerprinting (PMF) and MSMS fragmentation. Most of the proteins identified were metabolic and stress-related proteins and a high percentage of them (86%) matched with specific proteins of legume species. The behaviour pattern of the identified proteins suggests the existence of defence mechanisms operating during the early stages of infection that differed in both genotypes. Among these, several proteins were identified with protease activity which could play an important role in preventing the penetration and connection to the vascular system of the parasite. Our data are discussed and compared with those previously obtained in pea and Medicago truncatula. PMID:21920908

Castillejo, Ma Ángeles; Fernández-Aparicio, Mónica; Rubiales, Diego

2012-01-01

39

Precipitation of champagne base wine proteins prior to 2D electrophoresis.  

PubMed

Numerous methods have been employed to depict the protein content of wines. Among them, two-dimensional electrophoresis (2D-E) presents a powerful resolution, but has been poorly applied to wine. Furthermore, 2D-E was coupled with various extraction methods of proteins without any reference method for wine. Here, we describe a rapid method to extract proteins from a champagne base wine through ultrafiltration followed by precipitation with ethanol and trichloroacetic acid. More than 50 spots were visualized on 2D-gels (7 cm, pH 3-6) by colloidal Coomassie Brilliant Blue staining. PMID:24136561

Cilindre, Clara

2014-01-01

40

Analysis of protein changes using two-dimensional difference gel electrophoresis.  

PubMed

A protocol for protein analysis using two-dimensional difference gel electrophoresis (2D-DIGE) is described. 2D-DIGE is one of the most popular and versatile methods of protein separation among rapidly increasing proteomics technologies. Similar to traditional two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), the proteins are separated based on their charges and molecular weight by 2D-DIGE. Different from 2D-PAGE, proteins are pre-labeled with different fluorescent and different protein samples are run in one gel by this method. Therefore, 2D-DIGE not only carries the advantages of 2D-PAGE but also eliminates gel-to-gel variation and achieves high resolution, sensitivity, and reproducibility. PMID:24623216

Gao, Weimin

2014-01-01

41

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

Microsoft Academic Search

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

Gerard Muyzer; Kornelia Smalla

1998-01-01

42

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

Microsoft Academic Search

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

Gerard Muyzer; Kornelia Smalla

1998-01-01

43

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

44

Lanes Detection in PCR Gel Electrophoresis Images  

Microsoft Academic Search

This study aims at development of methods to track the center of and detect lanes as the first step of automated tooì to analyze rose DNA using PCR gel electrophoresis images. Although several research results have been previously reported using projection profiles in a whole image, it is still challenge to track the center of and detect bent lanes using

Sang Cheol Park; In Seop Na; Soo Hyung Kim; Guee Sang Lee; Kang Han Oh; Jeong Hwan Kim; Tae Ho Han

2011-01-01

45

The repton model of gel electrophoresis  

NASA Astrophysics Data System (ADS)

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

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

1997-02-01

46

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.

2007-04-05

47

Lipoprotein Agarose Gel Electrophoresis. Application in HDL-Cholesterol Methodology.  

National Technical Information Service (NTIS)

We perform agarose gel lipoprotein electrophoresis (AGLE) in this laboratory using glass microscope slides to support the agarose gel and 0.025 M barbital buffer for the electrophoresis. This report describes details, including special apparatus used to f...

E. L. Mosser D. A. Clark

1985-01-01

48

Electrophoresis for genotyping: microtiter array diagonal gel electrophoresis on horizontal polyacrylamide gels, hydrolink, or agarose.  

PubMed

Electrophoresis of DNA has been performed traditionally in either an agarose or acrylamide gel matrix. Considerable effort has been directed to improved quality agaroses capable of high resolution, but for small fragments, such as those from polymerase chain reaction (PCR) and post-PCR digests, acrylamide still offers the highest resolution. Although agarose gels can easily be prepared in an open-faced format to gain the conveniences of horizontal electrophoresis, acrylamide does not polymerize in the presence of air and the usual configurations for gel preparation lead to electrophoresis in the vertical dimension. We describe here a very simple device and method to prepare and manipulate horizontal polyacrylamide gels (H-PAGE). In addition, the open-faced horizontal arrangement enables loading of arrays of wells. Since many procedures are undertaken in standard 96-well microtiter plates, we have also designed a device which preserves the exact configuration of the 8 x 12 array and enables electrophoresis in tracks following a 71.6 degrees diagonal between wells (MADGE, microtiter array diagonal gel electrophoresis), using either acrylamide or agarose. This eliminates almost all of the staff time taken in setup, loading, and recordkeeping and offers high resolution for genotyping pattern recognition. The nature and size of the gels allow direct stacking of gels in one tank, so that a tank used typically to analyze 30-60 samples can readily be used to analyze 1000-2000 samples. The gels would also enable robotic loading. Electrophoresis allows analysis of size and charge, parameters inaccessible to liquid-phase methods: thus, genotyping size patterns, variable length repeats, and haplotypes is possible, as well as adaptability to typing of point variations using protocols which create a difference detectable by electrophoresis. PMID:7864363

Day, I N; Humphries, S E

1994-11-01

49

Comparison of distance measures according to suitability for 2D electrophoresis image registration using synthetic image data and SOFM  

NASA Astrophysics Data System (ADS)

This paper presents a comparative investigation of distance measures that may be used in image registration algorithms specialized for two-dimensional electrophoresis gel images. Standard image registration techniques employ correlationbased similarity measures but they are not robust against geometric and large intensity distortions. Exploitation of specific electrophoresis gel image patterns allows achieving better registration performance. In order to make comparison of distance measures synthetic electrophoresis images were generated. Synthetic data gave ability to control variation of amount of image dissimilarity specific to 2D electrophoresis images. Relation of distance measure response to variation of image dissimilarity was analyzed using self-organizing feature maps. Measures that gave desired responses of self-organizing feature maps were determined. These results were consistent with matching performance of real gel image regions. Computational needs of measures were tested additionally.

Matuzevi?ius, Dalius; Navakauskas, Dalius

2010-06-01

50

Two-dimensional gel electrophoresis: recent advances in sample preparation, detection and quantitation  

Microsoft Academic Search

A strength of two-dimensional polyacrylamide gel electrophoresis (2D PAGE) is its ability to resolve and investigate the abundance of several thousand proteins in a single sample. This enables identification of the major proteins in a tissue or subcellular fraction by mass spectrometric methods. In addition, 2D PAGE can be used to compare quantities of proteins in related samples, such as

Kathryn S Lilley; Azam Razzaq; Paul Dupree

2002-01-01

51

Photothermal densitometer for reading electrophoresis gels  

US Patent & Trademark Office Database

A densitometer apparatus for evaluating electrophoresis gel samples based on photothermal techniques. In accordance with this invention, electrophoresis gels are characterized by passing a heating light beam through the gel at a particular location. Light absorbed by the presence of staining dyes in that area causes heat evolution which generates a local index of refraction variation or a "thermal lens". A probe beam is passed through the sample in the area of the thermal lens a predetermined period of time after it is generated and the modification to the beam caused by the thermal lens is evaluated. For example, defocusing of the probe beam can be sensed by a detector which receives transmitted light through a limiting aperture. Various means of separating the heating and probe beams are disclosed, including use of separate lasers, crossed beams, modulation by plane of polarization, etc. One embodiment of this invention is particularly adapted for characterizing dry gels in which the heating beam is absorbed by the sample and the probe beam passes across the sample and is modified by a thermal lens generated in the air above the sample. Several embodiments are related to means for offsetting the probe beam from the heating beam for use with samples that are swept by the photothermal techniques in accordance with this invention offer advantages in terms of sensitivity over conventional transmission-type densitometers. These advantages enable increased sensitivity and facilitate the use of simplified staining techniques.

1990-07-03

52

"High-resolution" mini-two-dimensional gel electrophoresis automatically run and stained in less than 6 h with small, ready-to-use slab gels.  

PubMed

Although two-dimensional (2-D) gel electrophoresis is one of the most powerful techniques for analyzing protein mixtures, its application in routine clinical laboratories is currently limited, because it is time-consuming, complex, and relatively expensive. Here we describe a method for automatically running and staining "high-resolution" mini 2-D electrophoresis gels in less than 6 h, by using "ready-to-use" slab gels and a PhastSystem electrophoresis apparatus. We present 2-D gel electrophoretograms of 25 nL of plasma, as well as their automatic computer analysis. For comparison, a conventional 2-D gel electrophoresis profile of 200 nL of a plasma sample is shown. The technique is easy to perform, highly sensitive, rapid, and potentially useful in semi-routine clinical chemistry laboratories. PMID:2448065

Hochstrasser, D; Augsburger, V; Pun, T; Weber, D; Pellegrini, C; Muller, A F

1988-01-01

53

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

ERIC Educational Resources Information Center

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

Atkins, Thomas

1991-01-01

54

Hybrid slab-microchannel gel electrophoresis system  

DOEpatents

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

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

1998-01-01

55

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 âÂÂprecursor to DNA separationsâ and thus provides an important step in the subject matter. The lab provides for students: detailed instructions, background information, and a quiz and group questions. Answers to the questions, and also the general objective of the lab, are provided for the instructor. Overall, the lab is introductory in nature and perfect for any science classroom.

2008-12-05

56

Using Gel Electrophoresis To Illustrate Protein Diversity and Isoelectric Point.  

ERIC Educational Resources Information Center

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

Browning, Mark; Vanable, Joseph

2002-01-01

57

Gel Electrophoresis on a Budget to Dye For  

NSDL National Science Digital Library

Gel electrophoresis is one of the most important tools used in molecular biology and has facilitated the entire field of genetic engineering by enabling the separation of nucleic acids and proteins. However, commercial electrophoresis kits can cost up to

Yu, Julie H.

2010-01-01

58

Plasmid topoisomer separation by capillary gel electrophoresis  

NASA Astrophysics Data System (ADS)

We report capillary electrophoretic separation of pUC8 and pBr322 plasmid topoisomers in cross-linked polyacrylamide (PAA) gels in 1X TBE buffer. Plasmid topoisomers are supercoiled forms that have exactly the same chain length but differ in their number of superhelical turns. Because the size in base pairs is invariant, topoisomer mobilities reflect conformational details and differ by only small increments. In cross-linked PAA rapid topoisomer separation can be achieved by DC electrophoresis in capillary lengths as short as 3 cm and near-baseline resolution in longer capillaries. We propose that the separation depends upon the regular structure obtained when a gel is prepared intra-capillary. The isothermal environment promotes formation of a cross-linked polymer of low polydispersity. Such PAA is a sieving matrix of high resolving power, but usable over a relatively narrow DNA size range. It is also possible to prepare gels in which a wide base pair range of supercoiled and nicked plasmids as well as linear ds-DNA may be separated, but without topoisomers resolution. In this paper, we discuss the latest results in topoisomer resolution using a range of plasmids employed in molecular biology and gene therapy.

de Carmejane, Olivia; Schwinefus, Jeffrey J.; Morris, Michael D.

1999-05-01

59

Spot volume vs. amount of protein loaded onto a gel: a detailed, statistical comparison of two gel electrophoresis systems.  

PubMed

The long-term goal of this research program is to clarify the molecular mechanisms that participate in the formation of human pituitary macroadenomas. One approach to that goal is to characterize the differentially expressed proteins that are found by a comparison of the proteomes of control pituitary vs. macroadenoma tissues. In order to accurately perform a comparative proteomics study, based on the combination of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and PDQuest 2-D analysis software, a reproducible 2-DE separation system with a wide linear dynamic measure range is needed. A typical horizontal system is the Multiphor II system that analyzes one gel at a time, using a precast gradient gel (180 x 245 x 0.5 mm); a typical vertical system is the Dodeca system that analyzes up to 12 gels at a time on a single-concentration gel (190 x 205 x 1.0 mm). We have evaluated (Zhan and Desiderio, Electrophoresis 2003, 24, 1834-1846) the spatial and quantitative reproducibility of the two second-dimensional gel systems to separate a human pituitary proteome; that study showed a higher reproducibility for the Dodeca gel system. This present study investigated the relationship between the spot volume and the amount of protein loaded onto the gel for those two 2-D systems. The results demonstrated that the Dodeca gel system provides a wider linear dynamic range to measure the changes in the protein abundance in pituitary proteome. PMID:12783459

Zhan, Xianquan; Desiderio, Dominic M

2003-06-01

60

Hybrid slab-microchannel gel electrophoresis system  

DOEpatents

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

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

1998-05-05

61

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

PubMed

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

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

2014-06-01

62

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.

2010-01-01

63

ELECTROPHORESIS GEL BUFFER RECIRCULATOR FOR UNDER 20 DOLLARS  

EPA Science Inventory

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

64

Gel Electrophoresis on a Budget to Dye for  

ERIC Educational Resources Information Center

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

Yu, Julie H.

2010-01-01

65

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

PubMed Central

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

Hyrien, O; Mechali, M

1992-01-01

66

Scrambling of bands in gel electrophoresis of DNA.  

PubMed Central

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

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

1988-01-01

67

Gel Electrophoresis--The Easy Way for Students  

ERIC Educational Resources Information Center

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

VanRooy, Wilhelmina; Sultana, Khalida

2010-01-01

68

Detection of fluorescence dye-labeled proteins in 2-D gels using an Arthur 1442 Multiwavelength Fluoroimager.  

PubMed

Labeling of proteins with SYPRO Orange, SYPRO Red, and SYPRO Ruby after 2-D polyacrylamide gel electrophoresis (PAGE) using plastic-backed immobilized pH gradient (IPG) strips and precast SDS polyacrylamide gels was tested. Protein spots were detected using an Arthur 1442 Multiwavelength Fluoroimager. The labeling methods described allow detection of proteins both after isoelectric focusing (IEF) and PAGE with a sensitivity higher than or comparable to standard silver staining methods. In addition to the post-labeling methods mentioned above, pre-labeling with the cysteine-specific fluorophore monobromobimane before 2-D PAGE is a sensitive, fast, and cost-effective alternative to existing staining protocols. PMID:11464508

Herick, K; Jackson, P; Wersch, G; Burkovski, A

2001-07-01

69

Identification of viroids by gel electrophoresis.  

PubMed

Two-dimensional PAGE involving an initial fractionation under nondenaturing conditions followed by a second electrophoresis under denaturing conditions provides a powerful means to detect viroids and other small circular RNAs. This unit describes a method known as "R(eturn) PAGE" in which denaturation is achieved by simultaneously raising the temperature and lowering the ionic strength during the second electrophoresis. Under denaturing conditions, circular RNAs migrate more slowly than their corresponding linear forms. Following fractionation, RNAs are visualized by staining with ethidium bromide, SYBR Gold, or silver nitrate. Unlike nucleic acid hybridization or RT-PCR, viroid identification by R-PAGE requires no nucleotide sequence information. PMID:18729055

Owens, Robert A

2008-08-01

70

Agarose gel electrophoresis for the separation of DNA fragments.  

PubMed

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb(1). Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits(2). During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel's molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight(3). The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along(4). The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation(5); 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: Understand the mechanism by which DNA fragments are separated within a gel matrix Understand how conformation of the DNA molecule will determine its mobility through a gel matrix Identify an agarose solution of appropriate concentration for their needs Prepare an agarose gel for electrophoresis of DNA samples Set up the gel electrophoresis apparatus and power supply Select an appropriate voltage for the separation of DNA fragments Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands Determine the sizes of separated DNA fragments. PMID:22546956

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

2012-01-01

71

Optimization of Quantitative Proteomics Using 2-Dimensional Difference Gel Electrophoresis to Characterize Molecular Mechanisms of Chemical Warfare Nerve Agent Exposure in the Rat Brain.  

National Technical Information Service (NTIS)

This report describes the optimization and use of 2-dimensional difference gel electrophoresis (2-D DIGE) to investigate the proteomic changes induced by chemical warfare nerve agents (CWNAs) in whole cell homogenates and isolated mitochondria from rat br...

H. M. Hoard-Fruchey R. K. Kan

2010-01-01

72

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

Microsoft Academic Search

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

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

2010-01-01

73

Characterization of fish acid proteases by substrate-gel electrophoresis  

Microsoft Academic Search

Several analytical techniques based upon the use of substrate-polyacrylamide gel electrophoresis were evaluated to achieve characterization of aspartate proteases in fish stomach. Since aspartate proteases of fish are more stable at high pH than mammalian pepsins, the most accurate technique for activity assessment is electrophoresis at neutral pH and revealing of such activity at low pH with hemoglobin as substrate.

Manuel Diaz-Lopez; Francisco J. Moyano-Lopez; F. Javier Alarcon-Lopez; Fernando L. Garcia-Carreno; M. Angeles; Navarrete del Toro

74

Inexpensive and safe DNA gel electrophoresis using household materials.  

PubMed

Gel electrophoresis is the single most important molecular biology technique and it is central to life sciences research, but it is often too expensive for the secondary science classroom or homeschoolers. A simple safe low-cost procedure is described here that uses household materials to construct and run DNA gel electrophoresis. Plastic containers are fitted with aluminum foil electrodes and 9-V batteries to run food-grade agar-agar gels using aquarium pH buffers and then stained with gentian violet. This activity was tested in a high school biology classroom with significantly positive responses on postactivity reflective surveys. The electrophoresis activity addresses several Life Science Content Standard C criteria, including aspects of cell biology, genetics, and evolution. It also can be used to teach aspects of motion and force in the physical science classroom. PMID:22615228

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

2012-01-01

75

THERMAL DETECTION OF DNA AND PROTEINS DURING GEL ELECTROPHORESIS  

SciTech Connect

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

R. JOHNSTON

2000-08-01

76

Differences in the spatial and quantitative reproducibility between two second-dimensional gel electrophoresis systems.  

PubMed

Two-dimensional polyacrylamide gel electrophoresis (PAGE), together with 2-D gel electrophoresis (GE) analysis software, is a common technique to analyze a complex proteome. In order to accurately locate the differentially expressed proteins in human pituitary macroadenoma tissues in our long-term research program to clarify the molecular mechanisms of macroadenoma formation, a reproducible separation system is needed. An immobilized pH-gradient dry gel-strip (IPG strip) has been extensively used for first-dimensional isoelectric focusing (IEF), and has achieved a high degree of reproducibility in the IEF direction. For the second dimension (SDS-PAGE), different types of gel systems are available, including horizontal vs. vertical gel systems, and gradient vs. constant-percentage gels. A typical horizontal system is the Multiphor II system that analyzes one gel at a time, using a precast gradient gel (180 x 245 x 0.5 mm), and a typical vertical system is the Dodeca system, which analyzes up to 12 gels at a time, using usually a single-concentration gel (190 x 205 x 1 mm). The present study evaluated the spatial and quantitative reproducibility of the two systems for the separation of the complex human pituitary proteome. PDQuest software was used to analyze the digitized gel-image data, and SPSS statistical software was used to analyze the data. The results demonstrated a high percentage (>99%) of protein-spot matches within each electrophoretic system. The Dodeca gel system demonstrated better between-gel reproducibility for spot position, higher resolution in the Sodium dodecyl sulfate (SDS)-PAGE direction, lower gel background, better spot quality, and higher reproducibility of the spot volume. PMID:12783460

Zhan, Xianquan; Desiderio, Dominic M

2003-06-01

77

Comparison between agarose gel electrophoresis and capillary electrophoresis for variable numbers of tandem repeat typing of Mycobacterium tuberculosis  

Microsoft Academic Search

Variable numbers of tandem repeat (VNTR) typing of Mycobacterium tuberculosis was performed on 54 strains including 23 strains derived from 9 outbreaks. PCR amplicon sizes of 12 mycobacterial interspersed repetitive unit tandem repeat loci were measured using both agarose gel electrophoresis and capillary electrophoresis. Similarities using agarose gel electrophoresis of Euclidian distances among the 23 strains derived from the 9

Eiji Yokoyama; Kazunori Kishida; Masako Uchimura; Sadato Ichinohe

2006-01-01

78

Semiquantitative proteomic analysis of the human spliceosome via a novel two-dimensional gel electrophoresis method.  

PubMed

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, B(act), 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, B(act), 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; Odenwälder, Peter; Bessonov, Sergey; Will, Cindy L; Urlaub, Henning; Lührmann, Reinhard

2011-07-01

79

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.

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

2011-01-01

80

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

PubMed

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

2009-01-01

81

PCR and Gel Electrophoresis: Moving Beyond the Techniques  

NSDL National Science Digital Library

This college biology curriculum unit from the Wisconsin Program for Scientific Teaching helps students learn key molecular biology techniques in the context of a real-world issue (genetically modified organisms, or GMOs). It provides a set of novel exercises that integrate the biological concepts of DNA structure and replication with the techniques of polymerase chain reaction (PCR) and gel electrophoresis.

Jo Handelsman (University of WisconsinâÂÂMadison;); Allison Phillips (Stanford University;); Amber Robertson (University of Wisconsin-Madison;)

2010-05-28

82

Application of the single cell gel electrophoresis on yeast cells  

Microsoft Academic Search

In the present paper, we have applied the single cell gel electrophoresis (SCGE) assay on yeast cells treating Saccharomyces cerevisiae cells with hydrogen peroxide and methyl methanesulfonate (MMS), two DNA damaging agents. In order to overcome the problem with the yeast cell wall that prevented DNA to be extended by the electric field, we disintegrated the cell wall after embedding

George Miloshev; Ivailo Mihaylov; Boyka Anachkova

2002-01-01

83

'Laterally aggregated' polyacrylamide gels for electrophoresis.  

PubMed

A new method is described for producing highly porous polyacrylamide matrices: polymerization in presence of a preformed hydrophilic polymer. If a standard mixture of monomers (e.g., 5%T, 4%C) is polymerized in presence of, e.g., polyethylene glycol (PEG) 10 kDa, lateral chain aggregation occurs, with formation of large pore sizes. In PEG 10 kDa, the transition from a small- to a large-pore gel is clearly apparent at 0.5% PEG addition and reaches a plateau already at 2.5% PEG. Even with shorter PEG fragments (6.2 and 1 kDa) this transition occurs, but with progressively larger amounts of PEG in solution (up to 25% for the 1 kDa species). Other polymers such as hydroxymethyl cellulose (1000 kDa) and polyvinyl-pyrrolidone (360 kDa and 25 kDa) are also able to elicit this phenomenon. It appears that lateral chain aggregation (before the cross-linking event) is induced via intra-chain hydrogen bonding, since urea and temperature strongly inhibit it, whereas tetramethylurea (an agent quenching hydrophobic interactions) does not hamper it. By scanning electron microscope, it is found that the maximum pore size obtained in a 5%T, 4%C gel in presence of 2.5% PEG 10 kDA is of the order of 0.5 micron, whereas the same 5%T, 4%C control gel would have an average pore diameter of 5 nm. Thus, an increment of pore size of about 2 orders of magnitude is obtained: in these new matrices, a 21000 bp DNA fragment exhibits a much greater migration than in a control gel in which the sample is entrapped at the application site. PMID:1459071

Righetti, P G; Caglio, S; Saracchi, M; Quaroni, S

1992-01-01

84

The new horizon in 2D electrophoresis: new technology to increase resolution and sensitivity.  

PubMed

A principally new type of an electrophoresis setup for the second dimension of 2DE named HPE (high performance electrophoresis) has recently become available that provides excellent reproducibility much superior to traditional 2DE. It takes up ideas from early beginnings of 2DE which could not be satisfactory realized at that time. The new HPE system is in contrast to all other established systems a horizontal electrophoresis that employs a new type of precast polyacrylamide gels on film-backing and runs on a multilevel flatbed electrophoresis apparatus. In a systematic approach we compared its features to traditional 2DE for the cytosolic proteome of Bacillus subtilis. Not only the reproducibility is enhanced, but also nearly all qualitative parameters as resolution, sensitivity, the number of protein spots (25% more), and the number of different proteins (also additional 25%) are markedly increased. More than 200 proteins were exclusively found in HPE. This new electrophoresis system does not use buffer tanks. No glass plates are needed. Therefore handling of gels is greatly facilitated and very simple to use even for personnel with low technical skills. The new HPE system is technically at the beginnings and further development with increased performance can be expected. PMID:23494680

Moche, Martin; Albrecht, Dirk; Maaß, Sandra; Hecker, Michael; Westermeier, Reiner; Büttner, Knut

2013-06-01

85

Evaluation of proteinuria by two-dimensional polyacrylamide gel electrophoresis after kidney transplantation.  

PubMed

Urinary proteins were studied in patients after kidney transplantation during various functional states (normal function, acute rejection, chronic rejection) by two-dimensional polyacrylamide gel electrophoresis. In the first dimension, the proteins were separated according to their electric charge by isoelectric focusing, and in the second dimension according to their molecular weight by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis the proteins were visualized using a highly sensitive silver stain technique. The combination of these methods allowed the discrimination of up to 250 protein spots in human urine, most of them unidentified up to now. Functional changes of the kidney transplants were accompanied by complex changes of the protein pattern in urine. These changes cannot be simply compared to the tubular and glomerular pattern of proteinuria which can be identified by one-dimensional sodium dodecyl sulfate electrophoresis. The 2D electrophoresis of urinary proteins may develop into a useful tool in localizing of kidney damage and in the evaluation of renal disease. PMID:3915927

Bauer, H; Nagel, J; Wierer, D; Brunner, H; Franz, H E

86

Identification of intrinsically disordered proteins by a special 2D electrophoresis.  

PubMed

Intrinsically disordered proteins (IDPs) lack a well-defined three-dimensional structure under physiological conditions. They constitute a significant fraction of various proteomes and have significant roles in key cellular processes. Here we report the development of a two-dimensional electrophoresis technique for their de novo recognition and characterization. This technique consists of the combination of native and 8 M urea electrophoresis of heat-treated proteins where IDPs are expected to run into the diagonal of the gel, whereas globular proteins either precipitate upon heat treatment or unfold and run off the diagonal in the second dimension. PMID:22821526

Tantos, Agnes; Tompa, Peter

2012-01-01

87

Use of Two-Dimensional Gel Electrophoresis To Measure Changes in Synovial Fluid Proteins from Patients with Rheumatoid Arthritis Treated with Antibody to CD4  

Microsoft Academic Search

Synovial fluid proteins from microliter volumes of synovial fluid were resolved by two-dimensional poly- acrylamide gel electrophoresis and detected by silver staining to investigate the feasibility of using two- dimensional (2D) electrophoresis in the clinical research setting and provide global disease information of disease progression. Several hundred proteins could be resolved as spots, many of which displayed the characteristic pattern

MARJORIE A. SMITH; SATBINDER K. BAINS; JOANNA C. BETTS; ERNEST H. S. CHOY; E. D. Zanders

2001-01-01

88

Nanostructured Copolymer Gels for dsDNA Separation by Capillary Electrophoresis  

PubMed Central

Pluronics copolymers are triblock copolymers of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) and are able to form many different ordered nanostructures at appropriate polymer concentrations and temperatures in selective solvents. These nano-structured ‘gels’ showed desirable criteria when used as DNA separation media, especially in microchip electrophoresis, including dynamic coating ability and viscosity switchable property. A ternary system of F127 (E99P69E99)/TBE buffer/1-butanol was selected as a model system to test the sieving performance of different nanostructures in separating dsDNA by capillary electrophoresis. The lattice structures were determined by small-angle x-ray scattering with quasi-lattice crystal parameters being calculated according to the x-ray scattering data. Viscosity measurements showed the sol-gel transition phenomena. In addition to the cubic structure, successful electrophoretic separation of dsDNA in 2-D hexagonal packed cylinders was achieved. Results showed that without further optimization, ?X174 DNA-Hae III digest was well separated within 15 minutes in a 7-cm separation channel, by using F127/TBE/1-butanol gel with a 2-D hexagonal structure. A mechanism for DNA separations by those gels with both hydrophilic and hydrophobic domains is discussed.

Wan, Fen; Zhang, Jun; Lau, Angela; Tan, Sarah; Burger, Christian; Chu, Benjamin

2010-01-01

89

Thermally reversible gels in electrophoresis. I - Matrix characterization  

NASA Technical Reports Server (NTRS)

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

Righetti, Pier Giorgio; Snyder, Robert S.

1988-01-01

90

Photopatterned free-standing polyacrylamide gels for microfluidic protein electrophoresis.  

PubMed

Designed for compatibility with slab-gel polyacrylamide gel electrophoresis (PAGE) reagents and instruments, we detail development of free-standing polyacrylamide gel (fsPAG) microstructures supporting electrophoretic performance rivalling that of microfluidic platforms. For the protein electrophoresis study described here, fsPAGE lanes are comprised of a sample reservoir and contiguous separation gel. No enclosed microfluidic channels are employed. The fsPAG devices (120 ?m tall) are directly photopatterned atop of and covalently attached to planar polymer or glass surfaces. Leveraging the fast <1 h design-prototype-test cycle - significantly faster than mold based fabrication techniques - we optimize the fsPAG architecture to minimize injection dispersion for rapid (<1 min) and short (1 mm) protein separations. The facile fabrication and prototyping of the fsPAGE provides researchers a powerful tool for developing custom analytical assays. We highlight the utility of assay customization by fabricating a polyacrylamide gel with a spatial pore-size distribution and demonstrate the resulting enhancement in separation performance over a uniform gel. Further, we up-scale from a unit separation to an array of 96 concurrent fsPAGE assays in 10 min run time driven by one electrode pair. The fsPAG array layout matches that of a 96-well plate to facilitate integration of the planar free standing gel array with multi-channel pipettes while remaining compatible with conventional slab-gel PAGE reagents, such as staining for label-free protein detection. Notably, the entire fsPAGE workflow from fabrication, to operation, and readout uses readily available materials and instruments - making this technique highly accessible. PMID:23609800

Duncombe, Todd A; Herr, Amy E

2013-06-01

91

Separation of amino acid homopolymers by capillary gel electrophoresis.  

PubMed

Gel-filled capillaries utilizing highly concentrated and moderately cross-linked acrylamide-type gels in capillary electrophoresis were successfully applied to the separation of the individual oligomers of various poly(amino acids). Mixtures of both anionic and cationic nature were adequately resolved. While UV detection at 220 nm was mostly utilized, the polyanions with N-terminal groups can also be tagged with 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde (CBQCA) for a more sensitive detection by a laser-induced fluorescence detector. PMID:8452245

Dolník, V; Novotny, M V

1993-03-01

92

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.

2014-01-01

93

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

PubMed

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

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

2014-01-01

94

Analysis of kimchi microflora using denaturing gradient gel electrophoresis  

Microsoft Academic Search

A polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique was used to determine the microfloral composition during the fermentation of kimchi, a traditional Korean fermented vegetable food. The kimchi was fermented at 10 °C or 20 °C for 30 or 20 days, respectively. DGGE of the partially amplified 16S rDNA was performed and the most intense bands sequenced. The application

Jung-Sook Lee; Gun-Young Heo; Jun Won Lee; Yun-Jung Oh; Jeong A Park; Yong-Ha Park; Yu-Ryang Pyun; Jong Seog Ahn

2005-01-01

95

Detection of serum proteins by native polyacrylamide gel electrophoresis using Blue Sepharose CL6B-containing stacking gels  

Microsoft Academic Search

Analysis of serum proteins by native polyacrylamide gel electrophoresis is difficult because albumin is abundant in serum and interferes with the resolution of other proteins, especially ?-antitrypsin which has mobility that is very similar to that of albumin. We present here a method in which serum proteins are separated by polyacrylamide gel electrophoresis using stacking gels containing Blue Sepharose CL-6B,

Haruhiro Muratsubaki; Kaoru Satake; Yasuhisa Yamamoto; Keiichiro Enomoto

2002-01-01

96

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

Microsoft Academic Search

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

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

1998-01-01

97

Plant Nucleases: III. Polyacrylamide Gel Electrophoresis of Corn Ribonuclease Isoenzymes.  

PubMed

Isoenzymes of RNase were detected in plant extracts after polyacrylamide gel electrophoresis with a new buffer system. The gels were incubated in an RNA solution, then dipped for 30 seconds into 0.2% toluidine blue. The method is rapid and is sensitive to very small amounts of RNase. The effects of buffers and ethylenediaminetetraacetate on the different enzymes are illustrated by photographs and scans of the gels.RNase I, from endosperms and roots, was the fastest-moving corn RNase. Two isoenzymes of corn RNase II, from microsomes, were detected in the hybrid WF9 x M14, while each parental inbred had one of the isoenzymes. Three isoenzymes of corn Nuclease I, from crude mitochondria, had about the same mobility as the RNase II isoenzymes, but were inhibited by ethylenediaminetetraacetate. RNases were also detected in tobacco and wild carrot tissue cultures. PMID:16657737

Wilson, C M

1971-07-01

98

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

PubMed Central

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

2012-01-01

99

Comparative Study of Three Proteomic Quantitative Methods, DIGE, cICAT, and iTRAQ, Using 2D Gel or LC?MALDI TOF\\/TOF  

Microsoft Academic Search

A comparative study on the three quantitative methods frequently used in proteomics, 2D DIGE (difference gel electrophoresis), cICAT (cleavable isotope-coded affinity tags) and iTRAQ (isobaric tags for relative and absolute quantification), was carried out. DIGE and cICAT are familiar techniques used in gel- and LC-based quantitative proteomics, respectively. iTRAQ is a new LC-based technique which is gradually gaining in popularity.

Wells W. Wu; Guanghui Wang; Seung Joon Baek; Rong-Fong Shen

2006-01-01

100

Analysis of Inorganic Polyphosphates by Capillary Gel Electrophoresis  

PubMed Central

This paper describes the development of a method that uses Capillary Gel Electrophoresis (CGE) to analyze mixtures of inorganic polyphosphate ((Pi)n). Resolution of (Pi)n on the basis of n, the number of residues of dehydrated phosphate, is accomplished by CGE using capillaries filled with solutions of poly(N,N-dimethylacrylamide) (PDMA) and indirect detection by the UV-absorbance of a chromophore, terephthalate, added to the running buffer. The method is capable of resolving peaks representing (Pi)n with n up to ~70; preparation and use of authentic standards enables the identification of peaks for (Pi)n with n = 1 - 10. The main advantages of this method over previously reported methods for analyzing mixtures of (Pi)n (e.g., gel electrophoresis, CGE using polyacrylamide-filled capillaries) are its resolution, convenience, and reproducibility; gel-filled capillaries are easily regenerated by pumping in fresh, low-viscosity solutions of PDMA. The resolution is comparable to that of ion-exchange chromatography and detection of (Pi)n by suppressed conductivity. The method is useful for analyzing (Pi)n generated by the dehydration of Pi at low temperature (125 - 140 °C) with urea, in a reaction that may have been important in prebiotic chemistry. The method should also be useful for characterizing mixtures of other anionic, oligomeric or polymeric species without an intrinsic chromophore (e.g., sulfated polysaccharides, oligomeric phospho-diesters).

Lee, Andrew; Whitesides, George M.

2010-01-01

101

A new isoelectric focusing system for fast two-dimensional gel electrophoresis using a low-concentration polyacrylamide gel supported by a loose multifilament string.  

PubMed

A new isoelectric focusing (IEF) system for two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been proposed. In this system, a super-soft and tough IEF gel was achieved by casting polyacrylamide gel down to 2.0% T using a loose multifilament string (LMS) of nylon as a gel support. The IEF apparatus for the LMS-gel, fabricated from acrylic boards, had a cooling water chamber, and eliminated the need of electrode solutions by directly connecting the two ends of individual gels to platinum electrodes. The carrier ampholyte-generated pH gradients using the new IEF system was stable over a long duration of time and a wide range of voltages, and the IEF time became shorter using a 2.0% T gel than using a 4.0% T gel. Also, the LMS-gels prepared in different runs exhibited excellent reproducibility. The new IEF system was applied to 2-D PAGE of a chicken skeletal muscle extract, and it was found that the protein loading capacity, protein entry into the LMS-gels, and protein transfer efficiency from the first-dimensional to the second-dimensional gels were significantly improved by using a low-concentration (2.5% T) gel. Also, proteins of high molecular weight of more than 200 kDa were observed in the 2-D maps, and therefore the new IEF system has a very good potential to be applied for fast 2-D PAGE of high molecular-weight proteins. PMID:15636514

Li, Jinxiang; Ogasawara, Ayaka; Odake, Tamao; Umemura, Tomonari; Tsunoda, Kin-ichi

2004-12-01

102

Analysis of mutations using PCR and denaturing gradient gel electrophoresis  

SciTech Connect

Denaturing gradient gel electrophoresis (DGGE) separates DNA molecules based on primary sequence. Under the appropriate conditions, all base pair (bp) substitutions, frameshifts, and deletions less than about 10 bp can be resolved from the wild type sequence using DGGE. Polymerase chain reaction (PCR) permits facile amplification of a given region of the genome. The authors have combined PCR and DGGE to: (1) localize mutations in the X-linked human androgen receptor gene; (2) analyze thousands of thioguanine-resistant mutants simultaneously; (3) examine the fidelity of several DNA polymerases used in PCR.

Cariello, N.F.; Swenberg, J.A. (Univ. of North Carolina, Chapel Hill (United States) Duke Univ., Durham, NC (United States)); DeBellis, A.; Skopek, T.R. (Univ. of North Carolina, Chapel Hill (United States))

1991-01-01

103

GELBANK : A database of annotated two-dimensional gel electrophoresis patterns of biological systems with completed genomes.  

SciTech Connect

GELBANK is a publicly available database of two-dimensional gel electrophoresis (2DE) gel patterns of proteomes from organisms with known genome information (available at and ftp://bioinformatics.anl.gov/gelbank/). Currently it includes 131 completed, mostly microbial proteomes available from the National Center for Biotechnology Information. A web interface allows the upload of 2D gel patterns and their annotation for registered users. The images are organized by species, tissue type, separation method, sample type and staining method. The database can be queried based on protein or 2DE-pattern attributes. A web interface allows registered users to assign molecular weight and pH gradient profiles to their own 2D gel patterns as well as to link protein identifications to a given spot on the pattern. The website presents all of the submitted 2D gel patterns where the end-user can dynamically display the images or parts of images along with molecular weight, pH profile information and linked protein identification. A collection of images can be selected for the creation of animations from which the user can select sub-regions of interest and unlimited 2D gel patterns for visualization. The website currently presents 233 identifications for 81 gel patterns for Homo sapiens, Methanococcus jannaschii, Pyro coccus furiosus, Shewanella oneidensis, Escherichia coli and Deinococcus radiodurans.

Babnigg, G.; Giometti, C. S.; Biosciences Division

2004-01-01

104

Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry.  

PubMed

Two-dimensional difference gel electrophoresis (2-D DIGE) coupled with mass spectrometry (MS) was used to investigate tumor-specific changes in the proteome of human colorectal cancers and adjacent normal mucosa. For each of six patients with different stages of colon cancer, Cy5-labeled proteins isolated from tumor tissue were combined with Cy3-labeled proteins isolated from neighboring normal mucosa and separated on the same 2-D gel along with a Cy2-labeled mixture of all 12 normal/tumor samples as an internal standard. Over 1500 protein spot-features were analyzed in each paired normal/tumor comparison, and using DIGE technology with the mixed-sample internal standard, statistically significant quantitative comparisons of each protein abundance change could be made across multiple samples simultaneously without interference due to gel-to-gel variation. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and tandem (TOF/TOF) MS provided sensitive and accurate mass spectral data for database interrogation, resulting in the identification of 52 unique proteins (including redundancies due to proteolysis and post-translationally modified isoforms) that were changing in abundance across the cohort. Without the benefit of the Cy2-labeled 12 sample mixture internal standard, 42 of these proteins would have been overlooked due to the large degree of variation inherent between normal and tumor samples. PMID:14997500

Friedman, David B; Hill, Salisha; Keller, Jeffrey W; Merchant, Nipun B; Levy, Shawn E; Coffey, Robert J; Caprioli, Richard M

2004-03-01

105

DNA Electrophoresis in Agarose Gels: Mobility vs. Length Dependence  

NASA Astrophysics Data System (ADS)

Over the years, many different models have been applied to the migration of DNA fragments during gel electrophoresis. These models have been limited to describing DNA motion over specific size ranges. We propose a frictional and charge based model relating the electrophoretic mobility to length that fits data for DNA fragment lengths from 100 base pairs (bp) to 50 kilobase pairs (kbp). Excellent fits have been obtained from both published sources and experiments we have performed, with agarose gel concentrations of 0.5% to 1.5%. The length range of DNA for which the model works spans the range where a DNA fragment behaves like a semi-rigid rod to best described as a random coil polymer

Beheshti, Afshin; van Winkle, David; Rill, Randolph

2001-03-01

106

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

PubMed Central

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

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

1995-01-01

107

Deoxyribonuclease zymography adapted to the precast PhastGel electrophoresis media.  

PubMed

A set-up for casting fluorescent indicator agarose gels on ultrathin polyacrylamide microelectrophoresis gel media (Pharmacia PhastGel media) is described. The zymogram system allows a rapid and sensitive detection of deoxyribonuclease in various gel media, following isoelectric focusing, native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PMID:1692791

Straetkvern, K O; Raae, A J

1990-04-01

108

The gel edge electric field gradients in denaturing polyacrylamide gel electrophoresis.  

PubMed

It has previously been shown that zones of higher electric field form close to the loading end of the gel during denaturing polyacrylamide gel electrophoresis. Here we show that the field can reach up to three times its normal mean value a few cm in front of the loading wells when 44.5 mM Tris-44.5 mM boric acid-1 mM EDTA is used as the gel buffer. We also demonstrate that this electric field gradient is mostly due to the difference in ion transference numbers at the gel/buffer interface caused by the high viscosity of the urea solution contained in the gel. This field gradient leads to increased band widths and forces us to redefine both the electrophoretic mobility and the mean field intensity. We discuss some methods that can be used to minimize the effects of this gradient. PMID:9629888

Desruisseaux, C; Slater, G W; Drouin, G

1998-05-01

109

High-throughput genotyping of factor V Leiden mutation by ultrathin-layer agarose gel electrophoresis  

Microsoft Academic Search

Ultrathin-layer agarose gel electrophoresis is a novel combination of the established methodologies of slab gel electrophoresis and capillary gel electrophoresis. This new format provides a multilane separation platform with rapid analysis time and excellent sensitivity by using laser-induced fluorescence scanning detection system. Sample injection onto the ultrathin-layer separation platform is easily accomplished by membrane mediated loading technology. In this paper,

Timea Lengyel; Maria Sasvari-Szekely; Andras Guttman

1999-01-01

110

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

PubMed

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

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

2014-12-01

111

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

NASA Astrophysics Data System (ADS)

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

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

2013-06-01

112

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

PubMed

Rifaximin is a semi-synthetic rifamycin derivative that is used to treat different conditions including bacterial diarrhea and hepatic encephalopathy. Rifaximin is of particular interest because it is poorly adsorbed in the intestines and has minimal effect on colonic microflora. We previously demonstrated that rifaximin affected epithelial cell physiology by altering infectivity by enteric pathogens and baseline inflammation suggesting that rifaximin conferred cytoprotection against colonization and infection. Effects of rifaximin on epithelial cells were further examined by comparing the protein expression profile of cells pretreated with rifaximin, rifampin (control antibiotic), or media (untreated). Two-dimensional (2-D) gel electrophoresis identified 36 protein spots that were up- or down-regulated by over 1.7-fold in rifaximin treated cells compared to controls. 15 of these spots were down-regulated, including annexin A5, intestinal-type alkaline phosphatase, histone H4, and histone-binding protein RbbP4. 21 spots were up-regulated, including heat shock protein (HSP) 90? and fascin. Many of the identified proteins are associated with cell structure and cytoskeleton, transcription and translation, and cellular metabolism. These data suggested that in addition to its antimicrobial properties, rifaximin may alter host cell physiology that provides cytoprotective effects against bacterial pathogens. PMID:23922656

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

2013-01-01

113

Rifaximin-Mediated Changes to the Epithelial Cell Proteome: 2-D Gel Analysis  

PubMed Central

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

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

2013-01-01

114

Protein Separation by Capillary Gel Electrophoresis: A Review  

PubMed Central

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

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

2011-01-01

115

Electrophoresis gel media: the state of the art.  

PubMed

Some unique events have occurred in the last few years which might revolutionize the field of polyacrylamide gel electrophoresis. While it was widely recognized that such matrices could normally be cast with a small pore size distribution, typically of the order of a few nanometers diameter (for protein sieving), recent developments suggest that "macroporous" gels could also be produced in the domain of polyacrylamides. If constraints to chain motion are imposed during gel polymerization, large-pore structures can be grown. Such constraints can originate either from low temperatures or from the presence of preformed polymers in the gelling solution; in both cases, the growing chains are forced to "laterally aggregate" via inter-chain hydrogen bond formation. Upon consumption of pendant double bonds, such bundles are frozen in the three-dimensional space by permanent cross-links. As an additional development, a novel photopolymerization system is described, comprising a cationic dye (methylene blue) and a redox couple (sodium toluene sulfinate, a reducer, and diphenyliodonium chloride, a mild oxidizer). Methylene blue catalysis is characterized by a unique efficiency, ensuring >96% conversion of monomers, even in hydro-organic solvents and in the presence of surfactants, which normally quench or completely inhibit the persulphate-driven reaction. In addition, methylene blue-sustained photopolymerization can be operated in the entire pH 3-10 interval, where most other systems fail. Perhaps the most striking novelty in the field is the appearance of a novel monomer (N-acryloylaminopropanol, AAP) coupling a high hydrophilicity with a unique resistance to alkaline hydrolysis. Given the fact that a poly(AAP) matrix is 500 times more stable than a poly(acrylamide) gel, while being twice as hydrophilic, it is anticipated that this novel chemistry will have no difficulties in replacing the old electrophoretic anticonvective media. The review ends with a glimpse at novel sieving media in capillary zone electrophoresis: polymer networks. Such media, by providing an almost infinite range of pore sizes, due to the absence of a rigid support, allow sieving mechanisms to be operative over a wide interval of particle sizes, even up to genomic DNA. Viscous solutions of polymer networks, made with the novel poly(AAP) chemistry, allow repeated use of the same separation column, well above 50 injections. Silica-bound poly(AAP) chains provide effective quenching of electroosmosis and >200 analyses by isoelectric focusing. PMID:9392368

Righetti, P G; Gelfi, C

1997-10-10

116

Monitoring Piscirickettsia salmonis by denaturant gel electrophoresis and competitive PCR.  

PubMed

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

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

2000-05-25

117

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

Microsoft Academic Search

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

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

1989-01-01

118

Tissue-specific distribution of the Goodpasture antigen demonstrated by 2-D electrophoresis and western blotting.  

PubMed

The target of autoantibodies in Goodpasture's disease, the Goodpasture antigen has recently been characterized as the NC1 domain of the alpha 3 chain of type IV collagen. In order to study the Goodpasture antigen in different organs, NC1 domains were isolated from basement membranes (BM) of human glomeruli (GBM), tubules (TBM), alveoli (ABM), placenta (PBM) and aorta (VBM). NC1 preparations were separated by 2-D electrophoresis, and silver stained or immunoblotted to determine the subunit structure and antigenicity of different basement membranes. All basement membranes contained monomeric components of MW 26 kDa and 24 kDa, and associated dimers, corresponding to the 2-D location of alpha 1(IV) and alpha 2(IV) chains respectively. However, GBM, ABM, and to a lesser extent TBM possessed an extra set of monomeric components of MW 28 kDa and associated dimers corresponding to the proposed location of alpha 3 (IV) and alpha 4 (IV) chains. 2-D-separated polypeptides were Western blotted with autoantibodies from patients with Goodpasture's disease, a monoclonal antibody to the Goodpasture antigen (P1) and a monoclonal antibody to the bovine alpha 3 (IV) chain. The predominant binding of all these reagents was to cationic 28 kDa monomers of GBM, ABM and TBM, corresponding to the alpha 3 (IV) chain, although autoantibodies and Pl also bound to neutral 28 kDa monomers, corresponding to the alpha 4 (IV) chain. Autoantibodies bound weakly to more neutral components of PBM and VBM, but neither monoclonal antibody bound to these basement membranes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8084446

Derry, C J; Pusey, C D

1994-01-01

119

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

PubMed

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

2012-03-01

120

DNA Length Ranges Exhibiting Distinct Separation Mechanisms in Gel Electrophoresis  

NASA Astrophysics Data System (ADS)

Electrophoresis was performed on double stranded DNA ranging from 200 to 194,000 bp in agarose gel concentrations from 0.4% - 1.3%. The electric field was varied from 0.62 to 6.21 V/cm. A wide range of electric fields and gel concentrations were used to study how the new interpolation equation, frac1?(L) = frac1?L - (frac1?L - frac1?_s)e^-L/? (where ?_L, ?_s, and ? are independent free fitting parameters), helps to distinguish among different mechanisms of molecular transport. This exponential relation fits well when there is a smooth transition from Ogston sieving to reptation. These transitions are distinguished by so-called ``reptation plots" (plotting 3? L/?_rc vs. L) (J. Rousseau, G. Drouin, and G. W. Slater, Phys Rev Lett. 1997, 79, 1945-1948). Fits deviate from the data more than two characteristic trends are observed in the reptation plots. The failure of the fits to follow the data appears to be a consequence of another separation mechanism, ``entropic trapping," occurring between the sieving and reptation regimes. The boundaries between length and field ranges where different separation mechanisms dominate are extracted from reptation plots of the best fits and the data. ``Phase diagrams" expressing these boundaries are derived.

Beheshti, A.; van Winkle, D. H.; Rill, R. L.

2003-03-01

121

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

SciTech Connect

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

Koutney, L.B.

1992-09-09

122

Two dimensional difference gel electrophoresis analysis of cerebrospinal fluid in tuberculous meningitis patients.  

PubMed

Tuberculous meningitis (TBM) is a serious complication of tuberculosis that affects the central nervous system. Present methods to diagnose TBM are not suitable for early diagnosis. Molecular markers and sensitive methods to identify them in the early stage of infection of TBM are critically needed for efficient management. We have done the proteomic analysis of TBM cerebrospinal fluid (n=20) with 2-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry. We identified 11 human proteins and 8 mycobacterial proteins with changed expression levels in comparison to controls. Arachidonate 5-lipoxygenase and glial fibrillary acidic protein, two of the identified proteins, were validated with western blot technique on a larger set of disease and control samples (n=40). These two proteins were also analyzed in fungal meningitis samples. We suggest that arachidonate 5-lipoxygenase can be considered for validation as a potential marker for diagnosis of TBM. PMID:21723968

Kataria, Jitender; Rukmangadachar, Lokesh A; Hariprasad, Gururao; O, Jithesh; Tripathi, Manjari; Srinivasan, Alagiri

2011-09-01

123

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

PubMed

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

Hanneken, Marina; König, Simone

2014-04-01

124

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

PubMed

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

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

2007-01-01

125

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.

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

2007-01-01

126

Two-dimensional polyacrylamide gel electrophoresis of extracellular soybean pathogenesis-related proteins using PhastSystem.  

PubMed

Acidic and basic pathogenesis-related proteins (PR-Ps) were extracted from the intercellular fluid (IF) of soybean leaves, locally infected with tobacco necrosis virus and showing necrotic local lesions. Proteins were detected by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using PhastSystem and precast commercially available gels. Extracts from healthy leaves were run as controls. PR-Ps were first run under native PAGE conditions or isoelectric focusing (IEF), the gels stained with Coomassie Blue, then run under sodium dodecyl sulfate (SDS)-denaturing conditions and finally stained with silver. Ten major acidic PR-Ps were separated; their Mr's were close to those found by conventional PAGE. Their isoelectric points ranged from 3.5 to 5.0. Ten basic PR-Ps were separated and their Mr's estimated. None of these acidic or basic soybean PR-Ps was a glycoprotein. PAGE with PhastSystem and precast gels gives reliable results, comparable with those from conventional 2D-PAGE, with simpler experimental procedures. By electrophoresing Coomassie-stained gels with SDS in the second dimension, we were able to control the first-dimensional separation and to avoid laborious protocols generally adopted with unstained gels. PMID:2318193

Roggero, P; Pennazio, S

1990-01-01

127

Analysis of Tissue Proteomes of the Gulf Killifish, Fundulus grandis, by 2D Electrophoresis and MALDI-TOF/TOF Mass Spectrometry  

PubMed Central

The Gulf killifish, Fundulus grandis, is a small teleost fish that inhabits marshes of the Gulf of Mexico and demonstrates high tolerance of environmental variation, making it an excellent subject for the study of physiological and molecular adaptations to environmental stress. In the present study, two-dimensional (2D) gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry were used to resolve and identify proteins from five tissues: skeletal muscle, liver, brain, heart, and gill. Of 864 protein features excised from 2D gels, 424 proteins were identified, corresponding to a 49% identification rate. For any given tissue, several protein features were identified as the same protein, resulting in a total of 254 nonredundant proteins. These nonredundant proteins were categorized into a total of 11 molecular functions, including catalytic activity, structural molecule, binding, and transport. In all tissues, catalytic activity and binding were the most highly represented molecular functions. Comparing across the tissues, proteome coverage was lowest in skeletal muscle, due to a combination of a low number of gel spots excised for analysis and a high redundancy of identifications among these spots. Nevertheless, the identification of a substantial number of proteins with high statistical confidence from other tissues suggests that F. grandis may serve as a model fish for future studies of environmental proteomics and ultimately help to elucidate proteomic responses of fish and other vertebrates to environmental stress.

Abbaraju, Naga V.; Boutaghou, Mohamed Nazim; Townley, Ian K.; Zhang, Qiang; Wang, Guangdi; Cole, Richard B.; Rees, Bernard B.

2012-01-01

128

Analyzing Multiclonality of Staphylococcus aureus in Clinical Diagnostics Using spa-Based Denaturing Gradient Gel Electrophoresis?  

PubMed Central

We present a novel denaturing gradient gel electrophoresis (DGGE) method which characterizes multiclonal communities of Staphylococcus aureus. The spa PCR-based DGGE method simultaneously separates strains that differ in only one base, thereby revealing multiclonal colonization and infections.

Matussek, Andreas; Stark, Lisa; Dienus, Olaf; Aronsson, Joakim; Mernelius, Sara; Lofgren, Sture; Lindgren, Per-Eric

2011-01-01

129

Rapid (ten-minute) pore-gradient electrophoresis of proteins and peptides in Micrograd gels.  

PubMed

Precast gradient gels of short migration length (25 mm) have been developed to provide rapid electrophoretic separation without loss of resolution. These Micrograd gels have been prepared in gel ranges (conventional and unique) to match pore-gradient electrophoresis conditions to proteins/peptides ranging in size from several hundreds to millions. The Hylinx Micrograd gel combines an extreme gel range (6 to 48% polyacrylamide) with a novel crosslinker to provide sieving of polypeptides, and pore-limit electrophoresis of the smallest proteins (e.g. insulin monomer). All gel ranges (such as 3 to 30%) provide zone sharpening in routine analysis of conventional protein mixtures (e.g. serum) within 10 min electrophoresis at 200 to 300 volts. The gels are thin (1 mm) and thus stain quickly, but the gel cassette is of conventional overall width (83 mm), thus fitting many apparatus designs and accommodating 12 samples. The gels are finding valuable use in screening applications, requiring the electrophoretic analysis of many samples, and in cases where a rapid answer is needed, such as monitoring protein purification. The gels have proved particularly useful, in-house, for the latter application in developing Gradipore's new large-scale preparative electrophoresis system, the Gradiflow. PMID:1599958

Wrigley, C W; Margolis, J

1992-01-01

130

Analysis of Dictyostelium discoideum Inositol Pyrophosphate Metabolism by Gel Electrophoresis  

PubMed Central

The social amoeba Dictyostelium discoideum was instrumental in the discovery and early characterization of inositol pyrophosphates, a class of molecules possessing highly-energetic pyrophosphate bonds. Inositol pyrophosphates regulate diverse biological processes and are attracting attention due to their ability to control energy metabolism and insulin signalling. However, inositol pyrophosphate research has been hampered by the lack of simple experimental procedures to study them. The recent development of polyacrylamide gel electrophoresis (PAGE) and simple staining to resolve and detect inositol pyrophosphate species has opened new investigative possibilities. This technology is now commonly applied to study in vitro enzymatic reactions. Here we employ PAGE technology to characterize the D. discoideum inositol pyrophosphate metabolism. Surprisingly, only three major bands are detectable after resolving acidic extract on PAGE. We have demonstrated that these three bands correspond to inositol hexakisphosphate (IP6 or Phytic acid) and its derivative inositol pyrophosphates, IP7 and IP8. Biochemical analyses and genetic evidence were used to establish the genuine inositol phosphate nature of these bands. We also identified IP9 in D. discoideum cells, a molecule so far detected only from in vitro biochemical reactions. Furthermore, we discovered that this amoeba possesses three different inositol pentakisphosphates (IP5) isomers, which are largely metabolised to inositol pyrophosphates. Comparison of PAGE with traditional Sax-HPLC revealed an underestimation of the cellular abundance of inositol pyrophosphates by traditional methods. In fact our study revealed much higher levels of inositol pyrophosphates in D. discoideum in the vegetative state than previously detected. A three-fold increase in IP8 was observed during development of D. discoideum a value lower that previously reported. Analysis of inositol pyrophosphate metabolism using ip6k null amoeba revealed the absence of developmentally-induced synthesis of inositol pyrophosphates, suggesting that the alternative class of enzyme responsible for pyrophosphate synthesis, PP-IP5K, doesn’t’ play a major role in the IP8 developmental increase.

Pisani, Francesca; Livermore, Thomas; Rose, Giuseppina; Chubb, Jonathan Robert; Gaspari, Marco; Saiardi, Adolfo

2014-01-01

131

A versatile polyacrylamide gel electrophoresis based sulfotransferase assay  

PubMed Central

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

2010-01-01

132

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

PubMed Central

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

Serwer, Philip; Wright, Elena T.

2012-01-01

133

Efficient Quantitative Information Extraction from PCR-RFLP Gel Electrophoresis Images  

Microsoft Academic Search

For the purpose of PCR-RFLP analysis, as in the case of human papillomavirus (HPV) typing, quantitative information needs to be extracted from images resulting from one-dimensional gel electrophoresis by associating the image intensity with the concentration of biological material at the corresponding position on a gel matrix. However, the background intensity of the image stands in the way of quantifying

Christos Maramis; Anastasios Delopoulos

2010-01-01

134

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

SciTech Connect

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

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

1990-11-01

135

Lysine 58-cleaved ?2-microglobulin is not detectable by 2D electrophoresis in ex vivo amyloid fibrils of two patients affected by dialysis-related amyloidosis  

PubMed Central

The lysine 58 cleaved and truncated variant of ?2-microglobulin (?K58-?2m) is conformationally unstable and present in the circulation of a large percentage of patients on chronic hemodialysis, suggesting that it could play a role in the ?2-microglobulin (?2m) amyloid fibrillogenesis associated with dialysis-related amyloidosis (DRA). However, it has yet to be detected in the amyloid deposits of such patients. Here, we extracted amyloid fibrils, without denaturation or additional purification, from different amyloidotic tissues of two unrelated individuals suffering from DRA, and characterized them by high-sensitivity bidimensional gel electrophoresis (2D-PAGE), immunoblotting, MALDI time-of-flight mass spectrometry, and protein sequencing. To confirm whether or not this species could be identified by our proteomic approaches, we mapped its location in 2D-PAGE, in mixtures of pure ?K58-?2m, and extracts of amyloid fibrils from patients, to a discrete region of the gel distinct from other isoforms of ?2m. Using this approach, the two known principal isoforms found in ?2m amyloid were identified, namely, the full-length protein and the truncated species lacking six N-terminal amino acid residues (?N6-?2m). In contrast, we found no evidence for the presence of ?K58-?2m.

Giorgetti, Sofia; Stoppini, Monica; Tennent, Glenys A.; Relini, Annalisa; Marchese, Loredana; Raimondi, Sara; Monti, Maria; Marini, Sara; ?stergaard, Ole; Heegaard, Niels H.H.; Pucci, Piero; Esposito, Gennaro; Merlini, Giampaolo; Bellotti, Vittorio

2007-01-01

136

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

PubMed

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

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

2011-01-01

137

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

PubMed Central

A simple method for complete genome radiolabelling is described, involving long-wave UV exposure of agarose-embedded chromosomal DNA and [?-32P]dCTP incorporation mediated by the Klenow fragment. Experiments on the budding yeast genome show that the labelling procedure can be coupled with two new two-dimensional pulsed field gel electrophoresis (2D-PFGE) protocols of genome analysis: (i) the KARD (karyotype and restriction display)-PFGE which provides a complete view of the fragments resulting from a single restriction of the whole genome and (ii) the DDIC (double digestion of isolated chromosome)-PFGE which is the eukaryotic counterpart of complete/complete 2D-PFGE in bacterial genomics.

Brugere, Jean-Francois; Cornillot, Emmanuel; Metenier, Guy; Vivares, Christian P.

2000-01-01

138

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

PubMed

A simple method for complete genome radiolabelling is described, involving long-wave UV exposure of agarose-embedded chromosomal DNA and [alpha-(32)P]dCTP incorporation mediated by the Klenow fragment. Experiments on the budding yeast genome show that the labelling procedure can be coupled with two new two-dimensional pulsed field gel electrophoresis (2D-PFGE) protocols of genome analysis: (i) the KARD (karyotype and restriction display)-PFGE which provides a complete view of the fragments resulting from a single restriction of the whole genome and (ii) the DDIC (double digestion of isolated chromosome)-PFGE which is the eukaryotic counterpart of complete/complete 2D-PFGE in bacterial genomics. PMID:10773096

Brugère, J F; Cornillot, E; Méténier, G; Vivarès, C P

2000-05-15

139

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

PubMed

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

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

2014-06-01

140

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

PubMed

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

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

2013-01-01

141

Multiple Vibrio vulnificus strains in oysters as demonstrated by clamped homogeneous electric field gel electrophoresis.  

PubMed Central

Clamped homogeneous electric field gel electrophoresis and a computer program for managing electrophoresis banding patterns (ELBAMAP) were used to analyze genomic DNA of 118 Vibrio vulnificus strains, isolated from three oysters by direct plating. Analysis with SfiI resulted in 60 restriction endonuclease digestion profiles (REDP), while analysis with SrfI produced 53 different REDP. Similarities between REDP ranged from 7 to 93%. Principal-component analysis showed that the strains were heterogeneous.

Buchrieser, C; Gangar, V V; Murphree, R L; Tamplin, M L; Kaspar, C W

1995-01-01

142

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.

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

2010-01-01

143

Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots  

DOEpatents

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

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

1989-01-01

144

Identification of species of fish by gradient-gel electrophoresis.  

PubMed

An electrophoretic method is described for distinguishing between fish fillets according to their protein composition. Thaw fluid (4 microL) was applied to one of 14 sample positions of a precast gel, containing a gradient of polyacrylamide of either 2.5 to 27% or 3 to 40%. All reagents and gels are commercially available in ready-to-use form. Either gel provided a distinction between any of the 42 fish types, but the 3 to 40% gel gave better identification because of its superior molecular-sieving properties. Reproducible electrophoretic patterns were obtained for different samples of the same fish type, but small differences were shown for fish of widely different origin, for example Australian and New Zealand ling. PMID:3834902

Manusu, H P; Wrigley, C W

1985-11-01

145

Quantitation and characterization of rat tissue metallothioneins by gel electrophoresis  

SciTech Connect

A discontinuous gradient gel electrophoretic system was developed to quantitate and characterize metallothionein (MT) in rat tissue. Vertical slab separating gels (1.5 mm x 14 cm x 12 cm) consisted of a linear polyacrylamide gradient 7.5 to 30% T and 5% Bis. The stacking gels (3% T and 20% Bis) were photopolymerized using riboflavin as the catalyst. Liver cytosols were prepared from rats which received (i.p.) various amounts of Zn (5 mg/kg BW) or Cd (2.5 mg/kg BW). Purified MT was prepared by gel filtration and DEAE ion-exchange chromatography. Cytosols were heated (80/sup 0/C, 2 min) and centrifuged to obtain a supernatant. An appropriate amount of supernatant and various amounts of MT standard were electrophoresed (constant current, 20 mA per slab) for 9 hours. Gels were stained with Commassie Blue (R-250, 0.25%) for 12 hours and destained. Gels were scanned by densitometer and peaks heights were determined. Significantly linear standard curves (..mu..g MT vs. peak height) were established for both MTI and MTII. (Cd, Zn)-MTI migrated slower than Zn-MTI while mobilities for both (Cd, Zn)- and Zn-MTII were the same. The accumulation of MTI was consistently less than MTII in liver from both Zn- and Cd-injected rats. Their results suggest that electrophoretic analysis is an excellent system not only for quantitation but also for characterization of MT in rat tissue.

Lin, L.Y.; McCormick, C.C.

1986-03-05

146

Protamine extraction and analysis of human sperm protamine 1/protamine 2 ratio using Acid gel electrophoresis.  

PubMed

Protamines, sperm-specific nuclear proteins, are essential for sperm chromatin condensation and DNA stabilization. They are small, highly basic, and rich in disulfide bonds. Under reducing conditions, protamines, along with other basic proteins, are soluble in acid solutions. Because of their small and similar molecular weights, SDS-PAGE cannot resolve protamine 1 and protamine 2 well. Urea-acid gel electrophoresis separates proteins based on the level of the positive charge and is thus a suitable method for resolving protamines 1 and 2. Here, we describe the commonly used protamine extraction method and the Urea-acid gel electrophoresis for assessment of protamine 1/protamine 2 ratio. PMID:22992935

Liu, Lihua; Aston, Kenneth I; Carrell, Douglas T

2013-01-01

147

Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots  

DOEpatents

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.

1987-09-04

148

Tilapia (Oreochromis mossambicus) allergens characterized by ELISA, SDS-PAGE, 2D gels, Western blotting and MALDI-TOF mass spectrometry.  

PubMed

Parvalbumins have long been identi?ed as major allergens in fish, but our research has found that parvalbumins are not the main cause of allergic reactions to tilapia. After homogenization, proteins were extracted from freshwater tilapia to react with a pooled sera sample taken from patients (n = 20) tested to be allergic to tilapia. Enzyme-linked immunosorbent assay revealed immunoglobulin E antibody activity, followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis, Western blot, 2D electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to carry out protein separation and analysis. Protein identification against different databases yielded three known high molecular weight proteins as tilapia allergens: chromosome undetermined SCAF7145, fructose-bisphosphate aldolase A and enolase 3 (beta muscle). A fourth, new, unidentified protein with two T-cell receptors was discovered. PMID:21939426

Liu, Rong; Yang, Eleanor; Liu, Chuyi; Xue, Wentong

2012-05-01

149

Fluorophore-Labeled Primers Improve the Sensitivity, Versatility, and Normalization of Denaturing Gradient Gel Electrophoresis  

Microsoft Academic Search

Denaturing gradient gel electrophoresis (DGGE) is widely used in microbial ecology. We tested the effect of fluorophore-labeled primers on DGGE band migration, sensitivity, and normalization. The fluorophores Cy5 and Cy3 did not visibly alter DGGE fingerprints; however, 6-carboxyfluorescein retarded band migration. Fluorophore modification improved the sensitivity of DGGE fingerprint detection and facilitated normaliza- tion of samples from multiple gels by

Josh D. Neufeld; William W. Mohn

2005-01-01

150

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

NASA Astrophysics Data System (ADS)

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

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

2007-04-01

151

Preparative Gel Electrophoresis at High Sample Load. The Effect of Some Experimental Variables on Separation Performance  

Microsoft Academic Search

The separation of model protein pairs (hemoglobin\\/ albumin, trypsin\\/chymotrypsin, hemoglobin A\\/hemoglobin F) was studied in an apparatus for preparative gel electrophoresis at loads up to 40 mg\\/cm of the cross-sectional area of the gel bed. Separation was favored by higher ionic strength and by longer migration path. Under the conditions used and within the load range studied, increasing total protein

S. A. Saeed; T. R. C. Boyde

1980-01-01

152

Gel electrophoresis and diffusion of ring-shaped DNA  

NASA Astrophysics Data System (ADS)

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

Alon, Uri; Mukamel, David

1997-02-01

153

TGGE-STAR: PCR-primer design and melting analysis. Optimization of PCR-Gradient-Gel-Electrophoresis  

Microsoft Academic Search

Abstract PCR combined with temperature or denaturing gradient gel electrophoresis (TGGE) or (DGGE) are rapid and very sensitive screening method for point mutations. Computer aided design of PCR primers for denaturing gradient gel electrophoresis and the careful choice of a suitable gradient are the most important factors to guarantee the success of the screening. The program TGGE- STAR was written

Christoph Gille; Andreas Gille

154

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

PubMed Central

The gel electrophoresis mobility shift assay is widely used for qualitative and quantitative characterization of protein complexes with nucleic acids. Often it is found that complexes that are short-lived in free solution (t1/2 of the order of minutes) persist for hours under the conditions of gel electrophoresis. We have investigated the influence of polyacrylamide gels on the pseudo first-order dissociation kinetics of complexes containing the E.coli cyclic AMP receptor protein (CAP) and lactose promoter DNA. Within the gel matrix, kdiss decreased with increasing [polyacrylamide] and the order of the reaction was changed. In free solution, kdiss was proportional to [DNA]2, while in 5% gels, kdiss was proportional to [DNA]0.3. In gels of [polyacrylamide] > or = 10%, kdiss was nearly independent of [DNA] until fragment concentrations exceeded 0.1 microM. Even in the absence of competing DNA, kdiss(gel) < kdiss(solution). These results suggest that the lifetime of CAP-DNA complexes in free solution is limited by their encounter frequency with molecules of DNA or with protein-DNA complexes; some or all of the stabilization observed in gels may be due to a reduction in this frequency. Images

Fried, M G; Liu, G

1994-01-01

155

Higher resolution microplate array diagonal gel electrophoresis: application to a multiallelic minisatellite.  

PubMed

The 5' polymorphic region of the insulin (INS, MIM# 176730) gene contains a variable tandem repetition of 14-15 bp (a variable number of tandem repeats (VNTR) locus). After PCR amplification, we achieved precise sizing of class I alleles (range 641 to 843 bp) on 96-well open-face polyacrylamide microplate array diagonal gel electrophoresis (MADGE) gels, obtaining resolution of the 2% mobility difference which represents one tandem repeat. PCR products were run double-stranded, but no additional bands were generated except in the case of differences of three, two, and one repeat between alleles; none compromised allele identification, and in the latter case the heteroduplex was a useful confirmation signal. No end labelling of primers was required, as the sensitive Vistra Green intercalating dye for double strands was used for visualization of bands from diluted samples. Duracryl, a high mechanical-strength polyacrylamide derivative, proved to have good resolution properties for electrophoresis. A co-run ladder ensured precise binning without inter-lane variability. Simultaneous electrophoresis of gels in a thermostatically controlled tank allowed up to 1,000 samples to be run in 90 min. Gels were analyzed using a FluorImager 595 fluorescent scanning system, and alleles identified using a combination of Phoretix software for band migration measurement and Microsoft Excel to compute allele sizes. Unlike other systems for minisatellite allele sizing, throughput was not limited (in time or cost) by electrophoresis. PMID:10862086

O'Dell, S D; Chen, X; Day, I N

2000-01-01

156

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

PubMed Central

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

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

2003-01-01

157

Characterization of Carbohydrates Using Highly Fluorescent 2-Aminobenzoic Acid Tag Following Gel Electrophoresis of Glycoproteins  

Microsoft Academic Search

Application of the most sensitive fluorescent label 2-aminobenzoic acid (anthranilic acid, AA) for characterization of carbohydrates from the glycoproteins (?15 pmol) separated by polyacrylamide gel electrophoresis is described. AA label is used for the determination of both monosaccharide composition and oligosaccharide map. For the monosaccharide determination, bands containing the glycoprotein of interest are excised from the polyvinylidene fluoride (PVDF) membrane

Kalyan R. Anumula; Ping Du

1999-01-01

158

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

PubMed

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

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

2013-07-01

159

DETECTION OF GENETICALLY MODIFIED MAIZE BY PCR AND CAPILLARY GEL ELECTROPHORESIS (CGE) USING UNCOATED COLUMNS  

Microsoft Academic Search

In this work, analysis of genetically modified insect- resistant Bt maize is demostrated by combining amplification of a DNA fragment by PCR and subsequent detection by Capillary Gel Electrophoresis (CGE). A new CGE method is developed that allows obtaining reproducible separations of DNA fragments using bare fused silica capillaries. The method combines a washing routine of the column with 0.1

Virginia García-Cañas; Ramón González; Alejandro Cifuentes

160

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

Microsoft Academic Search

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

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

2003-01-01

161

Analysis of PCR-RFLP gel electrophoresis images for accurate and automated HPV typing  

Microsoft Academic Search

The identification of the types of the human papilomavirus (HPV) that have infected a woman provides valuable information as regards to her risk for developing cervical cancer. HPV typing is often performed by means of manually analyzing PCR-RFLP gel electrophoresis images. However, the typing procedure that is currently employed suffers from unsatisfactory accuracy and high time consumption. In order to

Christos F. Maramis; Anastasios N. Delopoulos; Alexandros F. Lambropoulos

2010-01-01

162

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

Microsoft Academic Search

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

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

2008-01-01

163

Comparison of gradient gel electrophoresis and zonal ultracentrifugation for quantitation of high density lipoproteins  

Microsoft Academic Search

The study was conducted to compare gradient gel electrophoresis (GGE) and zonal ultracentrifugation for quanti- tation of human plasma high density lipoproteins (HDL). Plasma samples were obtained from seven normal subjects consuming a high fat diet (65% total calories) followed by a high carbohydrate diet (65% total calories). HDL were fractionated into HDL2 and HDLS by zonal ultracentrifugation and lipid

Constance A. McNerney; Moti L. Kashyap; Roger L. Barnhart

164

Genomic Analysis of Clostridium botulinum Group II by Pulsed-Field Gel Electrophoresis  

Microsoft Academic Search

Pulsed-field gel electrophoresis (PFGE) was optimized for genomic analyses of Clostridium botulinum (non- proteolytic) group II. DNA degradation problems caused by extracellular DNases were overcome by fixation of cells with formaldehyde prior to isolation. A rapid (4-h) in situ DNA isolation method was also assessed and gave indistinguishable results. Genomic DNA from 21 strains of various geographical and temporal origins

SEBASTIAN HIELM; JOHANNA BJORKROTH; EIJA HYYTIA; HANNU KORKEALA

1998-01-01

165

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

NASA Astrophysics Data System (ADS)

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

Amirkhanian, Varoujan; Tsai, Shou-Kuan

2014-03-01

166

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

ERIC Educational Resources Information Center

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

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

2006-01-01

167

Single cell gel electrophoresis assay: a new technique for human biomonitoring studies  

Microsoft Academic Search

Human biomonitoring using the single cell gel electrophoresis (SCGE) or comet assay is a novel approach for the assessment of genetic damage in exposed populations. This assay enables the detection of various forms of DNA damage in individual cells with ease and speed and is, therefore, well suited to the analysis of a large group in a population. Here, application

Fekadu Kassie; Wolfram Parzefall; Siegfried Knasmüller

2000-01-01

168

Comparison of Restriction Enzymes for Pulsed-Field Gel Electrophoresis Typing of Moraxella catarrhalis  

PubMed Central

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

Puig, Carmen; Domenech, Arnau; Linares, Josefina; Ardanuy, Carmen

2013-01-01

169

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

PubMed

An analytical approach providing an insight into speciation of iodine in water insoluble fraction of edible seaweed (Nori) was developed. The seaweed, harvested in the Galician coast (Northwestern Spain), contained 67.7±1.3?gg(-1) iodine of which 25% was water soluble and could be identifies as iodide. Extraction conditions of water insoluble residue using urea, NaOH, SDS and Triton X-100 were investigated. The protein pellets obtained in optimized conditions (after precipitation of urea extracts with acetone), were digested with trypsin and protease XIV. Size exclusion chromatography-ICP-MS of both enzymatic digests demonstrated the occurrence of iodoaminoacids putatively present in proteins. Intact proteins could be separated by gel electrophoresis after an additional extraction of the protein extract with phenol. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) with laser ablation ICP-MS detection of (127)I indicated the presence of iodine in protein bands corresponding to molecular masses of 110kDa, 40kDa, 27kDa, 20kDa and 10kDa. 2D IEF-SDS PAGE with laser ablation ICP-MS (127)I imaging allowed the detection of 5 iodine containing protein spots in the alkaline pI range. PMID:24913873

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

2014-09-01

170

A technique for detecting antifungal activity of proteins separated by polyacrylamide gel electrophoresis.  

PubMed

A technique was developed for the detection of antifungal activity of proteins after discontinuous polyacrylamide gel electrophoresis under native conditions. The antifungal activity is detected as growth inhibition zones in a homogeneous fungal lawn, grown in an agar layer spread on top of the polyacrylamide gel. The position of proteins with antifungal activity can be determined on a diffusion blot prepared from the same gel. The technique is illustrated for three antifungal plant proteins, i.e. alpha-purothionin, Urtica dioica agglutinin, and tobacco chitinase. PMID:1889394

De Bolle, M F; Goderis, I J; Terras, F R; Cammue, B P; Broekaert, W F

1991-06-01

171

Model and computer simulations of the motion of DNA molecules during pulse field gel electrophoresis  

SciTech Connect

A model is presented for the motion of individual molecules of DNA undergoing pulse field gel electrophoresis (PFGE). The molecule is represented by a chain of charged beads connected by entropic springs, and the gel is represented by a segmented tube surrounding the beads. This model differs from earlier reptation/tube models in that the tube is allowed to leak in certain places and the chain can double over and flow out of the side of the tube in kinks. It is found that these kinks often lead to the formation of U shapes, which are a major source of retardation in PFGE. The results of computer simulations using this model are compared with real DNA experimental results for the following cases: steady field motion as seen in fluorescence microscopy, mobility in steady fields, mobility in transverse field alternation gel electrophoresis (TFAGE), mobility in field inversion gel electrophoresis (FIGE), and linear dichroism (LD) of DNA in agarose gels during PFGE. Good agreement between the simulations and the experimental results is obtained.

Smith, S.B.; Bustamante, C. (Univ. of Oregon, Eugene (United States)); Heller, C. (Univ. Konstanz (West Germany))

1991-05-28

172

Using in situ rheology to characterize the microstructure in photopolymerized polyacrylamide gels for DNA electrophoresis.  

PubMed

Photopolymerized cross-linked polyacrylamide hydrogels are attractive sieving matrix formulations for DNA electrophoresis owing to their rapid polymerization times and the potential to locally tailor the gel pore structure through spatial variation of illumination intensity. This capability is especially important in microfluidic systems, where photopolymerization allows gel matrices to be precisely positioned within complex microchannel networks. Separation performance is also directly related to the nanoscale gel pore structure, which is in turn strongly influenced by polymerization kinetics. Unfortunately, detailed studies of the interplay among polymerization kinetics, mechanical properties, and structural morphology are lacking in photopolymerized hydrogel systems. In this paper, we address this issue by performing a series of in situ dynamic small-amplitude oscillatory shear measurements during photopolymerization of cross-linked polyacrylamide electrophoresis gels to investigate the relationship between rheology and parameters associated with the gelation environment including UV intensity, monomer and cross-linker composition, and reaction temperature. In general, we find that the storage modulus G' increases with increasing initial monomer concentration, cross-linker concentration, and polymerization temperature. The steady-state value of G', however, exhibits a more complex dependence on UV intensity that varies with gel concentration. A simple model based on rubber elasticity theory is used to obtain estimates of the average gel pore size that are in surprisingly good agreement with corresponding data obtained from analysis of DNA electrophoretic mobility in gels cast under identical polymerization conditions. PMID:16892481

Wang, Jian; Ugaz, Victor M

2006-09-01

173

Fractionation of SWNT/nucleic acid complexes by agarose gel electrophoresis  

NASA Astrophysics Data System (ADS)

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

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

2006-08-01

174

New cellular automaton designed to simulate geometration in gel electrophoresis  

NASA Astrophysics Data System (ADS)

We propose a new kind of cellular automaton to simulate transportation of molecules of DNA through agarose gel. Two processes are taken into account: reptation at strong electric field E, described in the particle model, and geometration, i.e. subsequent hookings and releases of long molecules at and from gel fibres. The automaton rules are deterministic and they are designed to describe both processes within one unified approach. Thermal fluctuations are not taken into account. The number of simultaneous hookings is limited by the molecule length. The features of the automaton are: (i) the size of the cell neighbourhood for the automaton rule varies dynamically, from nearest neighbors to the entire molecule; (ii) the length of the time step is determined at each step according to dynamic rules. Calculations are made up to N=244 reptons in a molecule. Two subsequent stages of the motion are found. Firstly, an initial set of random configurations of molecules is transformed into a more ordered phase, where most molecules are elongated along the applied field direction. After some transient time, the mobility ? reaches a constant value. Then, it varies with N as 1/ N for long molecules. The band dispersion varies with time t approximately as Nt1/2. Our results indicate that the well-known plateau of the mobility ? vs. N does not hold at large electric fields.

Krawczyk, M. J.; Ku?akowski, K.; Maksymowicz, A. Z.

2002-08-01

175

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

PubMed Central

Background Small dense LDL is reported to be associated with increased coronary artery disease risk by various epidemiological studies. The gold standard for separation and identification of LDL subtypes in plasma is ultracentrifugation which is a lengthy procedure and difficult to perform. Various other methods like NMR, HPLC, gradient gel electrophoresis (GGE) have been reported for LDL sub fractionation all of which require specialized equipments and expertise. We report here a high throughput 3% polyacrylamide slab gel electrophoresis method (PASGE) for sub fractionation of LDL which was compared with GGE, a commonly used method for LDL sub fractionation. Results The 3% PASGE method compared well with the GGE method There was a good correlation between LDL particle diameter identified by the PASGE and GGE (Pearson correlation coefficient = 0.950). A 100% concordance was found when samples were classified as per LDL phenotypes in subjects with A and B phenotype by the two methods with the concordance being 66% in subjects with intermediate (I) phenotype. The electrophoresis apparatus was optimized and designed for running twenty eight samples at a time compared to twelve to fourteen by the conventional PASGE and eight to twelve by disc electrophoresis. Conclusion The rapid 3% polyacrylamide slab gel electrphoresis method developed is simple to perform, cost-effective and can be used for the identification LDL sub fractionation and phenotyping in large epidemiological studies.

Singh, Yogendra; Lakshmy, Ramakrishnan; Gupta, Ruby; Kranthi, Vemparala

2008-01-01

176

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

PubMed

Peanut allergy is triggered by several proteins known as allergens. In this study, the complexity the peanut allergome is investigated with proteomic tools. The strength of this investigation resides in combining the high-resolving power and reproducibility of fluorescence two-dimensional differential gel electrophoresis with specific immunological detection as well as polypeptide sequencing by high-resolution mass spectrometry. Matching of the peanut proteins in 2D gels was achieved by differential labelling whereby peanut proteins and purified allergens (Ara h 1, Ara h 2 or Ara h 3/4) were run on the same gel. Ten protein spots on a mass line of ca. 63-68 kDa were likely to correspond to Ara h 1. Two doublets on two different mass lines at ca. 16 and 18 kDa matched with purified allergen Ara h 2. The basic and acidic sub-units of Ara h 3/4 were observed at masses of ca. 25 kDa and 40-45 kDa, respectively. Subsequently the antibody-binding capacity of spots corresponding to peanut allergens was investigated by Western blotting of 2D gels using antibodies (IgY) raised against Ara h 1, Ara h 2 and the recombinant 40 kDa sub-unit of Ara h 3/4. Final confirmation of the identity of the protein spots matched after 2D electrophoresis and identified by Western blotting was obtained by in-gel digestion of protein spots and analysis by quadrupole time-of-flight mass spectrometry. By using the method developed in our work, the location and identification of two different isoforms of the allergen Ara h 1, the allergen Ara h 2 and six isoforms of the allergen Ara h 3/4 in 2D peanut protein maps was established. PMID:19223023

Chassaigne, Hubert; Trégoat, Virginie; Nørgaard, Jørgen V; Maleki, Soheila J; van Hengel, Arjon J

2009-04-13

177

Evaluation of total protein content in tears of dogs by polyacrylamide gel disk electrophoresis.  

PubMed

Concentration of total proteins was measured and sodium dodecyl sulfate-polyacrylamide gel disk electrophoresis was performed on tear and plasma samples obtained from 26 healthy dogs, and the results were compared. Mean +/- SEM concentration of total proteins in tears was 0.63 +/- 0.04 g/dl, and significant effects of age or gender were not found. The protein composition of tears in dogs was complex, and bands from light and heavy chains of immunoglobulins were identified by electrophoresis. PMID:1586012

Barrera, R; Jiménez, A; López, R; Mañé, M C; Rodríguez, J F; Molleda, J M

1992-04-01

178

Molecular Mechanisms of Reversible and Irreversible Trapping of >Large DNA Molecules Undergoing Gel Electrophoresis  

NASA Astrophysics Data System (ADS)

Megabase-size DNA becomes trapped in agarose gels during electrophoresis if the electric field is greater than 2 V/cm. Fluorescence microscopy reveals that megabase molecules invariably arrest during the U-shape phase of their caterpillar cycle, adhering to the gel near the vertex of the U. The electric field dependence of the molecular sizes trapped in the gel has been determined and indicates a critical force above which molecules trap. The size of unligated ?-ladders sheared during electrophoresis at a given field is the same as the size of molecules trapped at that field and suggests that both processes occur through nick melting near the vertex of the U. Consistently, molecules nicked by exposure to UV radiation trapped more readily than un-exposed ones. To further characterize the nature of the molecule-gel interaction near the vertex, the electric force on tethered DNA molecules within a gel has been measured using laser tweezers, yielding an effective charge of 0.16 e^-/phos. This figure translates into a critical tension at the vertex of 15 pN, a force sufficient to melt nicks bent around corners in the gel path near the vertex, and to trap a molecule.

Bustamante, Carlos

1998-05-01

179

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

PubMed

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

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

2004-08-01

180

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

PubMed Central

Polymerase chain reaction (PCR) and gel electrophoresis have become common techniques used in undergraduate molecular and cell biology labs. Although students enjoy learning these techniques, they often cannot fully comprehend and analyze the outcomes of their experiments because of a disconnect between concepts taught in lecture and experiments done in lab. Here we report the development and implementation of novel exercises that integrate the biological concepts of DNA structure and replication with the techniques of PCR and gel electrophoresis. Learning goals were defined based on concepts taught throughout the cell biology lab course and learning objectives specific to the PCR and gel electrophoresis lab. Exercises developed to promote critical thinking and target the underlying concepts of PCR, primer design, gel analysis, and troubleshooting were incorporated into an existing lab unit based on the detection of genetically modified organisms. Evaluative assessments for each exercise were aligned with the learning goals and used to measure student learning achievements. Our analysis found that the exercises were effective in enhancing student understanding of these concepts as shown by student performance across all learning goals. The new materials were particularly helpful in acquiring relevant knowledge, fostering critical-thinking skills, and uncovering prevalent misconceptions.

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

2008-01-01

181

Ixodes ricinus: the potential of two-dimensional gel electrophoresis as a tool for studying host-vector-pathogen interactions.  

PubMed

Ixodes ricinus is a three-host tick, with three active instars. For moulting to occur the tick has to find a host where it can take a blood meal. Throughout feeding I. ricinus can be infected or infect the host with different pathogens, e.g., Tick-Borne Encephalitis virus or Borrelia burgdorferi. The host-vector-pathogen interaction is very complex, making a detailed study difficult. Here we analyse the potential of two-dimensional gel electrophoresis (2DE) to study the host-vector-pathogen interaction. We examined 20 nymphs, which as larvae parasitised either mouse or hen. After moulting, they were kept alive for up to 30 weeks, to analyse whether tick ageing influenced host determination, and for comparison of the 2D-gels. Even though the number of proteins in the gel decreased during ageing, some proteins of the host determination persisted for all 30 weeks. We also discovered persisting proteins in relation to nymphs. These findings showed that 2DE is suitable as a tool for studying host-vector-pathogen interactions. PMID:16904668

Vennestrøm, J; Jensen, P M

2007-01-01

182

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

PubMed

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

Zhu, Xiaoming; Mason, Thomas G

2014-08-15

183

DNA Electrophoresis: how partially denatured DNA stops moving in a gel.  

NASA Astrophysics Data System (ADS)

During temperature gradient gel electrophoresis (TGGE), a DNA strand travels in a gel with a temperature gradient. As the strand travels in a position of higher temperature, the stability of the double helix is reduced resulting in melted domains. It has been experimentaly observed that in a gel, a partialy melted DNA strand exhibits a steep reduction in mobility ---perhaps even trapping. The sequence dependent melting of DNA can therefore be translated into a sequence dependent position at which the strand appears to stop. Thus, this can be used as an effective method for discriminating between strands that differ only in composition. However, the dominant blocking mechanisms remain unclear. Blocking/trapping events are re-created using Langevin dynamics simulations using the ESPResSo package to understand the physics behind the observed steep reduction in electrophoretic mobility. From these simulations a relation between gel parameters and the mobility of the strand is proposed.

Sean, David; Slater, Gary

2009-03-01

184

[Two-dimensional gel electrophoresis analysis of moderately halophilic bacterium Halobacillus dabanensis D-8T under hypoosmotic shock condition].  

PubMed

Halobacillus dabanensis D-8T was isolated from the saline deposits of Daban lake in Xinjiang of China, and is able to grow in complex medium containing 0.5% to 25% salt. To figure out the survival mechanisms of Gram-positive moderately halophilic bacteria under hypoosmotic shock conditions, two-dimensional gel electrophoresis (2-DE) was carried out to investigate differential protein expression profiles of H. dabanensis D-8T in response to low osmotic challenge. The 2-D gels were stored as dry gels and their images were taken by ImageScanner and analyzed by Imagemaster 2D Platinum software. About 650 protein spots were detected in 2-D gel. Most of proteins were distributed in molecular mass of 17.5 - 66kDa and the range of isoelectric point 4.0 - 5.9. A total of 34 protein spots were found to alter their expression after strain D-8T was subjected to hypoosmotic shock from 20% to 0% salinity for 5 min and 50 min. Among them, the expression of 20 protein spots is up-regulated including 6 new protein spots, while that of 14 protein spots is down-regulated in answer to sudden osmotic down-shift. Protein spots of interest were excised from the gels and digested by trypsin. By means of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) and MASCOT search engine, 4 up-regulated protein spots were identified with peptide mass fingerprint, and are similar to heat shock protein DanK, rod shape-determining protein, penicillin-binding protein (PBP-1A) and 5-enolpyruvoylshikimate-3-phosphate synthase, respectively. Noticeably, PBP-1A firstly was up-regulated after shock of 5 min but disappeared after shock of 50 min. This indicated that the strain activate a minor mechanism of peptidoglycan synthesis to compensate the major synthesis mechanism for cells survival through a down-shift challenge. In addition, this paper was the first report that heat shock proteins were up-regulated in response to sudden osmotic down-shift. PMID:17172020

Feng, De-Qin; Xie, Li-Shi; Li, Xiao-Hong; Yang, Su-Sheng

2006-10-01

185

Wheat germ lectin affinity electrophoresis of serum alkaline phosphatase with commercially available agarose gels.  

PubMed

We have modified a commercially available procedure involving precast agarose gels (Paragon Isopal System) to measure alkaline phosphatase (EC 3.1.3.1) isoenzymes. Including wheat germ lectin in the equilibration buffer improved the resolution of the bone and liver isoenzymes. Unlike previously described wheat germ lectin affinity electrophoresis methods, the procedure measures bone, liver, and biliary isoenzymes in a single step. There was good correlation between the affinity electrophoresis and the neuraminidase preincubation methods for the measurement of bone (r = 0.958) and liver (r = 0.962) alkaline phosphatase isoenzymes. However, the affinity electrophoresis method also quantified minor isoenzyme fractions that were poorly resolved by the neuraminidase method. The method is technically simple, reproducible, and capable of rapid handling of large workloads. PMID:8330397

Mattiazzo, M; Ramasamy, I

1993-07-01

186

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

PubMed

A simple non-gel sieving capillary electrophoresis (NGSCE) method was established to determine the MW of silk fibroin using CE. The background electrolyte with a pH of 8.8 was based on three components: polyethylene glycol, tris(hydroxymethyl)aminomethane, and sodium dodecyl sulfate (SDS). NGSCE showed a good linear relationship with satisfactory reproducibility between the migration time and the MW of standard proteins. It was found that the regenerated silk fibroin had an MW around 83 kDa with a wide MW distribution (MWD). This absolute value is lower than the result obtained from SDS-polyacrylamide gel electrophoresis due to the different principles of the methods, but their similar MWD shapes indicated that NGSCE could be a feasible, highly sensitive, rapid method for determination of the MW of silk fibroin. PMID:20922945

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

2010-01-01

187

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

NASA Astrophysics Data System (ADS)

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

Krawczyk, M. J.; Pasciak, P.; Dydejczyk, A.; Kulakowski, K.; Dulak, J.

2005-05-01

188

Southern elephant seal (Mirounga leonina)II. Studies of milk protein fractions by gel electrophoresis  

Microsoft Academic Search

Milk protein fractions during various stages of lactation in the southern elephant seal Mirounga leonina were analysed. Twelve milk samples were taken from ten females throughout the lactation period during 1990 and 1991 at Stranger\\u000a Point, King George Island, South Shetland Islands. Milk samples were subjected to polyacrylamide gel electrophoresis (PAGE).\\u000a Samples from different days of lactation gave similar qualitative

P. A. Ronayne de Ferrer; R. A. Gonzalez Colaso; M. E. I. Marquez; A. R. Carlini; D. F. Vergani; G. A. Daneri

1996-01-01

189

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.

1991-07-01

190

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

1991-01-01

191

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

Microsoft Academic Search

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

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

1988-01-01

192

A Computerized Methodology for Improved Virus Typing by PCR-RFLP Gel Electrophoresis  

Microsoft Academic Search

The analysis of digitized images from polymerase chain reaction-restriction fragment length polymorphism (PCR- RFLP) gel electrophoresis examinations is a popular method for virus typing, i.e., for identifying the virus type(s) that have in- fected an investigated biological sample. However, being mostly manual, the conventional virus typing protocol remains laborious, time consuming, and error prone. In order to overcome these short-

Christos F. Maramis; Anastasios N. Delopoulos; Alexandros F. Lambropoulos

2011-01-01

193

A system for automatic HPV typing via PCR-RFLP gel electrophoresis  

Microsoft Academic Search

The identification of the types of the human papil- lomavirus (HPV) that have infected a female patient provides valuable information as regards to her risk for developing cervical cancer. A widely used method for performing the above task (namely HPV typing) is PCR-RFLP gel electrophoresis. However, the conventional HPV typing protocol is error-prone and resource-ineffective due to lack of interaction

Christos F. Maramis; Anastasios N. Delopoulos; Alexandros F. Lambropoulos; Sokratis P. Katafigiotis

2011-01-01

194

Molecular profiling of Bacteroides spp. in human feces by PCR-temperature gradient gel electrophoresis  

Microsoft Academic Search

A group-specific PCR-based temperature gradient gel electrophoresis (TGGE) method was developed to study the population composition of genus Bacteroides in human gut. Highly reproducible and well-separated bands in TGGE fingerprints of ten unrelated human fecal samples showed complex and host-specific Bacteroides species composition. Dynamic monitoring over 22 months of samples from one healthy 10-year-old boy indicated a relatively stable population

Xiaoyan Pang; Dezhong Ding; Guifang Wei; Meiling Zhang; Linghua Wang; Liping Zhao

2005-01-01

195

Application of difference gel electrophoresis to the identification of inner medullary collecting duct proteins  

Microsoft Academic Search

In this study, we present a standardized approach to purification of native inner medullary collecting duct (IMCD) cells from rat kidney for proteomic analysis and apply the approach to identification of abundant proteins utilizing two-dimensional difference gel electrophoresis (DIGE) coupled with matrix-assisted laser desorption-ionization-time of flight mass spectrometry. Fractionation of inner medullary cell suspensions by low-speed centrifugation gave a highly

J. D. Hoffert; B. W. M. van Balkom; C. L. Chou; M. A. Knepper

2004-01-01

196

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

Microsoft Academic Search

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

Philippe Simoneau; Jacques Labarère

1988-01-01

197

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

Microsoft Academic Search

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

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

2005-01-01

198

Diversity of Proteolytic Clostridium botulinum Strains, Determined by a Pulsed-Field Gel Electrophoresis Approach  

Microsoft Academic Search

Pulsed-field gel electrophoresis (PFGE) was applied to the study of the similarity of 55 strains of proteolytic Clostridium botulinum (C. botulinum group I) types A, AB, B, and F. Rare-cutting restriction enzymes ApaI, AscI, MluI, NruI, PmeI, RsrII, SacII, SmaI, and XhoI were tested for their suitability for the cleavage of DNA of five proteolytic C. botulinum strains. Of these

Mari Nevas; Miia Lindstrom; Sebastian Hielm; K. Johanna Bjorkroth; Michael W. Peck; Hannu Korkeala

2005-01-01

199

Optimization of coomassie staining for quantitative densitometry of soybean storage proteins in gradient gel electrophoresis  

Microsoft Academic Search

Soybean (Glycine maxL. Merr., cv. Dare) protein subunits were separated by gradient gel electrophoresis and analyzed by two-dimensional densitometry\\u000a with computer-aided volume integration. Significant differences in the time required to achieve equilibrium staining with\\u000a Coomassie Blue were revealed among the various polypeptides. Bands corresponding to lipoxygenase reached staining equilibrium\\u000a in 2.7 h, whereas longer periods were required for polypeptides of

Prachuab Kwanyuen; Richard F. Wilson

2000-01-01

200

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

Microsoft Academic Search

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

C. Li; W. M. Moe

2004-01-01

201

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

Microsoft Academic Search

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

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

1966-01-01

202

Improved agarose gel electrophoresis method and molecular mass calculation for high molecular mass hyaluronan.  

PubMed

The molecular mass of the polysaccharide hyaluronan (HA) is an important determinant of its biological activity and physicochemical properties. One method currently used for the analysis of the molecular mass distribution of an HA sample is gel electrophoresis. In the current work, an improved agarose gel electrophoresis method for analysis of high molecular mass HA is presented and validated. HA mobility in 0.5% agarose minigels was found to be linearly related to the logarithm of molecular mass in the range from approximately 200 to 6000 kDa. A sample load of 2.5 ?g for polydisperse HA samples was employed. Densitometric scanning of stained gels allowed analysis of the range of molecular masses present in the sample as well as calculation of weight-average and number-average values. The method was validated for a polydisperse HA sample with a weight-average molecular mass of approximately 2000 kDa. Excellent agreement was found between the weight-average molecular mass determined by electrophoresis and that determined by rheological measurement of the solution viscosity. The revised method was then used to show that heating solutions of HA at 100°C, followed by various cooling procedures, had no effect on the HA molecular mass distribution. PMID:21683677

Cowman, Mary K; Chen, Cherry C; Pandya, Monika; Yuan, Han; Ramkishun, Dianne; LoBello, Jaclyn; Bhilocha, Shardul; Russell-Puleri, Sparkle; Skendaj, Eraldi; Mijovic, Jovan; Jing, Wei

2011-10-01

203

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

SciTech Connect

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

McGregor, D.A.

1993-07-01

204

Determination of association constants between 5'-guanosine monophosphate gel and aromatic compounds by capillary electrophoresis.  

PubMed

Hydro gel formed by 5'-guanosine monophosphate (GMP) in the presence of a potassium ion is expected to exhibit interesting selectivity in capillary electrophoretic separations. Here, we estimated the conditional association constants between the hydro gel (G-gel) and aromatic compounds by capillary electrophoresis in order to investigate the separation selectivity that is induced by the G-gel. Several aromatic compounds were separated in a solution containing GMP and potassium ion at different concentrations. The association constants were calculated by correlating the electrophoretic mobilities of the analytes obtained experimentally using a concentration of G-gel. During semi-quantitative estimation, naphthalene derivatives had larger association constants (Kass=10.3-16.8) compared with those of benzene derivatives (Kass=3.91-5.31), which means that the binding sites of G-gel match better to a naphthalene ring than to a benzene ring. A hydrophobic interaction was also found when the association constants for alkyl resorcinol were compared with those of different hydrocarbon chains. The association constants of nucleobases and tryptophan ranged from 6.05 to 12.6, which approximated the intermediate values between benzene and naphthalene derivatives. Consequently, the selective interaction between G-gel and aromatic compounds was classified as one of three types: (1) an intercalation into stacked planar GMP tetramers; (2) a hydrophobic interaction with a long alkyl chain; or, (3) a small contribution of steric hindrance and/or hydrogen bonding with functional groups such as amino and hydroxyl groups. PMID:23522259

Yamaguchi, Kaori; Takeyasu, Nobuyuki; Kaneta, Takashi

2013-05-01

205

Prototype for integrated two-dimensional gel electrophoresis for protein separation.  

PubMed

Two-dimensional gel electrophoresis practitioners have long waited for a fully automated system. This article presents an integrated platform that is capable of complete automation from sample introduction to spots detection. The strip gel for the first dimensional separation is fixed on the edge of a discrete planar stage before separation. A pair of platinum pin electrodes for isoelectric focusing (IEF) makes contact from underneath the stage. IEF is performed directly after rehydration and protein loading. After the first dimensional separation, sodium dodecyl sulfate (SDS) equilibration is done on the same stage without moving the gel. The IEF stage is then moved horizontally to couple with a precast second dimensional gel. The <0.5 mm gap between the two gels is filled with poly (ethylene oxide) solution. After SDS-polyacrylamide gel electrohporesis separation, a charge-coupled device camera is used to detect spots via protein native fluorescence excited by a Hg (Xe) lamp with the gel inside the running cell. Potential for full automation is demonstrated with 0.5 microg of Escherichia coli proteins on this miniaturized platform. More than 240 spots are detected in a total experiment time of <2.5 h. PMID:16130711

Xu, Aoshuang; Sluszny, Chanan; Yeung, Edward S

2005-09-16

206

The Use of Wool Fast Blue Bl for the Electrophoresis of Human Parotid Saliva Proteins in Acrylamide Gel.  

National Technical Information Service (NTIS)

Wool Fast Blue BL was examined as a stain to delineate the protein components separated by the acrylamide gel electrophoresis of parotid saliva. The tests were performed on samples of parotid saliva collected from young male naval personnel by stimulation...

T. S. Meyer B. L. Lamberts

1968-01-01

207

Ribosomal Ribonucleic Acids of Cultured Cells: a Preliminary Survey of Differences among Mammalian Species Detectable by Polyacrylamide Gel Electrophoresis.  

National Technical Information Service (NTIS)

Ribosomal ribonucleic acids (rRNA's) of cultured cells from various species were compared by polyacrylamide gel electrophoresis. Electrophoretic mobility of the 28 S RNA component varied according to species. Human cell 28 S rRNA was distinguishable from ...

M. E. Soergel F. L. Schaffer

1972-01-01

208

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

NASA Astrophysics Data System (ADS)

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

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

2008-11-01

209

A Sol-Gel-Modified Poly(methyl methacrylate) Electrophoresis Microchip with a Hydrophilic Channel Wall  

SciTech Connect

A sol-gel method was employed to fabricate a poly(methyl methacrylate) (PMMA) electrophoresis microchip that contains a hydrophilic channel wall. To fabricate such a device, tetraethoxysilane (TEOS) was injected into the PMMA channel and was allowed to diffuse into the surface layer for 24 h. After removing the excess TEOS, the channel was filled with an acidic solution for 3 h. Subsequently, the channel was flushed with water and was pretreated in an oven to obtain a sol-gel-modified PMMA microchip. The water contact angle for the sol-gel-modified PMMA was 27.4° compared with 66.3° for the pure PMMA. In addition, the electro-osmotic flow increased from 2.13×10-4 cm2 V-1 s-1 for the native-PMMA channel to 4.86×10-4 cm2 V-1 s-1 for the modified one. The analytical performance of the sol-gel-modified PMMA microchip was demonstrated for the electrophoretic separation of several purines, coupled with amperometric detection. The separation efficiency of uric acid increased to 74 882.3 m-1 compared with 14 730.5 m-1 for native-PMMA microchips. The result of this simple modification is a significant improvement in the performance of PMMA for microchip electrophoresis and microfluidic applications.

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

2007-07-27

210

Characterisation of soil-bound residue fractions of the fungicide dithianon by gel permeation chromatography and polyacrylamide gel electrophoresis.  

PubMed

The degradation of the (14)C-labelled fungicide dithianon in an orthic luvisol was investigated under standardized conditions in comparison to stimulated microbial activity by an amendment of maize straw. The compound is characterized by mineralization losses of approximately 33% and the formation of non-extractable bound residues of approximately 63% in 64 days. Despite the major role of microorganisms in mineralizing this compound, the formation of bound residues is not biotically induced. Gel permeation chromatography and polyacrylamide gel electrophoresis, as different size separation techniques of the humic acids fractions, showed differences in the distribution patterns of non-extractable residues depending on the addition of straw material. The results presented support the existence of humic substances in soil as a micellar system rather than as a biopolymer. PMID:15092966

Wanner, U; Burauel, P; Führ, F

2000-04-01

211

Routine diagnosis with PhastSystem compared to conventional electrophoresis: automated sodium dodecyl sulfate-polyacrylamide gel electrophoresis, silver staining and western blotting of urinary proteins.  

PubMed

The recent introduction of the PhastSystem, an automatic electrophoresis and staining system with precast gradient-gels, allows rapid and reproducible analysis of proteinuria in patients suffering from renal injury. A routine method for sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis (SDS-PAGE) and silver staining of unconcentrated urine specimens in the PhastSystem is described and compared to our conventional "macro"-method with self-cast SDS-polyacrylamide gradient gels. The method described for the PhastSystem using 0.3 microL sample volumes and an 8-25% polyacrylamide gradient gel leads to highly reproducible results within 1.5 h. Before electrophoresis urine specimens were neither concentrated nor dialyzed. Samples with a protein concentration exceeding 5 mg/mL had to be diluted 1:5 (v/v). Analysis and documentation of PhastGels appeared as easy as with our conventional SDS-PAGE. Protein bands could reliably be identified by Western blotting. Urine and serum proteins, separated in PhastGels, were electrophoretically transferred to nitrocellulose and detected with specific antibodies against human albumin, transferrin, alpha-1-antitrypsin and IgG. Comparison of several standard kits for molecular weight determination revealed considerable differences concerning the quality of protein separation patterns. Availability of precast gels and automatization of SDS-PAGE and staining allows easy standardization of urine SDS-PAGE among clinical routine laboratories. PMID:2469571

Scherberich, J E; Fischer, P; Bigalke, A; Stangl, P; Wolf, G B; Haimerl, M; Schoeppe, W

1989-01-01

212

Gel electrophoresis of DNA partially denatured at the ends: what are the dominant conformations?  

PubMed

Gel electrophoresis of a partially denatured dsDNA fragment is studied using Langevin Dynamics computer simulations. For simplicity, the denatured ssDNA sections are placed at the ends of the fragment in a symmetrical fashion. A squid-like conformation is found to sometimes cause the fragment to completely block in the gel. In fact, this conformation is the principal cause of the steep reduction in mobility observed in the simulations. As the field is increased, it is found that the occurrence of this conformation dominates the migration dynamics. Although the squid conformation seems to be more stable at high fields, the field can eventually force the fragments to thread through the gel pores regardless. We qualitatively explore the behavior of this squid-like conformation across a range of fields and degrees of denaturation, and we discuss the relevance of our findings for TGGE. PMID:23280692

Sean, David; Slater, Gary W

2013-03-01

213

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

PubMed

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

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

2013-01-01

214

Differentially regulated proteins in Prevotella intermedia after oxidative stress analyzed by 2D electrophoresis and mass spectrometry.  

PubMed

Prevotella intermedia is a rod-shaped, Gram-negative anaerobic bacterium found in human indigenous microbiota that plays an important role in opportunistic infections. The successful colonization depends on the ability of anaerobes to respond to oxidative stress (OS) in oxygenated tissues as well as to resist oxidative events from the host immune system until anaerobic conditions are present at the infection site. As knowledge of the mechanisms of protection against OS in Prevotella is limited, studies are needed to clarify aspects of molecular biology, physiology and ecology of this bacterium. The aim of this study was to access the proteins differentially regulated in P. intermedia after exposure to molecular oxygen by using two-dimensional gel electrophoresis (2DE) associated with the approach of MALDI-TOF/TOF Tandem Mass Spectrometry. The identity of the protein was evaluated by database search for homologous genomic sequences of P. intermedia strain 17 (TIGR). Twenty five out of 72 proteins found were identified as up-regulated (17) or down-regulated (9). These proteins were related to a variety of metabolic process, some of which could be associated to antioxidant and redox regulatory roles. Our data indicate that OS may stimulate an adaptive response in P. intermedia whose effect on its biology may be evidenced by the increase in aerotolerance and changes in protein abundance in the oxygen adapted cells. PMID:22193554

Santos, Simone G; Diniz, Cláudio G; Silva, Vânia L; Lima, Francisca L; Andrade, Hélida M; Chapeaurouge, Donat A; Perales, Jonas; Serufo, José Carlos; Carvalho, Maria Auxiliadora R; Farias, Luiz M

2012-02-01

215

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))

1990-03-01

216

High-throughput separation of DNA and proteins by three-dimensional geometry gel electrophoresis: feasibility studies.  

PubMed

We report a novel separation method that is applicable to both DNA and protein samples, based on electrophoresis in a three-dimensional (3-D) geometry. In contrast to conventional electrophoresis, samples are applied in a two-dimensional, planar array to one of the surfaces of a 3-D geometry separation medium. Loading onto a plane results in a very high sample capacity. Sample migration and separation occur along the third spatial dimension, which is perpendicular to the loading plane. The key problem of electrophoresis in a 3-D geometry separation setup is that temperature gradients are caused by Joule's heat, affecting the electrical conductivity and viscosity of the separation medium. A means of achieving straight sample migration under these circumstances is to force heat flow through the separation medium parallel to the axis of sample migration. This can be done by dissipating the heat via the electrode sides of the gel and blocking any other heat transfer. The separation of DNA and proteins by this method has been tested using agarose gel electrophoresis, polyacrylamide gel electrophoresis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Data were acquired off-line by conventional staining methods as well as on-line by detection of laser-induced fluorescence. We describe how to excise samples from the separation medium for preparative purposes. Possible unique applications of this 3-D geometry electrophoresis separation method are also discussed. PMID:14679562

Ventzki, Robert; Stegemann, Josef

2003-12-01

217

Two-dimensional (2D) infrared (IR) correlation spectroscopy for dynamic absorption behavior of oleic acid (OA) onto silica gel  

NASA Astrophysics Data System (ADS)

Dynamic absorption behavior of oleic acid (OA) onto silica gel was probed by infrared (IR) spectroscopy. Once OA is injected into silica gel placed on a horizontal attenuated total reflectance prism, the silica gel starts to absorb the OA molecules due to the molecular-level interaction based on hydrogen bonding between the COOH of OA and the OH of silica gel. The substantial level of variation of spectral feature is readily observed during the absorption of OA onto silica gel. 2D correlation analysis of the time-dependent IR spectra reveals fine details of absorption dynamics of OA molecules depending on the molecular structure. The predominant absorption of the monomers occurs at the onset of the absorption, and it is then quickly followed by the decrease in the dimers. In other words, the dissociation of the liquid crystals occurs via the disuniting of the tightly packed OA dimers.

Genkawa, Takuma; Kanematsu, Wataru; Shinzawa, Hideyuki

2014-07-01

218

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

PubMed Central

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

Wastling, Jonathan; Hunt, Ela

2003-01-01

219

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.

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

2013-01-01

220

Robust classification of DNA damage patterns in single cell gel electrophoresis.  

PubMed

Single cell gel electrophoresis, also known as comet assay, has been widely used for assessing the effect of genotoxicity and detecting DNA damage of individual eukaryotic cells. There exist established imaging techniques for cometassay analysis, but these platforms have limitations such as required user interventions, low throughput, and weakness to noise caused by incomplete dyeing of fluorescent materials and other experimental errors. To resolve these, we propose a novel procedure for analyzing comet assay images, which considers various DNA damage patterns and classifies them in a robust manner. We tested our approach with twenty golden data sets containing over 300 comets and achieved satisfactory classification accuracy. PMID:24110525

Lee, Taehoon; Lee, Sungmin; Sim, Woo Young; Jung, Yu Mi; Han, Sunmi; Chung, Chanil; Chang, Jay Junkeun; Min, Hyeyoung; Yoon, Sungroh

2013-01-01

221

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

PubMed

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

2014-06-15

222

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))

1993-04-01

223

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

NASA Technical Reports Server (NTRS)

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

Zieboll, Gerhard; Orgel, Leslie E.

1994-01-01

224

Detection of mutations in GC-rich DNA by bisulphite denaturing gradient gel electrophoresis.  

PubMed Central

Denaturing gradient gel electrophoresis (DGGE) in combination with PCR and 'GC-clamping' has proven highly efficient as a method for detection of DNA sequence differences. Due to strand dissociation phenomena, however, its use has been limited to the analysis of sequences with a relatively low content of GC pairs. This paper describes how treatment of template DNA with sodium bisulphite drastically lowers the melting temperature of very GC-rich sequences and renders them amenable to DGGE analysis. We demonstrate the use of bisulphite DGGE for rapid and efficient detection of mutations in the p16(INK4/CDKN2) tumour suppressor gene.

Guldberg, P; Gr?nbak, K; Aggerholm, A; Platz, A; thor Straten, P; Ahrenkiel, V; Hokland, P; Zeuthen, J

1998-01-01

225

Characterization of the Legionella anisa population structure by pulsed-field gel electrophoresis.  

PubMed

We analysed 38 French isolates of Legionella anisa by means of pulsed-field gel electrophoresis (PFGE) with single or double digestion. Double digestion was more discriminatory than single digestion, and can thus be useful for epidemiological studies of L. anisa. Several isolates from different parts of France clustered together on the basis of their PFGE patterns (similarity cutoff of 80%), suggesting that the L. anisa population structure is homogenous or that a few clones of L. anisa strains have spread widely in France. PMID:16640574

Akermi, Mongi; Doleans, Anne; Forey, Françoise; Reyrolle, Monique; Meugnier, Helene; Freney, Jean; Vandenesch, François; Etienne, Jerome; Jarraud, Sophie

2006-05-01

226

Quantitation of pyrimidine dimer contents of nonradioactive deoxyribonucleic acid by electrophoresis in alkaline agarose gels  

SciTech Connect

We have developed a method of quantitating the pyrimidine dimer content of nonradioactive DNAs. DNA samples are treated with the UV-endonuclease from Micrococcus luteus and then separated according to molecular weight by electrophoresis on alkaline agarose gels. From their migration relative to known molecular weight standards, their median molecular weight and thus the number of dimers per DNA molecule in each sample can be calculated. Results of action spectra for dimer formation in T7 bacteriophage measured by this method agree well with action spectra for T7 killing. In addition, the method gives dimer yields in good agreement with those obtained by others using alkaline sucrose gradient sedimentation.

Sutherland, B.M.; Shih, A.G.

1983-02-15

227

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

PubMed

Gel electrophoresis of fluorophore labeled saccharides provides a rapid and reliable method to screen enzymatic and/or chemical treatments of polysaccharides and glycoconjugates, as well as a sensitive and efficient microscale method to separate and purify oligosaccharides for further analysis. A simple and inexpensive method of derivatization and analysis using 2-aminobenzoic acid (anthranilic acid, AA) is described and applied to the extracellular polysaccharide released by the desiccation tolerant cyanobacterium Nostoc commune DRH-1. The results of these analyses suggest a possible protective functionality of two pendent groups, as well as a potential relationship between these groups and the desiccation tolerance of the organism. PMID:11005578

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

2000-08-18

228

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

PubMed

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

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

2004-04-01

229

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

PubMed

A protocol was established for two-dimensional gel electrophoresis (2-DE) of barley seed and malt proteins in the pH range of 6-11. Proteins extracted from flour in a low-salt buffer were focused after cup-loading onto IPG strips. Successful separation in the second dimension was achieved using gradient gels in a horizontal SDS-PAGE system. Silver staining of gels visualized around 380 (seed) and 500 (malt) spots. Thirty-seven different proteins from seeds were identified in 60 spots, among these 46 were visualized also in the malt 2-D pattern. Proteins were identified by peptide mass fingerprinting and by tandem MS sequencing after in-gel digestion by trypsin. In addition, the N-terminal sequence of 10 different proteins from 11 spots was determined after electroblotting to a polyvinylidene difluoride (PVDF) membrane. Five identified proteins (in 9 spots) are involved in glycolysis, 12 in defence against pathogens (21 spots), 4 in storage, folding, and synthesis of proteins, and in nitrogen metabolism (5 spots), 6 in carbohydrate metabolism (11 spots), and 4 in stress and detoxification (9 spots). Six proteins (7 spots) were not grouped in these categories, and 3 were not ascribed a function. The presented 2-D patterns and identifications will be used to describe proteome differences between cultivars and changes during malting. PMID:14997495

Bak-Jensen, Kristian Sass; Laugesen, Sabrina; Roepstorff, Peter; Svensson, Birte

2004-03-01

230

Miconazole oral gel increases exposure to oral oxycodone by inhibition of CYP2D6 and CYP3A4.  

PubMed

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 (AUC(0-?); 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

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

2011-03-01

231

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

PubMed

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

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

2011-07-01

232

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

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

1980-01-01

233

Studies on the bioactivity of radioiodinated highly purified bovine thyrotropin: analytical polyacrylamide gel electrophoresis  

SciTech Connect

Highly purified bovine TSH (stored in solution at -70 C) was radioiodinated by the stoichiometric chloroamine-T method. The iodinated material ws subjected to analytical polyacrylamide disc gel electrophoresis. TSH was eluted from gel slices (1 mm width) and was analyzed for radioactivity and bioactivity. The latter was determined using the cultured thyroid cell cAMP response assay. Radioactivity in the TSH preparation migrated separately from bioactivity, but concordant with the protein bands observed in gels run in parallel. Further studies performed on bovine TSH purified in our laboratory, as well as on a different TSH preparation of exceptionally high potency (both stored as lyophilized powder) revealed a different pattern, with TSH bioactivity and radioactivity eluting concurrently. Iodination of TSH did not alter its electrophoretic migration on disc gel electrophoresis. In all preparations polymorphism of TSH bioactivity was observed, with at least four separate protein bands containing TSH bioactivity being present in our preparation. The relationship between the degree of iodination and retention of TSH bioactivity was examined. Incorporation of /sup 125/I into TSH was greatly different at two different concentrations of chloramine-T. Despite this, however, the progressive loss of TSH bioactivity was similar at both concentrations, indicating that incorporation of iodine into the TSH molecule is not itself responsible for the decrease in bioactivity. These studies indicate variability among different TSH preparations in terms of their retention of bioactivity. Significant loss of TSH bioactivity appears to occur during storage in solution. The damage to the biological activity of TSH during the iodination procedure is more likely related to the oxidation process than to the incorporation of iodine.

Takai, N.A.; Filetti, S.; Rapoport, B.

1981-01-01

234

HP-Lattice QSAR for dynein proteins: experimental proteomics (2D-electrophoresis, mass spectrometry) and theoretic study of a Leishmania infantum sequence.  

PubMed

The toxicity and inefficacy of actual organic drugs against Leishmaniosis justify research projects to find new molecular targets in Leishmania species including Leishmania infantum (L. infantum) and Leishmaniamajor (L. major), both important pathogens. In this sense, quantitative structure-activity relationship (QSAR) methods, which are very useful in Bioorganic and Medicinal Chemistry to discover small-sized drugs, may help to identify not only new drugs but also new drug targets, if we apply them to proteins. Dyneins are important proteins of these parasites governing fundamental processes such as cilia and flagella motion, nuclear migration, organization of the mitotic splinde, and chromosome separation during mitosis. However, despite the interest for them as potential drug targets, so far there has been no report whatsoever on dyneins with QSAR techniques. To the best of our knowledge, we report here the first QSAR for dynein proteins. We used as input the Spectral Moments of a Markov matrix associated to the HP-Lattice Network of the protein sequence. The data contain 411 protein sequences of different species selected by ClustalX to develop a QSAR that correctly discriminates on average between 92.75% and 92.51% of dyneins and other proteins in four different train and cross-validation datasets. We also report a combined experimental and theoretic study of a new dynein sequence in order to illustrate the utility of the model to search for potential drug targets with a practical example. First, we carried out a 2D-electrophoresis analysis of L. infantum biological samples. Next, we excised from 2D-E gels one spot of interest belonging to an unknown protein or protein fragment in the region M<20,200 and pI<4. We used MASCOT search engine to find proteins in the L. major data base with the highest similarity score to the MS of the protein isolated from L. infantum. We used the QSAR model to predict the new sequence as dynein with probability of 99.99% without relying upon alignment. In order to confirm the previous function annotation we predicted the sequences as dynein with BLAST and the omniBLAST tools (96% alignment similarity to dyneins of other species). Using this combined strategy, we have successfully identified L. infantum protein containing dynein heavy chain, and illustrated the potential use of the QSAR model as a complement to alignment tools. PMID:18662882

Dea-Ayuela, María Auxiliadora; Pérez-Castillo, Yunierkis; Meneses-Marcel, Alfredo; Ubeira, Florencio M; Bolas-Fernández, Francisco; Chou, Kuo-Chen; González-Díaz, Humberto

2008-08-15

235

Real-time detection of allele-specific polymerase chain reaction products by automated ultra-thin-layer agarose gel electrophoresis  

Microsoft Academic Search

Ultra-thin-layer agarose gel electrophoresis, a novel combination of agarose slab gel electrophoresis and capillary gel electrophoresis was introduced in conjunction with laser-induced fluorescence (LIF) scanning detection for the analysis of polymerase chain reaction (PCR) products. Allele-specific fragments, amplified from genomic DNA of patients with congenital adrenal hyperplasia (most often caused by mutations of 21-hydroxylase gene, CYP-21), were used as a

András Guttman; Csaba Barta; Melinda Szöke; Mária Sasvári-Székely; Huba Kalász

1998-01-01

236

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.

2010-03-01

237

Human liver alkaline phosphatase purified by affinity chromatography, ultracentrifugation and polyacrylamide-gel electrophoresis.  

PubMed Central

A method is presented for the preparation of human liver alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1). The method gives a purification factor of 12.5 X 10(3) over the initial aq. butan-1-ol extract, a recovery of 6.0% and a specific activity for the preparation of 1450-1550 units/mg of protein, 1 unit being defined as the amount of enzyme catalysing the hydrolysis of 1mumol of p-nitrophenyl phosphate/min at 35 degrees C in 0.1 M-2-amino-2-methylpropan-1-ol/HCl buffer, pH 10.5, containing 10mM-p-nitrophenyl phosphate. Homogeneity was studied by ultracentrifugation, by immunoelectrophoresis and by polyacrylamide-gel electrophoresis. A single contaminating protein was present which was less than 5% of the total. Ultracentrifugation and equilibrium-gradient-pore electrophoresis techniques indicated a mol.wt. of 156000 and 160000 respectively. Equilibrium-gradient-pore electrophoresis indicated that the alkaline phosphatase molecule is possibly a dimer, comprising two subunits of about 80000 mol.wt. Amino acid analysis proved remarkably similar to that for alkaline phosphatase from other sources, regardless of species. Images PLATE 1

Latner, A L; Hodson, A W

1976-01-01

238

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

PubMed Central

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

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

2012-01-01

239

A novel universal primer-multiplex-PCR method with sequencing gel electrophoresis analysis.  

PubMed

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

Xu, Wentao; Zhai, Zhifang; Huang, Kunlun; Zhang, Nan; Yuan, Yanfang; Shang, Ying; Luo, Yunbo

2012-01-01

240

Secreted proteins of human monocytes. Analysis by two-dimensional gel electrophoresis and effect of lipopolysaccharide.  

PubMed

A monocyte-rich preparation from the adherent cell fraction of human peripheral blood leukocytes was incubated for 1-8 h with [35S]methionine or [3H]leucine in the presence and absence of bacterial lipopolysaccharide (LPS). The macromolecules released into the supernatant were analysed by two-dimensional gel electrophoresis and radioautography. A complex labelling pattern involving at least 20 easily demonstrable and apparently distinct products with a broad range of molecular masses and isoelectric points was observed. LPS or LPS plus actinomycin in combination markedly stimulated the labelling and release of at least twelve different macromolecules ranging in apparent Mr from 12,000 to 46,000. Studies with monocytes that had been additionally purified by centrifugal elutriation and with the monocyte-like human cell line U-937 indicated that monocytes rather than contaminating cells were the source of these products. The majority of the secreted products were unique and did not cross-react with antibodies to interleukin 1 or tumour necrosis factor. The high resolving capacity of two-dimensional gel electrophoresis may be useful to define further the diverse biological activities and potential monokines released from monocytes at various stages of their differentiation and activation. PMID:3257692

Panuska, J R; Fukui, K; Parker, C W

1988-01-15

241

Secreted proteins of human monocytes. Analysis by two-dimensional gel electrophoresis and effect of lipopolysaccharide.  

PubMed Central

A monocyte-rich preparation from the adherent cell fraction of human peripheral blood leukocytes was incubated for 1-8 h with [35S]methionine or [3H]leucine in the presence and absence of bacterial lipopolysaccharide (LPS). The macromolecules released into the supernatant were analysed by two-dimensional gel electrophoresis and radioautography. A complex labelling pattern involving at least 20 easily demonstrable and apparently distinct products with a broad range of molecular masses and isoelectric points was observed. LPS or LPS plus actinomycin in combination markedly stimulated the labelling and release of at least twelve different macromolecules ranging in apparent Mr from 12,000 to 46,000. Studies with monocytes that had been additionally purified by centrifugal elutriation and with the monocyte-like human cell line U-937 indicated that monocytes rather than contaminating cells were the source of these products. The majority of the secreted products were unique and did not cross-react with antibodies to interleukin 1 or tumour necrosis factor. The high resolving capacity of two-dimensional gel electrophoresis may be useful to define further the diverse biological activities and potential monokines released from monocytes at various stages of their differentiation and activation. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. Fig. 6. Fig. 7.

Panuska, J R; Fukui, K; Parker, C W

1988-01-01

242

Conformational Entropy Mechanism for Periodic Motion of DNA under Constant-Field Gel Electrophoresis  

NASA Astrophysics Data System (ADS)

Entropic elasticity of a single charged polymer undergoing gel electrophoresis is a fundamental theme of polymer statistical physics since the discovery of “periodic” behavior in constant field gel electrophoresis (CFGE). In the present work we address the problem numerically by two steps. In the first step, we carry out Brownian dynamics (BD) simulations on CFGE by solving semi-microscopic Langevin equations of a polymer consisting of beads separated by a mean distance much smaller than the Kuhn length. Results are analyzed based on coarse-graining over the Kuhn length scale. We show the averaged elongation-contraction motion involves asymmetric V-shaped configurations whose shorter arm length depends on the field and the temperature consistently with what is expected when the BD chain is described by the freely-jointed chain (FJC) model with a suitable Kuhn length. To our knowledge, this is the first numerical confirmation of the FJC model itself from a submicroscopic description of polymer motion. The saturation of chain mobility in high fields agrees well with the nonlinear dependence of this shorter arm length on the field. In the second step, we discuss the periodic elongation-contraction motion of the coarse-grained chain by such a simplified model as a one-dimensional chain consisting of beads, elastic strings, and obstacles. The results from these two chain models indicate that the periodic elongation-contraction motion of DNA under CFGE is self-organized by a balance between the field force and the conformational entropic force.

Azuma, Ryuzo; Takayama, Hajime

2006-06-01

243

The impact of two-dimensional pulsed-field gel electrophoresis techniques for the consistent and complete mapping of bacterial genomes: refined physical map of Pseudomonas aeruginosa PAO.  

PubMed

The SpeI/DpnI map of the 5.9 Mb Pseudomonas aeruginosa PAO (DSM 1707) genome was refined by two-dimensional (2D) pulsed-field gel electrophoresis techniques (PFGE) which allow the complete and consistent physical mapping of any bacterial genome of interest. Single restriction digests were repetitively separated by PFGE employing different pulse times and ramps in order to detect all bands with optimum resolution. Fragment order was evaluated from the pattern of 2D PFGE gels: 1. Partial-complete digestion. A partial restriction digest was separated in the first dimension, redigested to completion, and subsequently perpendicularly resolved in the second dimension. 2D-gel comparisons of the ethidium bromide stain of all fragments and of the autoradiogram of end-labeled partial digestion fragments was nearly sufficient for the construction of the macrorestriction map. 2. Reciprocal gels. A complete restriction digest with enzyme A was run in the first dimension, redigested with enzyme B, and separated in the second orthogonal direction. The order of restriction digests was reverse on the second gel. In case of two rare-cutters, fragments were visualized by ethidium bromide staining or hybridization with genomic DNA. If a frequent and a rare cutter were employed, linking fragments were identified by end-labeling of the first digest. 3. A few small fragments were isolated by preparative PFGE and used as a probe for Southern analysis.--38 SpeI and 15 DpnI fragments were positioned on the map. The zero point was relocated to the 'origin of replication'. The anonymous mapping techniques described herein are unbiased by repetitive DNA, unclonable genomic regions, unfavourable location of restriction sites, or cloning artifacts as frequently encountered in other top-down or bottom-up approaches. PMID:1905802

Römling, U; Tümmler, B

1991-06-25

244

Precision and reliability of paraprotein determinations by high-resolution agarose gel electrophoresis.  

PubMed

Agarose gel electrophoresis has recently replaced cellulose acetate electrophoresis as the preferred technique for monitoring paraprotein levels in patients with plasma cell dyscrasias. The authors studied the accuracy and precision of this method for paraprotein determination. Twenty-seven serum samples with paraprotein concentrations ranging from 5 to 73 g/L were aliquotted and assayed on 20 separate occasions, and the mean and standard deviation for the paraprotein concentration in each serum was established. Linear regression analysis showed that the standard deviation of paraprotein concentration (SD) increased as a function of paraprotein concentration (PC). For IgG paraproteins, the regression equation was SD = 0.041 (PC) + 1.06; R = 0.942; standard error = 0.32. For non-IgG paraproteins the equation was SD = 0.101 (PC) - 0.04; R = 0.851; standard error = 0.5. The accuracy of paraprotein determinations by the agarose gel electrophoretic technique was assessed by comparison with values obtained with the use of a previously validated enzyme-linked immunosorbent assay (ELISA) method for quantitation of IgG subclasses. Results obtained by the two methods were similar and highly correlated: (concentration by electrophoresis) = 0.921 (concentration by ELISA) + 0.46; R = 0.988; standard error = 0.34. The laser densitometric scanning procedure showed a loss of linearity above 60 g/L, indicating the need to dilute sera with very high paraprotein concentrations in order to obtain accurate results. A table is presented that should help pathologists who interpret such scans to determine whether small changes in paraprotein measurements occurring over time represent true changes in paraprotein concentration or merely reflect the analytic variability inherent in the technique. PMID:2929498

Stemerman, D; Papadea, C; Martino-Saltzman, D; O'Connell, A C; Demaline, B; Austin, G E

1989-04-01

245

Accurate evaluation of the sizes of DNA fragments (from 30 to 4700 kb) in pulse field gel electrophoresis.  

PubMed

Construction of long-range genomic maps by pulse field gel electrophoresis requires optimum resolution of large DNA fragments. Using the transverse-alternating field electrophoresis system, we describe a method to accurately evaluate the sizes of fragments generated by rare-cutter digestions within the 30-4700-kb range. A protocol generating large (greater than 1000 kb) molecules by partial digestion is also reported. PMID:1667081

Crété, N; Delabar, J M; Sinet, P M; Créau-Goldberg, N

1991-12-01

246

PCR-Denaturing Gradient Gel Electrophoresis of Complex Microbial Communities: A Two-Step Approach to Address the Effect of Gel-to-Gel Variation and Allow Valid Comparisons Across a Large Dataset  

Microsoft Academic Search

Denaturing gradient gel electrophoresis (DGGE) is widely used in microbial ecology to profile complex microbial communities\\u000a over time and in response to different stimuli. However, inherent gel-to-gel variability has always been a barrier toward\\u000a meaningful interpretation of DGGE profiles obtained from multiple gels. To address this problem, we developed a two-step methodology\\u000a to align DGGE profiles across a large dataset.

Panagiotis Tourlomousis; E. Katherine Kemsley; Karyn P. Ridgway; Michael J. Toscano; Thomas J. Humphrey; Arjan Narbad

2010-01-01

247

Characterization of selenium incorporation into wheat proteins by two-dimensional gel electrophoresis-laser ablation ICP MS followed by capillary HPLC-ICP MS and electrospray linear trap quadrupole Orbitrap MS.  

PubMed

A method has been developed for a rapid and precise location of selenium-containing proteins in large two-dimensional (2D) electrophoresis gels. A sample was divided into four aliquots which were analyzed in parallel by 1D isoelectric focusing electrophoresis (IEF)-laser ablation (LA) inductively coupled plasma mass spectrometry (ICP MS), 1D sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE)-LA ICP MS, and, in duplicate, by 2D IEF-PAGE. On the basis of the 1 D electropherograms obtained, areas supposed to contain the largest concentrations of Se were subjected to LA ICP MS imaging to locate precisely the position of Se-containing proteins which were then identified in the parallel 2D gel by electrospray Orbitrap MS/MS. The method was applied to the identification and semiquantitative determination of selenium storage proteins in wheat. MS evidence is presented for the Se-S substitution in plants not only in methionine but also in cysteine. PMID:23330978

Bianga, Juliusz; Govasmark, Espen; Szpunar, Joanna

2013-02-19

248

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

PubMed Central

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

Callahan, L T

1976-01-01

249

Colorful Electrophoresis  

NSDL National Science Digital Library

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

Utah, University O.

2012-01-01

250

Derivation of clones from the choroideremia locus by preparative field inversion gel electrophoresis.  

PubMed Central

By making use of preparative field inversion gel electrophoresis, we have constructed a lambda ZAP library that is highly enriched for sequences from the choroideremia locus. In vivo excision of pBluescript SK(-) constructs from lambda ZAP obviates the subcloning of DNA inserts and allows for rapid processing of several hundred recombinants. From a 625 kb Sfil fragment we isolated 7 clones that were physically mapped using microdeletions associated with the disease. One of these clones is located within, or just telomeric to, the choroideremia gene and detects two restriction fragment length polymorphisms (RFLPs). Another clone detects a RFLP which maps centromeric to the disease locus. Together these probes should improve the reliability of linkage analysis in choroideremia families and should pave the way for the isolation of the choroideremia gene. Images

van de Pol, T J; Cremers, F P; Brohet, R M; Wieringa, B; Ropers, H H

1990-01-01

251

Epidemiological investigation of Salmonella tilene by pulsed-field gel electrophoresis and polymerase chain reaction  

PubMed Central

Pulsed-field gel electrophoresis (PFGE) and DNA fingerprinting by the polymerase chain reaction (PCR) were performed on 11 isolates of Salmonella tilene. Five strains were from a cluster of human patients, six from sugar gliders and pygmy hedgehogs kept as family pets or from local pet retailers, and one isolate from the first North American case of S tilene described in Washington State in 1994. The PFGE restriction patterns showed all isolates to be similar. However, PCR using primers to the 16S and 23S rRNA genes of Escherichia coli demonstrated that the Washington State isolate differed from the rest of the other isolates, which were all similar based upon their DNA fingerprint. This study indicates that reliance on one technique alone may be insufficient to show nuances between strains that are, in many respects, closely related.

Anand, Chandar M; Fonseca, Kevin; Longmore, Ken; Rennie, Robert; Chui, Linda; Lingley, Mike; Woodward, David

1997-01-01

252

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

PubMed

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

2013-04-01

253

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

PubMed Central

A total of 153 strains of Listeria monocytogenes isolated from different sources (72 from sheep, 12 from cattle, 18 from feedstuffs, and 51 from humans) in Spain from 1989 to 2000 were characterized by pulsed-field gel electrophoresis. The strains of L. monocytogenes displayed 55 pulsotypes. The 84 animal, 51 human, and 18 feedstuff strains displayed 31, 29, and 7 different pulsotypes, respectively, indicating a great genetic diversity among the Spanish L. monocytogenes isolates studied. L. monocytogenes isolates from clinical samples and feedstuffs consumed by the diseased animals were analyzed in 21 flocks. In most cases, clinical strains from different animals of the same flock had identical pulsotypes, confirming the existence of a listeriosis outbreak. L. monocytogenes strains with pulsotypes identical to those of clinical strains were isolated from silage, potatoes, and maize stalks. This is the first study wherein potatoes and maize stalks are epidemiologically linked with clinical listeriosis.

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

2001-01-01

254

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

SciTech Connect

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

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

1987-06-05

255

Denaturing gradient gel electrophoresis shows that bacterial communities change with mid-ocean ballast water exchange.  

PubMed

Ships carry ballast water for better stability and to control trim. However, the discharge of ballast water near ports is known to transport invasive species from one coastal area to another. The exchange of ballast water on the high seas is supposed to reduce such invasions of exotic species. In this study, we used denaturing gradient gel electrophoresis (DGGE) to analyze the composition of the bacterial community in ballast water before and after such a mid-ocean exchange, and we also measured total bacterial counts. Our findings confirmed that the ballast water was replaced by the mid-ocean exchange, as indicated by the marked change in the composition of the bacterial community. There was also a significant decrease in bacterial abundance after the mid-ocean exchange. Finally, our findings support the incubation hypothesis, because the composition of the bacterial communities changed over time within the same ballast water. PMID:20022346

Tomaru, Akiko; Kawachi, Masanobu; Demura, Mikihide; Fukuyo, Yasuwo

2010-02-01

256

Karyotype studies on different strains of Candida molischiana by pulsed-field gel electrophoresis.  

PubMed

We used pulsed-field gel electrophoresis to compare the electrophoretic karyotype of a Candida molischiana mutant, de-repressed for beta-glucosidase production, with a wild-type strain and a reference strain. The chromosomal organization in this mutant yeast was found to be quite different. Hybridization patterns and the relative fluorescence of all bands indicated eight chromosomes in the mutant strain and seven in the other two. All three strains seemed to be haploid, with an estimated genome size of 12 Mb; the beta-glucosidase gene was on the same chromosome in all of them and the SfiI restriction patterns of this chromosome indicated that it is not affected by the mutation. PMID:8590466

Janbon, G; Magnet, R; Bigey, F; Arnaud, A; Galzy, P

1995-07-01

257

Evaluation of pulsed-field gel electrophoresis profiles for identification of Salmonella serotypes.  

PubMed

Pulsed-field gel electrophoresis (PFGE) is a standard typing method for isolates from Salmonella outbreaks and epidemiological investigations. Eight hundred sixty-six Salmonella enterica isolates from eight serotypes, including Heidelberg (n = 323), Javiana (n = 200), Typhimurium (n = 163), Newport (n = 93), Enteritidis (n = 45), Dublin (n = 25), Pullorum (n = 9), and Choleraesuis (n = 8), were subjected to PFGE, and their profiles were analyzed by random forest classification and compared to conventional hierarchical cluster analysis to determine potential predictive relationships between PFGE banding patterns and particular serotypes. Cluster analysis displayed only the underlying similarities and relationships of the isolates from the eight serotypes. However, for serotype prediction of a nonserotyped Salmonella isolate from its PFGE pattern, random forest classification provided better accuracy than conventional cluster analysis. Discriminatory DNA band class markers were identified for distinguishing Salmonella serotype Heidelberg, Javiana, Typhimurium, and Newport isolates. PMID:20631109

Zou, Wen; Lin, Wei-Jiun; Foley, Steven L; Chen, Chun-Houh; Nayak, Rajesh; Chen, James J

2010-09-01

258

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

NASA Technical Reports Server (NTRS)

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

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

1981-01-01

259

Gel electrophoresis of partially denatured DNA: split-ends, bubbles, and squids  

NASA Astrophysics Data System (ADS)

Gel electrophoresis separates partially denatured DNA fragments based on chemical sequence. Upon an increase in temperature, AT-rich regions melt into two strands which is thought to be the main contributor to the rapid reduction of the fragment's mobility. The reduction in mobility is often predicted from the average number of denatured bases regardless of their positions. We re-visit the theoretical basis of this approach and determine that the analysis only holds for denatured domains that occur at the ends. Langevin Dynamics simulations are used to study the effect that the placement of the melted regions has on the mobility by discriminating between denatured domains which occur in the middle of the fragment (bubbles) and at the ends (split-ends). It is found that the split-ends dominate the blocking mechanism. In addition, we find a novel conformation (the "squid") which seems to be responsible for the blocking at high fields.

Sean, David; Slater, Gary W.

2011-03-01

260

[Comparative studies of avian mycoplasmas by flat gel polyacrylamide electrophoresis (author's transl)].  

PubMed

The phenol-acetic-acid extraced cell proteins of Mycoplasma (M.) and Acholeplasma (A.) reference strains (PG31 (M. gallisepticum), PG 16 (M. gallinarum), PG30 (M iners), 17529 (M. meleagridis), WVU 1853 (M. synoviae), 1340 (M. anatis), PG8 and PG9 (A. laidlawii), CKK (Serovar C), DD (Serovar D), WR1 (Serogroup F), 695 (Serogroup I) and 694 (Serogroup L) were anlysed by the flat gel polyacrylamide electrophoresis. With exception of PG8 and PG9 the Coomassie Blue-stained protein patterns show that each of the strains produced reproducible characteristic electrophoretic pattern by which the reference strains could be differentiated. However, before the question could be answered whether the procedure described is suitable to replace the serological species differentiation of avian mycoplasmas, serological and electrophoretic studies of a relevant number of field strain are necessary. PMID:566006

Hinz, K H; Neumann, U

1978-04-01

261

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

PubMed Central

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

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

2005-01-01

262

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

PubMed Central

Numerous microorganisms, including bacteria, yeasts, and molds, constitute the complex ecosystem present in milk and fermented dairy products. Our aim was to describe the bacterial ecosystem of various cheeses that differ by production technology and therefore by their bacterial content. For this purpose, we developed a rapid, semisystematic approach based on genetic profiling by temporal temperature gradient electrophoresis (TTGE) for bacteria with low-G+C-content genomes and denaturing gradient gel electrophoresis (DGGE) for those with medium- and high-G+C-content genomes. Bacteria in the unknown ecosystems were assigned an identity by comparison with a comprehensive bacterial reference database of ?150 species that included useful dairy microorganisms (lactic acid bacteria), spoilage bacteria (e.g., Pseudomonas and Enterobacteriaceae), and pathogenic bacteria (e.g., Listeria monocytogenes and Staphylococcus aureus). Our analyses provide a high resolution of bacteria comprising the ecosystems of different commercial cheeses and identify species that could not be discerned by conventional methods; at least two species, belonging to the Halomonas and Pseudoalteromonas genera, are identified for the first time in a dairy ecosystem. Our analyses also reveal a surprising difference in ecosystems of the cheese surface versus those of the interior; the aerobic surface bacteria are generally G+C rich and represent diverse species, while the cheese interior comprises fewer species that are generally low in G+C content. TTGE and DGGE have proven here to be powerful methods to rapidly identify a broad range of bacterial species within dairy products.

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

2004-01-01

263

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

PubMed Central

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

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

1991-01-01

264

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

PubMed

New DNA amplification methods are continuously developed for sensitive detection and quantification of specific DNA target sequences for, e.g. clinical, environmental or food applications. These new applications often require the use of long DNA oligonucleotides as probes for target sequences hybridization. Depending on the molecular technique, the length of DNA probes ranges from 40 to 450 nucleotides, solid-phase chemical synthesis being the strategy generally used for their production. However, the fidelity of chemical synthesis of DNA decreases for larger DNA probes. Defects in the oligonucleotide sequence result in the loss of hybridization efficiency, affecting the sensitivity and selectivity of the amplification method. In this work, an enzymatic procedure has been developed as an alternative to solid-phase chemical synthesis for the production of long oligonucleotides. The enzymatic procedure for probe production was based on ligation of short DNA sequences. Long DNA probes were obtained from smaller oligonucleotides together with a short sequence that acts as bridge stabilizing the molecular complex for DNA ligation. The ligation reactions were monitored by capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) using a bare fused-silica capillary. The capillary gel electrophoresis-LIF method demonstrated to be very useful and informative for the characterization of the ligation reaction, providing important information about the nature of some impurities, as well as for the fine optimization of the ligation conditions (i.e. ligation cycles, oligonucleotide and enzyme concentration). As a result, the yield and quality of the ligation product were highly improved. The in-lab prepared DNA probes were used in a novel multiplex ligation-dependent genome amplification (MLGA) method for the detection of genetically modified maize in samples. The great possibilities of the whole approach were demonstrated by the specific and sensitive detection of transgenic maize at percentages lower than 1%. PMID:21404441

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

2011-05-01

265

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

2003-09-01

266

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

Microsoft Academic Search

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

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

1996-01-01

267

Effect of linear polymer additives on the electroosmotic characteristics of agarose gels in ultrathin-layer electrophoresis.  

PubMed

Electroosmotic properties of agarose gels with low, medium, high and super high electroendosmosis (EEO) were evaluated based on the apparent electric field mediated mobility of a neutral, fluorescent marker under constant field strength using ultrathin-layer separation configuration. Electroosmotic flow mobility values were measured in different gel concentrations and also in the absence and the presence of various linear polymer additives. Under ultrathin-layer separation conditions, a slight decrease in electroosmotic flow mobility was observed with increasing agarose gel concentration of 1 to 3% for all agarose gels investigated. When linear polymer additives, such as linear polyacrylamide, hydroxyethyl cellulose or polyethylene oxide were added to 1% low electroendosmosis agarose gel, significant reduction of the electroosmotic flow properties were observed with increasing additive concentration. Effect of the intrinsic electroosmotic properties of the various electroendosmosis agaroses on the apparent mobilities and separation performance of double-stranded DNA fragments during automated ultrathin-layer agarose gel electrophoresis was also investigated. PMID:10486760

Lengyel, T; Guttman, A

1999-08-20

268

Unfolding of hemoglobin variants—insights from urea gradient gel electrophoresis photon correlation spectroscopy and zeta potential measurements  

Microsoft Academic Search

The unfolding pattern of crystal human hemoglobin and variants of hemoglobin obtained from hemolysate were studied using transverse urea gradient gel electrophoresis (TUGGE). A smooth sigmoid like increase of electrophoretic mobility was observed with increasing urea concentrations. A decrease in electrophoretic mobility resulted, if the protein was unfolded with guanidium hydrochloride (GdnHCl). The anomaly was resolved after the Stoke’s radii

Jaydeep Bhattacharya; Ranjita GhoshMoulick; Utpal Choudhuri; Prantar Chakrabarty; Pranab K. Bhattacharya; Prabir Lahiri; Bikas Chakraborti; Anjan Kr. Dasgupta

2004-01-01

269

Use of capillary sodium dodecyl sulfate gel electrophoresis to detect the prion protein extracted from scrapie-infected sheep  

Microsoft Academic Search

Scrapie in sheep and in goats is the prototype of a group of transmissible spongiform encephalopathies (TSE). A feature of these diseases is the accumulation in the brain of rod shaped fibrils that form from an aggregated protein that is a protease-resistant form of a modified normal host cell protein. In this study, we compared SDS gel capillary electrophoresis to

Mary Jo Schmerr; Allen Jenny; Randall C. Cutlip

1997-01-01

270

ASSESSMENT OF DNA DAMAGE IN BRAIN, LIVER, KIDNEY AND TESTIS FROM MALNOURISHED RATS BY SINGLE CELL GEL ELECTROPHORESIS ASSAY  

Microsoft Academic Search

The purpose of this study was to determine if severe malnutrition induced during the lactation period (21 days of age) of rats increases DNA damage in brain, kidney, testis and liver cells. DNA damage was measured by using the alkaline single cell gel electrophoresis assay. The results indicate that severe malnutrition is associated with a significant increase in DNA damage

Miguel BETANCOURT; Edith CORTÉS; Patricia PÉREZ-VERA; Cristina GONZÁLEZ; Rocío ORTIZ

271

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

PubMed Central

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

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

2006-01-01

272

Differentiation of dextran-producing Leuconostoc strains from fermented rice cake ( puto) using pulsed-field gel electrophoresis  

Microsoft Academic Search

Lactic acid bacteria were isolated from puto, a fermented rice cake consumed as a breakfast and snack food in the Philippines. The microflora was dominated by dextran-producing leuconostocs, and these were differentiated into four groups using pulsed-field gel electrophoresis of restriction enzyme digested chromosomal DNA, in conjunction with taxonomic tests. The four groups corresponded to the species Leuconostoc mesenteroides subsp.

W. J. Kelly; R. V. Asmundson; G. L. Harrison; C. M. Huang

1995-01-01

273

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

Microsoft Academic Search

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

Luca Settanni; Sara Valmorri; Douwe van Sinderen; Giovanna Suzzi; Antonello Paparella; A. Corsetti

2006-01-01

274

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

Microsoft Academic Search

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

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

2003-01-01

275

Pulsed-Field Gel Electrophoresis Analysis of Vibrio vulnificus Strains Isolated from Taiwan and the United States  

Microsoft Academic Search

Vibrio vulnificus is a marine bacterium that causes human wound infections and septicemia with a high mortality rate. V. vulnificus strains from different clinical and environmental sources or geographic regions have been successfully characterized by ribotyping and several other methods. Pulsed-field gel electrophoresis (PFGE) is a highly discriminative method, but previous studies suggested that it was not suitable for examining

Hin-chung Wong; Shau-Yan Chen; Meng-Yi Chen; James D. Oliver; Lien-I Hor; Wen-Cherng Tsai

2004-01-01

276

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

SciTech Connect

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

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

1986-01-01

277

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

PubMed

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

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

2014-06-01

278

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

PubMed

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

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

2012-12-01

279

The application of amine-terminated silicon quantum dots on the imaging of human serum proteins after polyacrylamide gel electrophoresis (PAGE).  

PubMed

Novel amine-terminated silicon (Si) quantum dots (QDs) were synthesized and applied for the detection of human serum proteins on gels directly after polyacrylamide gel electrophoresis (PAGE). The diameter of these stable amine-terminated Si?QDs was in the range of 0.5-2.0 nm. In this study, the fluorescent imaging conditions, such as the buffer solution, pH value, buffer concentration and quantity of Si?QDs, were optimized and the possible mechanisms of Si?QDs-protein interaction were analyzed. The mode of Si?QDs and human serum albumin association was found to occur by hydrogen bond interactions; this was probably attributed to the interaction between the amino group of amine-terminated Si?QDs and the carboxyl group of proteins. Meanwhile, human serum proteins separated by native 1D and native 2D electrophoresis were detected by Si QD-based fluorescent imaging. Some proteins, such as isoform 1 of ?-1-antitrypsin, complement C3 (Fragment) and hemopexin, which were identified by mass spectrometry (MS), were easily detected by using Si?QDs, but not with CBB-R250 staining. The Si?QDs-based fluorescent imaging technique with high resolution is a sensitive and dependable method for direct detection of human serum proteins, and has enormous potential in clinical diagnosis. PMID:22249969

Liu, Pingping; Na, Na; Huang, Lingyun; He, Dacheng; Huang, Changgang; Ouyang, Jin

2012-01-27

280

Rapid fabrication of a poly(dimethylsiloxane) microfluidic capillary gel electrophoresis system utilizing high precision machining.  

PubMed

In this work, we demonstrate a rapid protocol to address one of the major barriers that exists in the fabrication of chip devices, creating the micron-sized structures in the substrate material. This approach makes it possible to design, produce, and fabricate a microfluidic system with channel features >10 microm in poly(dimethylsiloxane)(PDMS) in under 8 hours utilizing instrumentation common to most machine shops. The procedure involves the creation of a master template with negative features, using high precision machining. This master is then employed to create an acrylic mold that is used in the final fabrication step to cast channel structures into the PDMS substrate. The performance of the microfluidic system prepared using this fabrication procedure is evaluated by constructing a miniaturized capillary gel electrophoresis (micro-CGE) system for the analysis of DNA fragments. Agarose is utilized as the sieving medium in the micro-CGE device and is shown to give reproducible (RSD (n= 34) approximately 5.0%) results for about 34 individual separations without replenishing the gel. To demonstrate the functionality of the micro-CGE device, a DNA restriction ladder (spanning 26-700 base pairs) and DNA fragments generated by PCR are separated and detected with laser-induced fluorescence (LIF). The microchip is shown to achieve a separation efficiency of 2.53 x 10(5) plates m(-1). PMID:15100789

Zhao, Dong S; Roy, Binayak; McCormick, Matthew T; Kuhr, Werner G; Brazill, Sara A

2003-05-01

281

Genome analysis of the fungal plant pathogen, Leptosphaeria maculans using pulsed field gel electrophoresis.  

PubMed

Pulsed field gel electrophoresis (PFGE), or electrophoretic karyotyping, separates chromosomal-sized pieces of DNA in agarose gels where the orientation of the electric field is periodically altered. This technique has revealed that many fungi have a high degree of chromosomal length polymorphisms. Often the only isolates with identical karyotypes are derived from a single clone, thus PFGE provides a 'genetic fingerprint' for them. The size range and number of chromosomes within isolates of a particular species are usually constant, hence PFGE can distinguish between morphologically similar fungi. This technique can also be used to follow inheritance of chromosomal length polymorphisms and shows that in some fungi novel-sized chromosomes are produced during meiosis. As well as resolving the nuclear (A-type) chromosomes, it can also resolve dispensable (B-type) chromosomes and cytoplasmic genomes including mitochondrial DNA and linear plasmids. The application of this technique to Australian isolates of Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus), is discussed. PMID:9378119

Howlett, B J

1997-08-01

282

A comparison of LDL size determination using gradient gel electrophoresis and light-scattering methods.  

PubMed

This study compared gradient gel electrophoresis (GGE) and light-scattering (LS) methods of determining low density lipoprotein (LDL) particle size. LDL was isolated from 27 fasting subjects. Peak particle size was determined by GGE on 3-13% gradient gels (Gradipore, Sydney, Australia) and by LS using a Zetasizer 3000 (Malvern Instruments, Malvern, UK). Repeated measurements on a single specimen indicated a coefficient of variation (CV) of 0.3%. A correlation was noted (P < 0.0001; r = 0.78) when comparing LDL particle size determined by LS methodology and GGE. Particle diameter results obtained by LS were smaller than those obtained by GGE (23.1 +/- 0.1 vs. 26.1 +/- 0.1 nm; P < 0.0001). LDL particle size determined by LS methodology correlated inversely with the log of triglyceride level (P < 0.0001; r = -0.77) and positively with high density lipoprotein (HDL) cholesterol level (P < 0.002; r = 0.57). PMID:9788255

O'Neal, D; Harrip, P; Dragicevic, G; Rae, D; Best, J D

1998-10-01

283

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

PubMed

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

Singh, Sanjay Kumar; Ramaiah, Nagappa

2011-05-01

284

Theory of DNA electrophoresis in physical gels and entangled polymer solutions  

NASA Astrophysics Data System (ADS)

A scaling theory is presented for the electrophoretic mobility of DNA in sieving media that form dynamically evolving meshworks, such as physical gels and solutions of entangled polymers. In such media, the topological constraints on the DNA's motion are perpetually changing as cross links break and rejoin or as the polymers diffuse. It is shown that if the rate of constraint release falls within a certain range (which depends on the field strength), fractionation can be extended to higher molecular weights than would be feasible using a permanent gel of equivalent pore size. This improvement is a consequence of the disruptive effect that constraint release has on the mechanism of molecular orientation. Numerical simulations support the predictions of the theory. The possibility of realizing such a system in practice, with the aim of improving on current electrophoresis methods, is commented upon. It is suggested that semidilute polymer solutions may be a versatile medium for the rapid separation of long single-stranded DNA molecules, and the particular quality of solution required is identified.

Duke, Thomas; Viovy, Jean Louis

1994-03-01

285

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

PubMed

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

1991-10-18

286

2D-electrophoresis and multiplex immunoassay proteomic analysis of different body fluids and cellular components reveal known and novel markers for extended fasting  

Microsoft Academic Search

Background  Proteomic technologies applied for profiling human biofluids and blood cells are considered to reveal new biomarkers of exposure\\u000a or provide insights into novel mechanisms of adaptation.\\u000a \\u000a \\u000a \\u000a \\u000a Methods  Both a non-targeted (classical 2D-electrophoresis combined with mass spectrometry) as well as a targeted proteomic approach\\u000a (multiplex immunoassay) were applied to investigate how fasting for 36 h, as compared to 12 h, affects the

Freek G Bouwman; Baukje de Roos; Isabel Rubio-Aliaga; L Katie Crosley; Susan J Duthie; Claus Mayer; Graham Horgan; Abigael C Polley; Carolin Heim; Susan LM Coort; Chris T Evelo; Francis Mulholland; Ian T Johnson; Ruan M Elliott; Hannelore Daniel; Edwin CM Mariman

2011-01-01

287

Precast commercial polyacrylamide gels for separation of DNA amplificates by temperature gradient gel electrophoresis: application to clonality analysis of lymphomas.  

PubMed

The third complementary determining region (CDR-III) of the rearranged immunoglobulin heavy chain (IgH) genes represents a unique marker for a lymphocyte and its clonal descendants and can be amplified by the polymerase chain reaction (PCR) technique. This approach has markedly enhanced the sensitivity for detection of clonal lymphocyte populations in patients with malignant B-lymphoid neoplasias. To monitor minimal residual disease (MRD) in tissue specimens during or after antineoplastic treatment, the problem of detecting the presence of a few clonal (malignant) lymphocytes in coexistence with a majority of polyclonal lymphocytes has to be addressed. Semi-nested PCR amplification of CDR-III rearrangements from specimen infiltrated by tumor cells generates clonal signals in front of a polyclonal background, and therefore high resolution electrophoretic techniques for separation of DNA fragments are required. Temperature gradient gel electrophoresis (TGGE) resolving DNA homo- and heteroduplexes according to their thermal stability has been successfully applied for this purpose using special electrophoretic equipment. We describe an adjustment to this technique by using a commercially available precast 0.5 mm thick polyacrylamide gel and by changing a standard horizontal electrophoretic device into a TGGE device. By this means we screened patients with B-cell lymphoma undergoing high-dosage radiochemotherapy followed by autologous transplantation for continuous presence of clonal (tumor-specific) CDR-III rearrangements. Specimens from blood and bone marrow were collected on diagnosis as well as before and after autologous transplantation. In addition, the autograft (bone marrow or peripheral blood hematopoietic stem cells) was analyzed. Tumor cells were easily detected in the transplants and in specimens collected during follow-up examinations. The clinical value of these findings remains unclear as yet because the number of cases investigated was small and the follow-up time is still too short. However, we conclude that the technique of combining the sensitivity of PCR with the specificity of high resolution TGGE is easy to use, making it possible to handle, in a clinical routine, a great number of samples within a short time in order to monitor MRD in patients with B-cell neoplasias. PMID:8738325

Suttorp, M; von Neuhoff, N; Tiemann, M; Dreger, P; Schaub, J; Löffer, H; Parwaresch, R; Schmitz, N

1996-04-01

288

The use of biphasic linear ramped pulsed field gel electrophoresis to quantify DNA damage based on fragment size distribution  

SciTech Connect

The development of biphasic linear pulse ramping gel electrophoresis has permitted resolution of DNA fragments from 200 Kbp to 6 Mbp in a single gel. We used this technique to measure radiation-induced DNA damage based on fragment size. Human colon cancer cells (HT29 and LS174T) and Chinese hamster ovary cells were embedded in agarose, deproteinized, irradiated with 5-80 Gy, and assessed for DNA double strand breakage using pulsed field gel electrophoresis. The frequency of DNA double strand breakage determined using a previously published method was compared to the breakage frequency calculated using the fragment size distribution. Both methods produced similar estimates for breakage frequency of approximately 5 {times} 10{sup {minus}9} breaks Gy{sup {minus}1} bp{sup {minus}1}. These findings suggest that biphasic linear pulse ramping gel electrophoresis can yield a quantitative estimate of DNA fragment distribution resulting from irradiation. The ability to quantify the distribution of DNA fragment sizes produced by irradiation should yield information concerning the mechanisms of both DNA double strand break induction and repair. 16 refs., 5 figs.

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

1993-10-20

289

Enzymatic assessment of cholesterol on electrophoresis gels for estimating HDL size distribution and plasma concentrations of HDL subclasses[S  

PubMed Central

The aim of this study was to develop an enzymatic cholesterol staining method to determine HDL subclasses in a polyacrylamide gradient gel electrophoresis, which further allows staining by protein in the same electrophoresis lane. HDLs from 120 healthy individuals were separated through nondenaturing PAGE. HDLs were stained for cholesterol using an enzymatic semisolid mixture. Once the gels were unstained, they were stained again for proteins with Coomassie blue. The proportions of HDL subclasses were determined by densitometry. HDL subclasses were transformed to concentrations using as reference HDL-cholesterol plasma levels. This method is comparable in linearity and reproducibility to Coomassie blue staining, although it provides quantitative data. As expected, HDL size distribution shifted toward larger particles when determined by cholesterol as compared with protein. With this method, we observed different proportions of HDL subclasses between men and women as compared with Coomassie blue staining. We described a method to determine HDL size distribution by enzymatic cholesterol staining on polyacrylamide gels. The method allows the quantification of the cholesterol plasma concentration of each HDL subclass with the possibility to further stain the protein in the same sample. The combination of HDL staining by cholesterol and protein on electrophoresis gels provides information that may have clinical relevance.

Toledo-Ibelles, Paola; Garcia-Sanchez, Cynthia; Avila-Vazzini, Nydia; Carreon-Torres, Elizabeth; Posadas-Romero, Carlos; Vargas-Alarcon, Gilberto; Perez-Mendez, Oscar

2010-01-01

290

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

PubMed

Two-dimensional non-equilibrium pH gel electrophoresis (2D-NEPHGE) analysis was used to evaluate the effects of dietary protein depletion on the protein composition of mouse liver cytosol. Analysing the cytosol from both normal and protein depleted liver, the position in gels of more than three hundred protein spots was determined. After 5 days of protein depletion, about 20% of the spots either increased or decreased more than 2 fold. Five spots of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were recognised by specific antibodies. The glutathione S-transferase (GSTs) subunits Ybl, Yc and Yf were identified by the simultaneous analysis of both glutathione-binding cytosolic proteins and the corresponding standards. As estimated by internal optical density (IOD) of spots, the changes caused by protein depletion in GAPDH and GST subunit contents were similar to those obtained by other methods. By means of mass spectrometric analysis of tryptic peptides generated from spots and/or comparison of two-dimensional gel electrophoretic patterns, carbonic anhydrase III (CAIII), Cu, Zn superoxide dismutase (CuZnSOD) and a cytochrome P450 cytosolic protein (cyt P450) were identified. These three proteins, as well as GSTs, are related with intracellular detoxification and free radical scavenging systems. Their contents were regulated by dietary protein restriction in a manner indicative of diminished liver defence against oxidising agents. PMID:11451382

Sanllorenti, P M; Rosenfeld, J; Ronchi, V P; Ferrara, P; Conde, R D

2001-04-01

291

Optimization of 2-dimensional gel electrophoresis for proteomic studies of solid tumor tissue samples.  

PubMed

The method of 2?dimensional gel electrophoresis (2-DE) has been widely used for the proteomic profiling of solid biological samples, however, the analytical conditions have not been optimized. The present study optimized the major conditions of 2?DE for determining the protein contents of solid tumor tissues, through enhancement of the separation efficiency and resolution. Three major analytical conditions of 2?DE analysis, namely protein extraction, focusing time for isoelectric focusing (IEF), and pre?reduction and alkylation prior to IEF, were carefully examined so that the optimal parameters and procedures were achieved. The use of a bead mill for protein extraction resulted in a higher protein yield in a minimal processing time. An optimal focusing time for IEF was established which improved the 2?DE image quality and reproducibility. Furthermore, reduction and alkylation of the protein sample prior to IEF reduced the horizontal streaking caused by oxidation and improved the resolution at the cathode. The optimized 2?DE analysis enabled the detection of 20% more protein spots compared with the previous reported conditions, with higher image quality and reproducibility. Accordingly, the optimized conditions may be used in the 2?DE analysis of tumor tissue samples, by which novel biomarkers of cancerous diseases and molecular targets of drugs are expected to be identified. PMID:24270922

Liang, Xu; Wang, Jing-Rong; Wong, Kam-Wai V; Hsiao, Wen Luan; Zhou, Hua; Jiang, Zhi-Hong; Kam, Kin Ting R; Liu, Liang

2014-02-01

292

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.

1990-12-10

293

Characterization of carbohydrates using highly fluorescent 2-aminobenzoic acid tag following gel electrophoresis of glycoproteins.  

PubMed

Application of the most sensitive fluorescent label 2-aminobenzoic acid (anthranilic acid, AA) for characterization of carbohydrates from the glycoproteins ( approximately 15 pmol) separated by polyacrylamide gel electrophoresis is described. AA label is used for the determination of both monosaccharide composition and oligosaccharide map. For the monosaccharide determination, bands containing the glycoprotein of interest are excised from the polyvinylidene fluoride (PVDF) membrane blots, hydrolyzed in 20% trifluoroacetic acid, derivatized, and analyzed by C-18 reversed-phase high-performance liquid chromatography. For the oligosaccharide mapping, bands were digested with peptide N-glycosidase F (PNGase F) in order to release the N-linked oligosaccharides, derivatized, and analyzed by normal-phase anion-exchange chromatography. For convenience, the PNGase F digestion was performed in 1:100 diluted ammonium hydroxide overnight. The oligosaccharide yield from ammonium hydroxide-PNGase F digestion was better or equal to all the other reported procedures, and the presumed "oligosaccharide-amine" product formed in the reaction mixture did not interfere with labeling of the oligosaccharides under the conditions used for derivatization. Sequencing of oligosaccharides can be performed using the same mapping method following treatment with an array of glycosidases. In addition, the mapping method is useful for determining the relative and simultaneous distribution of sialic acid and fucose. PMID:10552910

Anumula, K R; Du, P

1999-11-15

294

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

PubMed

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

Haliem, E A; Al-Huqail, A A

2013-01-01

295

DNA fingerprinting of isolates of Streptococcus mutans by pulsed-field gel electrophoresis.  

PubMed

Forty isolates and five standard laboratory strains, representing serotypes c, e and f of Streptococcus mutans were analyzed by pulsed-field gel electrophoresis (PFGE) after digestion of the genomic DNA with BssH II. The digestion patterns of standard laboratory strains were characteristic of serotypes c, e and f. Serotypes c and f generated diagnostic DNA fragments of approximately 145 kbp and of approximately 130-175 kbp in length, respectively. Serotype e generated a ladder of at least 14 fragments of 15-155 kbp in length. The digestion patterns of isolates were essentially similar to those of the standard laboratory strains. The patterns of almost all isolates obtained from a single individual were identical, but patterns of a few different types were also observed among isolates obtained from two individuals. Digestion with BssH II revealed differences among isolates obtained from different individuals. We used differences in banding patterns among isolates to construct a dendrogram. The dendrogram included two major clusters, one that consisted of isolates of serotypes c and f, and an other that consisted of isolates of serotype e. Our results indicate that BssH II is a useful enzyme for distinguishing among isolates of S. mutans and that digestion patterns obtained by PFGE can be used for chromosomal DNA fingerprinting. PMID:16870412

Mineyama, R; Yoshino, S; Maeda, N

2007-01-01

296

The Limitations of Pulsed-Field Gel Electrophoresis for Analysis of Yersinia enterocolitica Isolates.  

PubMed

This study describes the analysis of 432 isolates of Yersinia enterocolitica by pulsed-field gel electrophoresis (PFGE). PFGE had a high level of discrimination with biotype 1A isolates (Simpson's Diversity Index 0.997), but with the clinically important biotypes 2, 3 and 4, the discriminatory ability of PFGE was so low as to severely limit its usefulness (DI <0.6). For biotypes 2, 3 and 4, 79% or more of isolates of each biotype were of just three different PFGE profiles. Because of this, four known outbreaks of yersiniosis would not have been identified by PFGE analysis. However, a previously unrecognized potential outbreak of yersiniosis caused by biotype 4 isolates was identified on the basis of a rare PFGE genotype with spatial and temporal clustering. We conclude that PFGE has a very limited application to the genotyping of Y. enterocolitica biotypes 2, 3 and 4, and inferences based on finding indistinguishable PFGE profiles among cases or between cases and sources need to be substantiated using alternative typing tools, or strong epidemiological evidence. PMID:24237638

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

2014-09-01

297

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

PubMed Central

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

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

2012-01-01

298

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

PubMed

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

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

2012-01-01

299

A program for selecting DNA fragments to detect mutations by denaturing gel electrophoresis methods.  

PubMed Central

A computer program was developed to automate the selection of DNA fragments for detecting mutations within a long DNA sequence by denaturing gel electrophoresis methods. The program, MELTSCAN, scans through a user specified DNA sequence calculating the melting behavior of overlapping DNA fragments covering the sequence. Melting characteristics of the fragments are analyzed to determine the best fragment for detecting mutations at each base pair position in the sequence. The calculation also determines the optimal fragment for detecting mutations within a user specified mutational hot spot region. The program is built around the statistical mechanical model of the DNA melting transition. The optimal fragment for a given position is selected using the criteria that its melting curve has at least two steps, the base pair position is in the fragment's lowest melting domain, and the melting domain has the smallest number of base pairs among fragments that meet the first two criteria. The program predicted fragments for detecting mutations in the cDNA and genomic DNA of the human p53 gene.

Brossette, S; Wartell, R M

1994-01-01

300

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

PubMed Central

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

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

1995-01-01

301

Prenatal diagnosis of neural tube defects: experiences with acetylcholinesterase gel electrophoresis.  

PubMed

We assessed the qualitative test for AChE in addition to AFP for the diagnosis of NTD in 420 AF samples selected from 16,000 second trimester amniocenteses. Three-hundred and thirty were from normal and ninety from abnormal pregnancies. There were three false positives by this test, two from stored samples containing fetal blood and one from a clear specimen; all had normal outcomes and two had had abnormal AFPs. Forty-seven of forty-seven NTDs were positive by AChE and included 26 cases of anencephaly, 16OSBs, and 5 encephaloceles. In addition, about half of the cases of omphalocele, fetal demise, cystic hygroma, and hydrops gave positive results. One case of congenital nephrosis was negative. We conclude that AChE gel electrophoresis is a useful adjunctive test when AFP levels measure +3 SD above the mean. Although no false negative occurred in this series identified by elevated AFP or increased risk for NTDs, the number in unselected pregnancies is not known. PMID:6180640

Crandall, B F; Kasha, W; Matsumoto, M

1982-07-01

302

Prediction system for rapid identification of Salmonella serotypes based on pulsed-field gel electrophoresis fingerprints.  

PubMed

A classification model is presented for rapid identification of Salmonella serotypes based on pulsed-field gel electrophoresis (PFGE) fingerprints. The classification model was developed using random forest and support vector machine algorithms and was then applied to a database of 45,923 PFGE patterns, randomly selected from all submissions to CDC PulseNet from 2005 to 2010. The patterns selected included the top 20 most frequent serotypes and 12 less frequent serotypes from various sources. The prediction accuracies for the 32 serotypes ranged from 68.8% to 99.9%, with an overall accuracy of 96.0% for the random forest classification, and ranged from 67.8% to 100.0%, with an overall accuracy of 96.1% for the support vector machine classification. The prediction system improves reliability and accuracy and provides a new tool for early and fast screening and source tracking of outbreak isolates. It is especially useful to get serotype information before the conventional methods are done. Additionally, this system also works well for isolates that are serotyped as "unknown" by conventional methods, and it is useful for a laboratory where standard serotyping is not available. PMID:22378901

Zou, Wen; Lin, Wei-Jiun; Hise, Kelley B; Chen, Hung-Chia; Keys, Christine; Chen, James J

2012-05-01

303

Prediction System for Rapid Identification of Salmonella Serotypes Based on Pulsed-Field Gel Electrophoresis Fingerprints  

PubMed Central

A classification model is presented for rapid identification of Salmonella serotypes based on pulsed-field gel electrophoresis (PFGE) fingerprints. The classification model was developed using random forest and support vector machine algorithms and was then applied to a database of 45,923 PFGE patterns, randomly selected from all submissions to CDC PulseNet from 2005 to 2010. The patterns selected included the top 20 most frequent serotypes and 12 less frequent serotypes from various sources. The prediction accuracies for the 32 serotypes ranged from 68.8% to 99.9%, with an overall accuracy of 96.0% for the random forest classification, and ranged from 67.8% to 100.0%, with an overall accuracy of 96.1% for the support vector machine classification. The prediction system improves reliability and accuracy and provides a new tool for early and fast screening and source tracking of outbreak isolates. It is especially useful to get serotype information before the conventional methods are done. Additionally, this system also works well for isolates that are serotyped as “unknown” by conventional methods, and it is useful for a laboratory where standard serotyping is not available.

Lin, Wei-Jiun; Hise, Kelley B.; Chen, Hung-Chia; Keys, Christine; Chen, James J.

2012-01-01

304

High Resolution Melt Analysis (HRMA); a Viable Alternative to Agarose Gel Electrophoresis for Mouse Genotyping  

PubMed Central

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

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

2012-01-01

305

Pulsed-Field Gel Electrophoresis Analysis of Nasopharyngeal Flora in Children Attending a Day Care Center  

PubMed Central

To investigate how bacterial pathogens spread from child to child in a day care center, we monitored six children, two boys and four girls, born between August 1995 and November 1997, attending a day care center and analyzed nasopharyngeal samples from them using pulsed-field gel electrophoresis (PFGE). We obtained nasopharyngeal cultures from all of the affected children and almost all of the unaffected children between September 1998 and March 1999 after some children presented simultaneously with purulent rhinorrhea. Moreover, when a child was found to have acute otitis media, nasopharyngeal secretions from the child were independently cultured during treatment. During this period, 28 isolates of Moraxella catarrhalis, 13 of Streptococcus pneumoniae, and 4 of Haemophilus influenzae were recovered. PFGE gave 8 patterns for M. catarrhalis, 10 for S. pneumoniae, and 1 for H. influenzae. PFGE patterns demonstrated spread of M. catarrhalis between children. However, each occurrence of clusters of infection with M. catarrhalis lasted 2 to 6 weeks, with a change in PFGE pattern between occurrences of clusters. The M. catarrhalis strain infecting each child also changed. Similarly, the S. pneumoniae strain in each child also changed. In contrast, infection with H. influenzae persisted for about 3 months in an affected child.

Yano, Hisakazu; Suetake, Mitsuko; Kuga, Akio; Irinoda, Kazuhiko; Okamoto, Ryoichi; Kobayashi, Toshimitsu; Inoue, Matsuhisa

2000-01-01

306

Imaging of kinked configurations of DNA molecules undergoing orthogonal field alternating gel electrophoresis by fluorescence microscopy  

SciTech Connect

The dynamics of individual DNA molecules undergoing orthogonal field alternating gel electrophoresis (OFAGE) have been studied by use of T2 DNA molecules labeled with a dye and visualized with a fluorescence microscope. The mechanism of reorientation used by a molecule to align itself in the direction of the new orthogonal field depends on the degree of extension of the chain immediately before the application of this field. The formation of kinks is promoted when time is allowed between the application of the two orthogonal fields so that the molecule attains a partially relaxed configuration. In this case, the chain appears bunched up in domains moving along the contour of the molecule. These regions are found to be the locations where the kinks are formed upon application of the second field perpendicular to the chain. The formation of kinks provides a significative retardation of the reorientation of the molecules, relative to molecules that do not form kinks, and appears to play an important role in the fractionation attained with ORAGE. A classification of various reorientation mechanisms observed in molecules that form kinks is presented.

Gurrieri, S.; Beach, D.; Bustamante, C. (Univ. of New Mexico, Albuquerque (USA)); Rizzarelli, E. (Universita di Catania (Italy))

1990-04-03

307

Genetic diversity of atypical Aeromonas salmonicida studied by pulsed-field gel electrophoresis.  

PubMed Central

Pulsed-field gel electrophoresis (PFGE) pattern analysis with XbaI restriction enzyme was used to study the genetic heterogeneity of 88 atypical Aeromonas salmonicida strains which were earlier or during this study characterized phenotypically, by ribotyping (ClaI/PstI) and by plasmid profile analysis. The strains of certain'ribotypes were also analysed by digestion with SpeI. The strains represented different geographic locations: Finland (72 strains), Iceland (5 strains), Norway (5 strains), Sweden (4 strains) and Denmark (2 strains), and they were from 17 fish species during 1981 97. Thirty-one PFGE genotypes found among these strains correlated well with the ribotypes, and in most cases PFGE pattern analysis subdivided ribotypes into several PFGE genotypes, and further within a PFGE genotype into subtypes. XbaI and SpeeI digests produced concordant results. In most cases, PFGE patterns of strains with the same ribotype shared many fragments, suggesting genetic relatedness. PFGE patterns of most Norwegian and Icelandic strains isolated during an approximately 10-year period had the same ribotype and their PFGE patterns shared most fragments, suggesting close genetic relatedness. Moreover, atypical strains of ribotypes B/B and H/H isolated from the same Finnish fish farms had closely related patterns suggesting genetic stability and persistence of these genotypes. Genotype 29 of Achromogenic strains was strongly associated with disease of Finnish arctic char and grayling. PFGE was shown to be a distinguishing method to study the genetic heterogeneity of atypical A. salmonicida. epidemiology of these infections.

Hanninen, M. L.; Hirvela-Koski, V.

1999-01-01

308

Suitability of PCR Fingerprinting, Infrequent-Restriction-Site PCR, and Pulsed-Field Gel Electrophoresis, Combined with Computerized Gel Analysis, in Library Typing of Salmonella enterica Serovar Enteritidis  

Microsoft Academic Search

Strains of Salmonella enterica (n 5 212) of different serovars and phage types were used to establish a library typing computerized system for serovar Enteritidis on the basis of PCR fingerprinting, infrequent-restriction- site PCR (IRS-PCR), or pulsed-field gel electrophoresis (PFGE). The rate of PCR fingerprinting interassay and intercenter reproducibility was low and was only increased when DNA samples were extracted

JAVIER GARAIZAR; NURIA LOPEZ-MOLINA; IDOIA LACONCHA; DORTE LAU BAGGESEN; AITOR REMENTERIA; ANA VIVANCO; ANA AUDICANA; ILDEFONSO PERALES

2000-01-01

309

Phenotyping breast cancer cell lines EM-G3, HCC1937, MCF7 and MDA-MB-231 using 2-D electrophoresis and affinity chromatography for glutathione-binding proteins  

PubMed Central

Background Transformed phenotypes are common to cell lines derived from various cancers. Proteome profiling is a valuable tool that may reveal uncharacteristic cell phenotypes in transformed cells. Changes in expression of glutathione S-transferases (GSTs) and other proteins interacting with glutathione (GSH) in model cell lines could be of particular interest. Methods We compared the phenotypes of breast cell lines EM-G3, HCC1937, MCF7 and MDA-MB-231 using 2-D electrophoresis (2-DE). We further separated GSH-binding proteins from the cell lines using affinity chromatography with GSH-Sepharose 4B, performed 2-DE analysis and identified the main protein spots. Results Correlation coefficients among 2-DE gels from the cell lines were lower than 0.65, pointing to dissimilarity among the cell lines. Differences in primary constituents of the cytoskeleton were shown by the 2-D protein maps and western blots. The spot patterns in gels of GSH-binding fractions from primary carcinoma-derived cell lines HCC1937 and EM-G3 were similar to each other, and they differed from the spot patterns of cell lines MCF7 and MDA-MB-231 that were derived from pleural effusions of metastatic mammary carcinoma patients. Major differences in the expression of GST P1-1 and carbonyl reductase [NADPH] 1 were observed among the cell lines, indicating differential abilities of the cell lines to metabolize xenobiotics. Conclusions Our results confirmed the applicability of targeted affinity chromatography to proteome profiling and allowed us to characterize the phenotypes of four breast cancer cell lines.

2010-01-01

310

Antigens of scrub typhus rickettsiae: separation by polyacrylamide gel electrophoresis and identification by enzyme-linked immunosorbent assay.  

PubMed Central

Antigens of plaque-purified Rickettsia tsutsugamushi strains Gilliam, Karp, and Kato were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were analyzed by an enzyme-linked immunosorbent assay. Six antigens were identified in each of the three prototype strains; in strain Gilliam, these antigens were located in the cell envelope fraction of the organisms. Reactivity of these isolated antigens with homologous or heterologous immune sera indicated that different macromolecules existed in all three strains, although they exhibited very similar mobilities during electrophoresis. Antigens of strain Gilliam reacted equally well with antibodies directed against Gilliam, Karp, or Kato rickettsiae. However, strains Karp and Kato each had two distinct antigens which did not react with heterologous antisera. R. tsutsugamushi antigens retained immunogenicity after electrophoresis, and antisera raised against them reacted with intact organisms and exhibited specificity in reactions with isolated antigens.

Eisemann, C S; Osterman, J V

1981-01-01

311

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, Stéphane; Deves, Guillaume; Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis; Ortega, Richard

2009-11-01

312

Improved banding pattern of rat plasma lipoproteins developed by agarose gel electrophoresis at pH 7.0.  

PubMed

The use of a conventional agarose gel electrophoretic method to separate rat plasma lipoproteins resulted in a rather poor resolution of lipoproteins of lower density. Therefore, attempts were made to improve the resolution by running the electrophoresis under different conditions. It was shown that rat plasma lipoproteins could be separated into at least three fractions by agarose gel electrophoresis in phosphate buffer at pH 7.0. Using rat plasma lipoproteins isolated by sequential flotation as standards, these fractions were shown to correspond to high-density lipoprotein (HDL2), very-low-density lipoprotein (VLDL) and a mixed low-density lipoprotein (LDL)/high-density lipoprotein (HDL1) fraction. Since VLDL is completely separated from the other lipoprotein classes the method could be used to monitor changes in plasma VLDL. PMID:3718995

Eklund, A; Sjöblom, L

1986-06-11

313

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

PubMed Central

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

Steger, G

1994-01-01

314

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

PubMed

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, María G; Casanueva, Enrique; Cane, Alejandro; Moroni, Mirian P; Brengi, Silvina P; Pichel, Mariana G

2013-01-01

315

Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis Monitoring of Fecal Bifidobacterium Populations in a Prebiotic and Probiotic Feeding Trial  

Microsoft Academic Search

A culture-independent approach based on genus-specific PCR and denaturing gradient gel electrophoresis (DGGE) was used to monitor qualitative changes in fecal bifidobacterial communities in a human feeding trial. DNA was extracted directly from feces and bifidobacterial 16S rDNA sequences were amplified using genus-specific PCR. The PCR fragments were subsequently separated in a sequence-specific manner by DGGE in order to obtain

Reetta M. Satokari; Elaine E. Vaughan; Antoon D. L. Akkermans; Maria Saarela; Willem M. de Vos

2001-01-01

316

Two-Dimensional Gel Electrophoresis Analysis of the Response ofPseudomonas putidaKT2442 to 2Chlorophenol  

Microsoft Academic Search

The effects of exposure ofPseudomonas putidaKT2442 to 2-chlorophenol as a model for the chemical stress response were examined by two-dimensional polyacrylamide gel electrophoresis. Individual protein concen- trations were determined at 45, 65, and 95 min following the addition of 2-chlorophenol at a concentration of 1.63 mM to exponentially growing cultures ofP. putidaKT2442 by silver staining the separated proteins. The changes

CLAUDIO G. LUPI; TERESA COLANGELO; ANTHONY MASON

1995-01-01

317

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

Microsoft Academic Search

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

Christoph Helma; Maria Uhl

2000-01-01

318

A high-efficiency, two-dimensional gel electrophoresis platform for mature leaves of grass pea ( Lathyrus sativus L.)  

Microsoft Academic Search

Grass pea (Lathyrus sativus L.) is the most drought-tolerant legume crop rich in dietary protein. However, little is known about the molecular mechanisms\\u000a of its drought resistance. Two-dimensional gel electrophoresis (2-DE) is an important experiment technique in proteomics,\\u000a which has been applied extensively in studies on plant resistance to abiotic stress. To establish an effective 2-DE platform\\u000a and further study

Qingfeng WuChun; Chun Li; Lanming Ke; Chengjin Jiao; Jinglong Jiang; Xiaoyan Sun; Fengmin Li; Chongying Wang

319

Automated Ribotyping and Pulsed-Field Gel Electrophoresis for Rapid Identification of Multidrug-Resistant Salmonella Serotype Newport  

PubMed Central

In a series of 116 Salmonella enterica Newport isolates that included 64 multidrug-resistant (MDR) isolates, automated ribotyping and pulsed-field gel electrophoresis (PFGE) discriminated MDR S. Newport with a sensitivity of 100% and 98% and specificity of 76% and 89%, respectively. Clustering of PFGE patterns (but not ribotyping) linked human and bovine cases. Automated ribotyping rapidly identified the MDR strain, and PFGE detected associations that aided epidemiologic investigations.

Stout, Alison; Bolstorff, Barbara; Timperi, Ralph

2003-01-01

320

Phylogenetic diversity of nitrogen-fixing bacteria in mangrove sediments assessed by PCR–denaturing gradient gel electrophoresis  

Microsoft Academic Search

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

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

2008-01-01

321

Analysis of microbial communities in doenjang, a Korean fermented soybean paste, using nested PCR-denaturing gradient gel electrophoresis  

Microsoft Academic Search

Doenjang is a traditional Korean fermented soybean paste that provides a major source of protein. The microbial diversity of 10 samples of doenjang (5 commercially manufactured products and 5 homemade products) was investigated using nested PCR-denaturing gradient gel electrophoresis (DGGE). In the first step, the nearly complete 16S rRNA and 18S rRNA genes were amplified using universal primers. Subsequently, these

Tae-Woon Kim; Jun-Hwa Lee; Sung-Eon Kim; Min-Hee Park; Hae Choon Chang; Hae-Yeong Kim

2009-01-01

322

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

Microsoft Academic Search

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

Chiyoko Satoh; Norio Takahashi; Junichi Asakawa; Keiko Hiyama; Meiko Kodaira

1993-01-01

323

Optimized PCR-Temporal Temperature Gel Electrophoresis compared to cultivation to assess diversity of gut microbiota in neonates  

Microsoft Academic Search

Temporal Temperature Gel Electrophoresis of amplified 16S rRNA gene sequences (16S rDNA PCR-TTGE) constitutes a culture-independent molecular method used to study bacterial communities. All the technical steps are crucial for quality and exhaustiveness of the results obtained by such approach. Careful optimization of the protocols used is ideally needed for each ecosystem studied. We present here the strategy used to

Laurent Roudière; Aurélien Jacquot; Hélène Marchandin; Fabien Aujoulat; Raymonde Devine; Isabelle Zorgniotti; Hélène Jean-Pierre; Jean-Charles Picaud; Estelle Jumas-Bilak

2009-01-01

324

16S rDNA PCR-denaturing gradient gel electrophoresis in determining proportions of coexisting Actinobacillus actinomycetemcomitans strains  

Microsoft Academic Search

Certain serotypes of Actinobacillus actinomycetemcomitans seem to prefer coexistence in vivo. The 16S rDNA PCR-denaturing gradient gel electrophoresis (DGGE) was tested for its capability to distinguish coexisting A. actinomycetemcomitans strains of different serotypes or genetic lineages and to determine their proportions in vitro. The migration pattern of the PCR amplicon from serotype c differed from those of the other serotypes.

Riikka Ihalin; Sirkka Asikainen

2006-01-01

325

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

Håkan Sjunnesson; Tobias Fält; Erik Sturegård; Waleed Abu Al-Soud; sa Ljungh; Torkel Wadström

2003-01-01

326

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

2000-01-01

327

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

1997-01-01

328

Protein concentration of cerebrospinal fluid by precipitation with Pyrogallol Red prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis  

Microsoft Academic Search

The Pyrogallol Red Molybdate (PRM) and Coomassie Brilliant Blue (CBB) protein dye-binding assays have been applied to samples of cerebrospinal fluid (CSF) to investigate protein concentration by dye precipitation prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration values of the CSF samples (N=62) showed good agreement between the PRM and CBB assays as indicated by linear regression

Katherine M Williams; Thomas Marshall

2001-01-01

329

DNA damage by hydroquinone in human white blood cells: analysis by alkaline single-cell gel electrophoresis  

Microsoft Academic Search

The genotoxicity of hydroquinone (HQ) in human white blood cells was investigated by means of alkaline single-cell gel electrophoresis (SCGE). The exposure of purified lymphocytes to HQ (0.5–50 ?g\\/ml) produced significant and dose-related increases in DNA migration; conversely, no induction of DNA damage was observed in leukocytes after in vitro treatment of whole blood samples (100–500 ?g\\/ml). Similar differences in

Cristina Andreoli; Sabrina Rossi; Paola Leopardi; Riccardo Crebelli

1999-01-01

330

Effects of ochratoxin A on DNA evaluated in vitro with the single cell gel electrophoresis (Comet assay)  

Microsoft Academic Search

In cell cultures of Madin Darby canine kidney (MDCK) cells, the mycotoxin ochratoxin A (OTA) induced single strand breaks\\u000a (ssb) in a concentration dependent manner detected with the single cell gel electrophoresis (Comet assay). When an external\\u000a metabolizing enzyme system (S9-mix from rat liver) was added, this genotoxic effect was significantly stronger. By addition\\u000a of methotrexate (MT), a substrate of

S Lebrun; W Föllmann

2001-01-01

331

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

NOELLE BARBIER; PATRICK SAULNIER; ELISABETH CHACHATY; SANDRINE DUMONTIER; ANDANTOINE ANDREMONT

1996-01-01

332

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

Microsoft Academic Search

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

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

1998-01-01

333

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

Microsoft Academic Search

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

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

2002-01-01

334

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

PubMed

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

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

2012-10-01

335

Sequence analysis of 5'[32P] labeled mRNA and tRNA using polyacrylamide gel electrophoresis.  

PubMed Central

Sequence analysis of 5'-[32P] labeled tRNA and eukaryotic mRNA using an adaptation of a method recently described by Donis-Keller, Maxam and Gilbert for mapping guanines, adenines and pyrimidines from the 5'-end of an RNA is described. In addition, a technique utilizing two-dimensional polyacrylamide gel electrophoresis for identification of pyrimidines within a sequence is described. 5'-[32P] Labeled rabbit beta-globin mRNA and N. crassa mitochondrial initiator tRNA were partially digested with T1- RNase for cleavage at G residues, with U2-RNase for cleavage at A residues, with an extracellular RNase from B. cereus for cleavage at pyrimidine residues and with T2-RNase or with alkali for cleavage at all four residues. The 5'-[32P] labeled partial digestion products were separated according to their size, by electrophoresis in adjacent lanes of a polyacrylamide slab gel and the location of G's, A's and of pyrimidines extending 60-80 nucleotides from the 5'-end of the RNA determined. Two-dimensional polyacrylamide gel electrophoresis was used to separate the 5'-[32P] labeled fragments present in partial alkali digests of a 5'-[32P] labeled mRNA. The mobility shifts corresponding to the difference of a C residue were distinct from those corresponding to a U residue and this formed the basis of a method for distinguishing between the pyrimidines. Images

Lockard, R E; Alzner-Deweerd, B; Heckman, J E; MacGee, J; Tabor, M W; RajBhandary, U L

1978-01-01

336

OFFGEL isoelectric focusing and polyacrylamide gel electrophoresis separation of platinum-binding proteins.  

PubMed

In this work a 2D electrophoretic separation procedure able to maintain the integrity of platinum-protein bonds has been developed. The method is based on the use of sequential OFFGEL isoelectric focussing (IEF) and PAGE. A systematic study of the reagents used for PAGE, for OFFGEL-IEF separation, and post-separation treatment of gels (such as enzymatic digestion and sample preparation for MS analysis) was tackled regarding their suitability for the identification of platinum binding proteins using standard proteins incubated with cisplatin. The distribution of platinum in high and low molecular weight fractions (separated by cut-off filters) was determined by ICP-MS, which allows evaluating platinum-protein bond stability under the conditions studied. SDS-PAGE in the absence of ?-mercaptoethanol or dithiotreitol preserved the platinum-protein bonds. In addition, neither the influence of the electric field during the electrophoretic separation, nor the processes of fixing, staining and destaining of proteins in the gel did result in the loss of platinum from platinum binding proteins. SDS-PAGE under non-reducing conditions provides separation of platinum-binding proteins in very narrow bands with quantitative recoveries. Different amounts of platinum-bound proteins covering the range 0.3-2.0 ?g were separated and mineralised for platinum determination, showing good platinum linearity. Limits of detection for a mixture of five standard proteins incubated with cisplatin were between the range of 2.4 and 13.9 pg of platinum, which were satisfactory for their application to biological samples. Regarding OFFGEL-IEF, a denaturing solution without thiourea and without dithiotreitol is recommended. The suitability of the OFFGEL-IEF for the separation of platinum binding proteins of a kidney cytosol was demonstrated. PMID:21255782

Mena, Ma Luz; Moreno-Gordaliza, Estefanía; Moraleja, Irene; Cañas, Benito; Gómez-Gómez, Ma Milagros

2011-03-01

337

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

PubMed

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

Eiler, Alexander; Bertilsson, Stefan

2006-11-01

338

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

PubMed

Using pulsed-field gel electrophoresis (PFGE), between 1987 and 1996 we analyzed Salmonella enteritidis isolates from gastroenteritis cases in four Caribbean countries: Barbados, Saint Kitts and Nevis, Saint Lucia, and Trinidad and Tobago. We also determined the resistance of the isolates to 12 antimicrobial agents. Of the 129 isolates of S. enteritidis available for testing, DNA digested by XbaI revealed 13 distinctive PFGE patterns. The most prevalent XbaI PFGE patterns were group 1 (88 of 129 isolates, 68.2%) and group 2 (26 of 129, 20.2%). The patterns found among S. enteritidis isolates correlated with the geographical origin of the isolates. Of the 28 isolates from Barbados, 20 of them (71.4%) belonged to XbaI PFGE group 2, and of the 93 isolates from Trinidad and Tobago, 78 of them (83.9%) belonged to group 1. SpeI digestion of S. enteritidis genome was not as discriminatory as XbaI. Overall, of the 129 isolates, 67 of them (51.9%) exhibited resistance to one or more of the 12 antimicrobial agents that we tested. The prevalence of resistance was 53.8% for the S. enteritidis isolates tested from Trinidad and Tobago, 50.0% for those from Barbados, 28.6% for those from Saint Lucia, and 100.0% for one isolate from the island of Saint Kitts. Resistance was highest to triple sulfur (59 of 129 isolates, 45.7%), followed by furadantoin (10 of 129, 7.8%), ampicillin (7 of 129, 5.4%), and carbamycin (5 of 129, 3.9%). PMID:11190971

Adesiyun, A; Carson, A; McAdoo, K; Bailey, C

2000-11-01

339

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

PubMed Central

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

Najdenski, H; Iteman, I; Carniel, E

1994-01-01

340

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

PubMed Central

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

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

2010-01-01

341

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

PubMed

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

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

2014-08-01

342

Effect of Antibiotics on Group A Streptococcus Exoprotein Production Analyzed by Two-Dimensional Gel Electrophoresis  

PubMed Central

High-dose clindamycin (CLDM) and benzylpenicillin (PCG) are the recommended chemotherapeutic remedies for toxic shock-like syndrome caused by group A streptococci. One reason for this is that it has been shown that CLDM suppresses the expression of some exoproteins, e.g., SpeB, SpeA, and streptolysin O (Slo). We analyzed the effects of antibiotics on the production of whole exoproteins by two-dimensional gel electrophoresis. Unexpectedly, we found that the levels of several exoproteins, Slo, NAD+-glycohydrolase (Nga), M protein, and Sic, were increased by CLDM treatment, although we also confirmed previous findings that the levels of various exoproteins, including SpeB, were decreased. The increases in exoprotein levels were also detected by using other protein synthesis inhibitor antibiotics: erythromycin, kanamycin, tetracycline, chloramphenicol, and linezolid. Peptidoglycan synthesis inhibitors (such as PCG, cefazolin, and imipenem), DNA replication inhibitors (such as gatifloxacin), and an RNA polymerase inhibitor (rifampin) did not have significant effects on exoprotein production. The combination of CLDM and PCG had no advantageous effects with regard to exoprotein production compared to the effect achieved with CLDM alone. We also analyzed the transcriptional levels of slo and nga by reverse transcription-PCR and found that this change was also detected at the transcriptional level. Furthermore, the phenomenon was seen not only in strains of the M1 serotype but also in strains of the other M serotypes. Our study suggests that the clinical effectiveness of CLDM might be due to the inhibition of the production of a limited number of exoproteins.

Tanaka, Megumi; Hasegawa, Tadao; Okamoto, Akira; Torii, Keizo; Ohta, Michio

2005-01-01

343

Reference System for Characterization of Bordetella pertussis Pulsed-Field Gel Electrophoresis Profiles  

PubMed Central

Pulsed-field gel electrophoresis (PFGE) has been used as an epidemiological tool for surveillance studies of Bordetella pertussis since the early 1990s. To date there is no standardized procedure for comparison of results, and therefore it has been difficult to directly compare PFGE results between laboratories. We propose a profile-based reference system for PFGE characterization of B. pertussis strain variation and to establish traceability of B. pertussis PFGE results. We initially suggest 35 Swedish reference strains as reference material for PFGE traceability. This reference material is deposited at the Culture Collection of the University of Gothenburg, Gothenburg, Sweden. Altogether, 1,810 Swedish clinical isolates from between 1970 and 2003 were studied, together with the Swedish Pw vaccine strain, six reference strains, and two U.S. isolates. Our system provides evidence that profiles obtained by using only one enzyme, i.e., XbaI, give enough data to analyze the epidemiological relationship between them. Characterization with one enzyme is far less labor intensive, yielding results in half the time than when a two-enzyme procedure is used. Also, we can see that there is a correlation between PFGE profile and pertactin type. One common PFGE profile, BpSR11 (n = 455), showed 100% prn2 and 100% Fim3 when analyzed for pertactin type and serotype. On the other hand, strains with the same profile may express various serotypes when isolated over longer periods of time. Subculturing of the same isolate eight times or lyophilization caused no change in PFGE profile.

Advani, Abdolreza; Donnelly, Declan; Hallander, Hans

2004-01-01

344

Identification and Characterization of Pathogenic Yersinia enterocolitica Isolates by PCR and Pulsed-Field Gel Electrophoresis  

PubMed Central

Approximately 550 to 600 yersiniosis patients are reported annually in Sweden. Although pigs are thought to be the main reservoir of food-borne pathogenic Yersinia enterocolitica, the role of pork meat as a vehicle for transmission to humans is still unclear. Pork meat collected from refrigerators and local shops frequented by yersiniosis patients (n = 48) were examined for the presence of pathogenic Yersinia spp. A combined culture and PCR method was used for detection, and a multiplex PCR was developed and evaluated as a tool for efficient identification of pathogenic food and patient isolates. The results obtained with the multiplex PCR were compared to phenotypic test results and confirmed by pulsed-field gel electrophoresis (PFGE). In all, 118 pork products (91 raw and 27 ready-to-eat) were collected. Pathogenic Yersinia spp. were detected by PCR in 10% (9 of 91) of the raw pork samples (loin of pork, fillet of pork, pork chop, ham, and minced meat) but in none of the ready-to-eat products. Isolates of Y. enterocolitica bioserotype 4/O:3 were recovered from six of the PCR-positive raw pork samples; all harbored the virulence plasmid. All isolates were recovered from food collected in shops and, thus, none were from the patients' home. When subjected to PFGE, the six isolates displayed four different NotI profiles. The same four NotI profiles were also present among isolates recovered from the yersiniosis patients. The application of a multiplex PCR was shown to be an efficient tool for identification of pathogenic Y. enterocolitica isolates in naturally contaminated raw pork.

Thisted Lambertz, S.; Danielsson-Tham, M.-L.

2005-01-01

345

The effect of pre-existing maternal obesity on the placental proteome: two-dimensional difference gel electrophoresis coupled with mass spectrometry.  

PubMed

Our aim was to study the protein expression profiles of placenta obtained from lean and obese pregnant women with normal glucose tolerance at the time of term Caesarean section. We used two-dimensional difference gel electrophoresis (2D-DIGE), utilising narrow-range immobilised pH gradient strips that encompassed the broad pH range of 4-5 and 5-6, followed by MALDI-TOF mass spectrometry of selected protein spots. Western blot and quantitative RT-PCR (qRT-PCR) analyses were performed to validate representative findings from the 2D-DIGE analysis. Eight proteins were altered (six down-regulated and two up-regulated on obese placentas). Annexin A5 (ANXA5), ATP synthase subunit beta, mitochondria (ATPB), brain acid soluble protein 1 (BASP1), ferritin light chain (FTL), heterogeneous nuclear ribonucleoprotein C (HNRPC) and vimentin (VIME) were all lower in obese patients. Alpha-1-antitrypsin (A1AT) and stress-70 protein, mitochondrial (GRP75) were higher in obese patients. Western blot analysis of ANXA5, ATPB, FTL, VIME, A1AT and GRP75 confirmed the findings from the 2D-DIGE analysis. For brain acid soluble protein 1 and HNRPC, qRT-PCR analysis also confirmed the findings from the 2D-DIGE analysis. Immunohistochemical analysis was also used to determine the localisation of the proteins in human placenta. In conclusion, proteomic analysis of placenta reveals differential expression of several proteins in patients with pre-existing obesity. These proteins are implicated in a variety of cellular functions such as regulation of growth, cytoskeletal structure, oxidative stress, inflammation, coagulation and apoptosis. These disturbances may have significant implications for fetal growth and development. PMID:22301947

Oliva, Karen; Barker, Gillian; Riley, Clyde; Bailey, Mark J; Permezel, Michael; Rice, Gregory E; Lappas, Martha

2012-04-01

346

Optimized sequence retrieval from single bands of temperature gradient gel electrophoresis profiles of the amplified 16S rDNA fragments from an activated sludge system  

Microsoft Academic Search

Sequence retrieval from single bands of polymerase chain reaction (PCR)-denaturing gel electrophoresis (DGE) profiles is an important but often difficult step for molecular diversity analysis of complex microbial communities such as activated sludge systems. We analyzed the temperature gradient gel electrophoresis (TGGE) profiles of PCR-amplified 16S rDNA fragments from an activated sludge sample of a coking wastewater treatment plant. Single

Xueli Zhang; Xing Yan; Pingping Gao; Linghua Wang; Zhihua Zhou; Liping Zhao

2005-01-01

347

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

PubMed Central

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

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

1996-01-01

348

Rapid protein separations in ultra-short microchannels: microchip sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing.  

PubMed

We have developed novel protein gel electrophoresis techniques, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) in short microchannels (approximately millimeters) that take less than a minute. A photopatterning technique was used to cast in situ crosslinked polyacrylamide gel in a microchannel to perform SDS-PAGE. A fluorescent protein marker sample (Mr range of 20,000-200,000) was separated in less than 30 s in less than 2 mm of channel length. Crosslinked polyacrylamide gel, patterned in channels using UV light, provides higher sieving power and sample stacking effect, therefore yielding faster and higher-resolution separation in a chip. IEF of proteins was also achieved in a microchannel, and several proteins were focussed within tens of seconds in mm-length channels. As resolution in IEF is independent of separation distance, focusing in ultra-short channels results in not only faster separation but also more concentrated bands potentially allowing detection of low-concentration species. PMID:15499934

Han, Jongyoon; Singh, Anup K

2004-09-17

349

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

PubMed

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

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

2011-12-21

350

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.

2002-10-01

351

An optimized procedure for sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of hydrophobic peptides from an integral membrane protein.  

PubMed

A procedure for successful analysis of the hydrophobic tryptic peptides of the Neurospora crassa plasma membrane H+-ATPase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is described. The features of this procedure that are essential for the best results include (i) treatment of the hydrophobic peptide samples with neat trifluoroacetic acid, (ii) dissolution and disaggregation of the hydrophobic peptide samples with SDS at 0 degrees C, (iii) SDS-PAGE of the hydrophobic peptide samples in gels containing a 200:1 ratio of acrylamide to bisacrylamide and a 5-20% convex acrylamide gradient, and (iv) silver-staining of the gels after electrophoresis. This method results in the reproducible resolution and visualization of the H+-ATPase hydrophobic tryptic peptides, which range in size from ca. 5 to 21 kDa, as well as other peptides and proteins ranging in size from ca. 2.5 to 150 kDa. The methods described should also prove useful in other studies where resolution and visualization of hydrophobic peptides of integral membrane proteins are required. PMID:2525882

Hennessey, J P; Scarborough, G A

1989-02-01

352

Agarose and Polyacrylamide Gel Electrophoresis Methods for Molecular Mass Analysis of 5-500 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 5–500 kDa have been investigated. For agarose-based systems, the suitability of different agarose types, agarose concentrations, and buffers systems were determined. Using chemoenzymatically synthesized HA standards of low polydispersity, the molecular mass range was determined for each gel composition, over which the relationship between HA 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 µg (for polyacrylamide) to 2.5 µg (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.

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

2011-01-01

353

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

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

1993-01-01

354

DNA fingerprinting by pulsed-field gel electrophoresis to investigate a nosocomial pneumonia caused by Legionella bozemanii serogroup 1.  

PubMed Central

We typed 18 isolates of Legionella bozemanii obtained from clinical and environmental sources by pulsed-field gel electrophoresis. Each of the unrelated strains showed individual restriction patterns of the genomic DNA when either the SfiI or NotI restriction enzyme was used. One strain isolated from a patient with nosocomial legionellosis and two strains from the corresponding hospital water supply were indistinguishable, arguing for a transmission of L. bozemanii from the water supply to the patient. In conclusion, macrorestriction analysis is a valuable tool for studies of the molecular epidemiology of L. bozemanii.

Luck, P C; Helbig, J H; Hagedorn, H J; Ehret, W

1995-01-01

355

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

NASA Technical Reports Server (NTRS)

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

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

2003-01-01

356

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.

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

2012-01-01

357

The use of 2D-electrophoresis and de novo sequencing to characterize inter- and intra-cultivar protein polymorphisms in an allopolyploid crop.  

PubMed

Polyploidy and allopolyploidy have played an important role in the evolution of many plants and crops. Several techniques exist to characterize allopolyploid varieties. Analyzing the consequences of genomic reorganization at the gDNA level is a prerequisite but a better insight into the consequences for the phenotype is also primordial. As such, protein polymorphism analysis is important in understanding plant and crop biodiversity and is a driving force behind crop improvement. Our strategy to analyze protein isoforms and to detect possible gene silencing or deletion in bananas was based on protein analysis. Bananas are a good representative of a complex allopolyploid and important crop. We combined two-dimensional electrophoresis (2DE) and 2D DIGE with de novo MS/MS sequence determination to characterize a range of triploid varieties. Via Principal Component Analysis (PCA) and hierarchical clustering we were able to blindly classify the different varieties according to their presumed genome constitution. We report for the first time the application of an automated approach for the derivatization of peptides for facilitated MS/MS de novo sequence determination. We conclude that the proteome does not always correspond to the presumed genome formulae and that proteomics is a powerful tool to characterize varieties. The observations at the protein level provide good indications for a more complex genome structure and genomic rearrangement in some banana varieties. PMID:21109271

Carpentier, Sebastien Christian; Panis, Bart; Renaut, Jenny; Samyn, Bart; Vertommen, Annelies; Vanhove, Anne-Catherine; Swennen, Rony; Sergeant, Kjell

2011-07-01

358

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

PubMed

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

2013-12-01

359

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

PubMed

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

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

2012-01-01

360

Horizontal two-dimensional electrophoresis of complex DNA samples using precast gel systems.  

PubMed

We have developed a simplified procedure for the separation of enzyme-digested genomic DNA, or of complex mixtures of cloned DNA, into two dimensions. The procedure relies on the use of precast gels for horizontal electrophoretic separations. Precast agarose-type gels are used for the first-dimensional separation of fragments based on size. Precast polyacrylamide gels are used for the second-dimensional separation of fragments. The separated fragments are subjected to enzymatic digestion in situ prior to their transfer to the second-dimensional gel. Applications of this procedure include the analysis of DNA libraries, analysis of yeast artificial chromosomes (YACs) as well as other similar preparations, and the screening of genomic DNA for the occurrence of multi-copy DNA fragments as in the case of genomic amplifications in cancer. PMID:10380763

Schickle, H; Lamb, B J; Hanash, S M

1999-06-01

361

Measurement of DNA Damage Using Agarose Gel Electrophoresis and Electronic Imaging.  

National Technical Information Service (NTIS)

Damage done to DNA by ultraviolet (uv) light, gamma rays and other carcinogens can be quantified using agarose gel electrophororesis. Agents that either produce strand breaks directly or that produce lesions that can be enzymatically or chemically convert...

J. C. Sutherland A. M. Bergman C. Z. Chen D. C. Monteleone J. Trunk

1988-01-01

362

Temperate Bacteriophages Affect Pulsed-Field Gel Electrophoresis Patterns of Campylobacter jejuni  

Microsoft Academic Search

The recently sequenced genome of Campylobacter jejuni RM1221 revealed the presence of three integrated bacteriophage-like elements. In this study, genes from the first element, a Mu-like bacteriophage, were amplified by PCR and used to probe pulsed-field gels of clinical C. jejuni strains obtained from a waterborne outbreak (Ontario, Canada, 2000). These highly similar strains differed only by their pulsed-field gel

Connie Barton; Lai-King Ng; Shaun D. Tyler; Clifford G. Clark

363

Temperate Bacteriophages Affect Pulsed-Field Gel Electrophoresis Patterns of Campylobacter jejuni  

Microsoft Academic Search

The recently sequenced genome of Campylobacter jejuni RM1221 revealed the presence of three integrated bacteriophage-like elements. In this study, genes from the first element, a Mu-like bacteriophage, were amplified by PCR and used to probe pulsed-field gels of clinical C. jejuni strains obtained from a waterborne outbreak (Ontario, Canada, 2000). These highly similar strains differed only by their pulsed-field gel

Connie Barton; Lai-King Ng; Shaun D. Tyler; Clifford G. Clark

2007-01-01

364

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

1995-01-01

365

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

PubMed

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

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

1993-04-01

366

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

PubMed

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

Sean, David; Slater, Gary W

2010-10-01

367

2D-electrophoresis and multiplex immunoassay proteomic analysis of different body fluids and cellular components reveal known and novel markers for extended fasting  

PubMed Central

Background Proteomic technologies applied for profiling human biofluids and blood cells are considered to reveal new biomarkers of exposure or provide insights into novel mechanisms of adaptation. Methods Both a non-targeted (classical 2D-electrophoresis combined with mass spectrometry) as well as a targeted proteomic approach (multiplex immunoassay) were applied to investigate how fasting for 36 h, as compared to 12 h, affects the proteome of platelets, peripheral blood mononuclear cells (PBMC), plasma, urine and saliva collected from ten healthy volunteers. Results Between-subject variability was highest in the plasma proteome and lowest in the PBMC proteome. Random Forests analysis performed on the entire dataset revealed that changes in the level of the RhoGDI2 protein in PBMC and plasma ApoA4 levels were the two most obvious biomarkers of an extended fasting. Random Forests (RF) analysis of the multiplex immunoassay data revealed leptin and MMP-3 as biomarkers for extended fasting. However, high between-subject variability may have masked the extended fasting effects in the proteome of the biofluids and blood cells. Conclusions Identification of significantly changed proteins in biofluids and blood cells using a non-targeted approach, together with the outcome of targeted analysis revealed both known and novel markers for a 36 h fasting period, including the cellular proteins RhoGDI2 and CLIC1, and plasma proteins ApoA4, leptin and MMP-3. The PBMC proteome exhibited the lowest between-subject variability and therefore these cells appear to represent the best biosamples for biomarker discovery in human nutrigenomics.

2011-01-01

368

A Marker-Free Watershed Approach for 2D-GE Protein Spot Segmentation  

Microsoft Academic Search

Two-dimensional gel electrophoresis (2D-GE) is the key technique in large-scale protein identification from complex protein mixtures. The 2D-GE images, which represent protein signals as spots of various intensities and sizes, may yield a lot of information that can help the biologists for exploring the elements affecting human health. Automatic analysis for the gel images can help saving time and labor

Minh-Tuan Trong Hoang; Yonggwan Won

2007-01-01

369

DNA double-strand breaks measured in individual cells subjected to gel electrophoresis  

Microsoft Academic Search

Microscopic examination of individual mammalian cells embedded in agarose, subjected to electrophoresis, and stained with a fluorescent DNA-binding dye provides a novel way of measuring DNA damage and more importantly, of assessing heterogeneity in DNA damage within a mixed population of cells. With this method, DNA double-strand breaks can be detected in populations of cells exposed to X-ray doses as

Peggy L. Olive; Danuta Wlodek; J. P. Banath

1991-01-01

370

Hybridization Assay by Time-Resolved Capillary Gel Electrophoresis with a Lanthanide Chelate  

Microsoft Academic Search

Capillary electrophoresis of crude biological samples with time-resolved fluorescence (TRF) detection enables elimination\\u000a of interference from organic fluorophores and from light scattering. Because the fluorescence lifetime of biological substances\\u000a and impurities overlaps the fluorescence lifetime of conventional labeling dyes, TRF detection with conventional organic labeling\\u000a dyes suffers from background fluorescence. In this work, we synthesized a luminescent lanthanide chelating reagent

Keiko Sumitomo; Takahisa Ito; Motoyasu Sasaki; Yoshinori Yamaguchi

2008-01-01

371

The Laboratory Technology of Discrete Molecular Separation: The Historical Development of Gel Electrophoresis and the Material Epistemology of Biomolecular Science, 1945–1970  

Microsoft Academic Search

Preparative and analytical methods developed by separation scientists have played an important role in the history of molecular\\u000a biology. One such early method is gel electrophoresis, a technique that uses various types of gel as its supporting medium\\u000a to separate charged molecules based on size and other properties. Historians of science, however, have only recently begun\\u000a to pay closer attention

Howard Hsueh-Hao Chiang

2009-01-01

372

Genotoxicity evaluation of acute doses of endosulfan to freshwater teleost Channa punctatus (Bloch) by alkaline single-cell gel electrophoresis.  

PubMed

The Indian freshwater air-breathing teleost fish Channa punctatus (Bloch) was exposed to acute concentrations of the organochlorine pesticide endosulfan. In flow-through bioassays the 24, 48, 72, and 96 h LC(50) values were estimated as 19.67, 12.95, 10.15, and 7.75 ppb, respectively. DNA damage (single-strand breaks) was also studied in gill and kidney tissues at single-cell levels in the specimens, exposed to different acute doses of endosulfan, by applying single-cell gel electrophoresis or comet assay. Dose-dependent responses were observed in DNA damage in both tissues. A comparison of DNA damage in both tissues at different doses indicated that the gill cells were more sensitive to the pesticide exposure than the kidney cells. This study explored the utility of the comet assay for in vivo laboratory studies using fish for screening the genotoxic potential of various agents. PMID:16095691

Pandey, Sanjay; Nagpure, N S; Kumar, Ravindra; Sharma, Shilpi; Srivastava, Satish K; Verma, Mahendra S

2006-09-01

373

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

PubMed

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

Urzainqui, A; Tabarés, E; Carrasco, L

1987-09-01

374

Cost-Effective Application of Pulsed-Field Gel Electrophoresis to Typing of Salmonella enterica Serovar Typhimurium  

PubMed Central

Salmonella enterica serovar Typhimurium is frequently isolated from humans and animals. Phage typing is historically the first-line reference typing technique in Europe. It is rapid and convenient for laboratories with appropriate training and experience, and costs of consumables are low. Phage typing and pulsed-field gel electrophoresis (PFGE) were performed on 503 isolates of serovar Typhimurium. Twenty-nine phage types and 53 PFGE patterns were observed. Most isolates of phage types DT104, DT104b, and U310 are not distinguishable from other members of their phage type by PFGE. By contrast, PFGE of isolates of phage types DT193 and U302 shows great heterogeneity. Analysis of experience with PFGE and phage typing can facilitate the selective application of PFGE to maximize the yield of epidemiologically relevant additional information while controlling costs.

Doran, Geraldine; Morris, Dearbhaile; O'Hare, Colette; DeLappe, Niall; Bradshaw, Bernard; Corbett-Feeney, Geraldine; Cormican, Martin

2005-01-01

375

Evaluation of genotoxicity after application of Listerine(R) on human lymphocytes by micronucleus and single cell gel electrophoresis assays.  

PubMed

Listerine (LN) is one of the most commonly used mouth rinses worldwide although very limited information is available concerning its genotoxicity. In another view, the biological safety profile of oral care products is frequently assumed on the basis of simplistic test models. Therefore, the present study was undertaken to investigate the in vitro genotoxic potential of LN using micronucleus and single cell gel electrophoresis tests as genetic endpoints. Different concentrations of LN (0-100% of ml/culture, v/v) were applied to whole human blood cultures (n = 5). The result of the present study showed that there were no statistically significant differences (p > 0.05) between the control group and the groups treated with LN alone in both analysed endpoints. In conclusion, our result first demonstrated the absence of genotoxicity of LN on human lymphocytes. PMID:22033428

Türkez, Hasan; Togar, Basak; Arabaci, Taner

2012-04-01

376

Protein concentration of cerebrospinal fluid by precipitation with Pyrogallol Red prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis.  

PubMed

The Pyrogallol Red Molybdate (PRM) and Coomassie Brilliant Blue (CBB) protein dye-binding assays have been applied to samples of cerebrospinal fluid (CSF) to investigate protein concentration by dye precipitation prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration values of the CSF samples (N=62) showed good agreement between the PRM and CBB assays as indicated by linear regression analysis (y(PRM)=1.033x(CBB)+1.004 in units of mg/l, r=0.99) but the PRM assay was optimal for protein concentration as the PRM protein-dye complex was less soluble allowing protein recovery over a wider working range. Dye precipitation using PRM is recommended as a simple, rapid and economic method for protein concentration of samples of CSF prior to SDS-PAGE. PMID:11245891

Williams, K M; Marshall, T

2001-02-26

377

Pulsed field gel electrophoresis analysis of Borrelia burgdorferi sensu lato isolated in Japan and taxonomic implications with Lyme disease spirochetes.  

PubMed

Genomic DNAs of Borrelia burgdorferi sensu lato isolates obtained in Japan sharing different rRNA gene ribotypes were digested with rare-cutter restriction endonucleases and the fragments obtained were separated by pulsed field gel electrophoresis (PFGE). The sizes of large restriction cleavage bands with MluI endonuclease were quite similar among isolates in each ribotype group. On the other hand, the PFGE profiles obtained with the other enzymes (NruI, Sal I or SplI) were rather divergent, and Japanese isolates were distinguishable from the United States and European isolates. The Japanese isolates classified as ribotypes group II (Borrelia garinii) and III (B. afzelii) showed different PFGE patterns from that of European isolates. The isolates grouped into ribotype IV revealed distinctively different PFGE profiles. These results indicate that the Japanese isolates may be genetically divergent and distinct from the United States and European isolates. PMID:7531811

Fukunaga, M; Takahashi, Y

1994-01-01

378

An outbreak of Campylobacter jejuni infections associated with food handler contamination: the use of pulsed-field gel electrophoresis.  

PubMed

In 1998, an outbreak of Campylobacter jejuni infections occurred in Kansas among persons attending a school luncheon; community cases were also reported. In a cohort study of luncheon attendees, 27 (17%) of 161 persons reported illness. Consuming gravy (relative risk [RR], 4.2; 95% confidence interval [CI], 1.5-11.7) or pineapple (RR, 2.4; 95% CI, 1.0-5.7) was associated with illness. Both foods were prepared in a kitchen that served 6 other schools where no illness was reported. A cafeteria worker at the luncheon had a diarrheal illness and was the likely source of the outbreak. The pulsed-field gel electrophoresis (PFGE) patterns of the isolates from the food handler and those of 8 lunch attendees were indistinguishable. Isolates from 4 community patients differed. This was the first use of PFGE in a Campylobacter outbreak in the United States; its use was critical in determining that community cases were not linked. PMID:11078485

Olsen, S J; Hansen, G R; Bartlett, L; Fitzgerald, C; Sonder, A; Manjrekar, R; Riggs, T; Kim, J; Flahart, R; Pezzino, G; Swerdlow, D L

2001-01-01

379

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

PubMed

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

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

2014-05-31

380

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

PubMed Central

The epidemiology of urinary tract infections (UTI) by Staphylococcus saprophyticus has not been fully characterised and strain typing methods have not been validated for this agent. To evaluate whether epidemiological relationships exist between clusters of pulsed field gel-electrophoresis (PFGE) genotypes of S. saprophyticus from community-acquired UTI, a cross-sectional surveillance study was conducted in the city of Rio de Janeiro, Brazil. In total, 32 (16%) female patients attending two walk-in clinics were culture-positive for S. saprophyticus. Five PFGE clusters were defined and evaluated against epidemiological data. The PFGE clusters were grouped in time, suggesting the existence of community point sources of S. saprophyticus. From these point sources, S. saprophyticus strains may spread among individuals.

de Sousa, Viviane Santos; Rabello, Renata Fernandes; Dias, Rubens Clayton da Silva; Martins, Ianick Souto; dos Santos, Luisa Barbosa Gomes da Silva; Alves, Elisabeth Mendes; Riley, Lee Woodford; Moreira, Beatriz Meurer

2013-01-01

381

Human cytomegalovirus-induced immediate early antigens: analysis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis after immunoprecipitation.  

PubMed Central

Immediate early antigen (IEA) induced in human lung fibroblasts by human cytomegalovirus was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis after immunoprecipitation with IEA-positive human sera. Two polypeptides of 76,000 daltons (76K) and 82K were detectable within 90 min after infection. Polypeptides of similar molecular weight were also found in immunoprecipitates of human cytomegalovirus-infected cells nonpermissive for virus replication. IEA is located within the nucleus, although some of the 76K material appears to be located on the outer nuclear membrane. Raising salt concentrations in the extraction buffer increased antigen extraction. The contribution of these IEA polypeptides to IEA nuclear fluorescent staining is discussed. Images

Michelson, S; Horodniceanu, F; Kress, M; Tardy-Panit, M

1979-01-01

382

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

PubMed

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

Furukawa, Takashi; Suzuki, Yoshihiro

2013-01-01

383

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.

Furukawa, Takashi; Suzuki, Yoshihiro

2013-01-01

384

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

PubMed

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

Duncombe, Todd A; Herr, Amy E

2012-10-16

385

Evaluation of Pulsed-Field Gel Electrophoresis in Epidemiological Investigations of Meningococcal Disease Outbreaks Caused by Neisseria meningitidis Serogroup C  

PubMed Central

Since 1990, the frequency of Neisseria meningitidis serogroup C (NMSC) outbreaks in the United States has increased. Based on multilocus enzyme electrophoresis (MEE), the current molecular subtyping standard, most of the NMSC outbreaks have been caused by isolates of several closely related electrophoretic types (ETs) within the ET-37 complex. We chose 66 isolates from four well-described NMSC outbreaks that occurred in the United States from 1993 to 1995 to evaluate the potential of pulsed-field gel electrophoresis (PFGE) to identify outbreak-related isolates specific for each of the four outbreaks and to differentiate between them and 50 sporadic isolates collected during the outbreak investigations or through active laboratory-based surveillance from 1989 to 1996. We tested all isolates collected during the outbreak investigations by four other molecular subtyping methods: MEE, ribotyping (ClaI), random amplified polymorphic DNA assay (two primers), and serotyping and serosubtyping. Among the 116 isolates, we observed 11 clusters of 39 NheI PFGE patterns. Excellent correlation between the PFGE and the epidemiological data was observed, with an overall sensitivity of 85% and specificity of 71% at the 95% pattern relatedness breakpoint using either 1.5 or 1.0% tolerance. For all four analyzed outbreaks, PFGE would have given public health officials additional support in declaring an outbreak and making appropriate public health decisions.

Popovic, Tanja; Schmink, Susanna; Rosenstein, Nancy A.; Ajello, Gloria W.; Reeves, Michael W.; Plikaytis, Brian; Hunter, Susan B.; Ribot, Efrain M.; Boxrud, David; Tondella, Maria L.; Kim, Chung; Noble, Corie; Mothershed, Elizabeth; Besser, John; Perkins, Bradley A.

2001-01-01

386

Characterization of antithrombin III from human plasma by two-dimensional gel electrophoresis and capillary electrophoretic methods.  

PubMed

The isoforms distribution of the glycoprotein antithrombin III (ATIII) derived from human plasma was investigated by means of isoelectric focusing (IEF) in polyacrylamide gels with immobilized pH gradients (IPG) and two-dimensional gel electrophoresis (2-DE) as well as capillary electrophoretic methods. It turned out that the presence of high concentrations of chaotropics (urea, thiourea) and zwitterionic detergents (3-[(3-cholamidepropyl)dimethylammonio]-1-propanesulfonate (CHAPS)) was decisive for attaining good resolution of the protein isoforms. Resolution by IPG-IEF was obtained with excellent reproducibility and pI differences down to 0.01 pH units could be distinguished. ATIII-alpha and ATIII-beta-fractions preseparated by heparin affinity chromatography showed an analogous but shifted spot pattern consisting each of one major and three minor isoforms. The main isoforms of ATIII-alpha and ATIII-beta exhibit pI values of 5.18 and 5.32, respectively, both values determined in the presence of high concentrations of urea. The pI difference of 0.14 pH units correspond to the effect of two sialic acids absent in ATIII-beta. The formation and occurrence of ATIII dimers and trimers turned out to be dependent on the sample preparation. The results obtained by 2-DE were compared with those of capillary zone electrophoresis (CZE) and capillary IEF (CIEF). Quantitative analysis regarding the CZE separated isoforms of plasma derived ATIII yielded a content of about 70% ATIII-alpha main isoform and about 6.6% of ATIII-beta. The pI values of ATIII determined by CIEF with internal calibration were in fair agreement with the pI values of the main isoforms achieved with 2-DE. PMID:14679575

Kremser, Leopold; Brückner, Andrea; Heger, Andrea; Grunert, Tom; Buchacher, Andrea; Josic, Djuro; Allmaier, Günter; Rizzi, Andreas

2003-12-01

387

Detection of trypsin- and chymotrypsin-like proteases using p-nitroanilide substrates after sodium dodecyl sulphate polyacrylamide gel electrophoresis.  

PubMed

Specific chromogenic p-nitroanilide substrates have proved useful for localizing proteolytic enzymes, such as trypsin, chymotrypsin and elastase after separation by agarose gel electrophoresis and when immobilized on nitrocellulose. This procedure was further developed for use with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). After SDS-PAGE, proteins were transferred electrophoretically to a nitrocellulose membrane. The membrane was incubated for 10-60 min with Bz-Ile-Glu-Gly-Arg-p-nitroanilide as a substrate for detection of trypsin-like proteases and with MeO-Suc-Arg-Pro-Tyr-p-nitroanilide for detection of chymotrypsin. The yellow p-nitroanilide released at the site of proteolytic activity was converted into a visible and stable red azo dye. By this method was identified and determined the molecular weight of a trypsin-like protease that occurs at high concentrations in mucinous ovarian tumour cyst fluid together with its specific inhibitor peptide, tumour-associated trypsin inhibitor (TATI). The method was also used to visualize trypsin and chymotrypsin in human pancreatic juice. Using the trypsin substrate, three proteolytic bands, corresponding to Mr of 22,000, 24,000 and 26,000 daltons, were visualized in pancreatic juice, while the proteolytic zones in cyst fluid had Mr of 25,000 and 28,000 daltons. With the chymotrypsin substrate, a band of 29,000 daltons was visualized in pancreatic juice, whereas no activity was detected in cyst fluid. By incubation of the blotted cyst fluid proteins with 125I-labelled TATI, a pattern of bands at 25,000 and 28,000 daltons was detected identical to that obtained with the chromogenic substrate. PMID:2768384

Koivunen, E

1989-05-26

388

Detection of specific sequences among DNA fragments separated by gel electrophoresis  

Microsoft Academic Search

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

E. M. Southern

1975-01-01

389

One-dimensional SDS-polyacrylamide gel electrophoresis (1D SDS-PAGE).  

PubMed

This protocol describes a denaturing polyacrylamide gel system utilizing sodium dodecyl sulfate (SDS) to separate protein molecules based on size as first described by Laemmli (1970). SDS-PAGE can be used to monitor protein purifications, check the purity of samples, and to estimate molecular weights for unknown proteins. PMID:24674069

Brunelle, Julie L; Green, Rachel

2014-01-01

390

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

PubMed Central

By means of agarose-gel electrophoresis, endonuclease R·EcoRI-generated fragments of DNA from various viruses were separated, their molecular weights were determined, and complete or partial fragment maps for lambda, ?80, and hybrid phages were constructed. Images

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

1974-01-01

391

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

2002-01-01

392

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

2003-01-01

393

Fast molecular diagnostics of canine T-cell lymphoma by PCR and capillary gel electrophoresis with laser-induced fluorescence detector  

Microsoft Academic Search

Lymphoma is the most common hematopoietic tumor in dogs and manifests as a proliferation of malignant lymphoid cells primarily affecting the lymph nodes or solid visceral organs. We describe the use of capillary gel electrophoresis (CGE) with a laser-induced fluorescence (LIF) detector based on polymerase chain reaction (PCR) to rapidly detect a disorder of the canine T-cell receptor ? (TCR?)

Seonsook Jeon; Mi-Jin Lee; Jinho Park; Seong Ho Kang

2007-01-01

394

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

Microsoft Academic Search

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

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

1996-01-01

395

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

Microsoft Academic Search

The microbial community structure of cork with marked musty-earthy aromas was analyzed using dena- turing gradient gel electrophoresis of amplified ribosomal DNA. Cork stoppers and discs were used for DNA extraction and were analyzed by using selective primers for bacteria and fungi. Stoppers clearly differed from discs harboring a different fungal community. Moreover, musty-earthy samples of both types were shown

Chantal Prat; Olaya Ruiz-Rueda; Rosalia Trias; Enriqueta Antico; Dimitra Capone; Mark Sefton; Lluís Baneras

2009-01-01

396

Use of 18S rDNA PCR-Denaturing Gradient Gel Electrophoresis to Study Composition of Fungal Community in Two Patients with Intestinal Transplants  

Microsoft Academic Search

Fungi form a diverse microbial community in the human intestine. Little is known about the succession of species after intestinal transplantation. We investigated the alterations of the gut fungal population in two patients with intestinal allografts. The ileal effluent and feces were fingerprinted using denaturing gradient gel electrophoresis (DGGE), with confirmation by DNA sequencing. Analysis of 18S rDNA indicated that

Qiurong Li; Chenyang Wang; Qiang Zhang; Chun Tang; Ning Li; Bing Ruan; Jieshou Li

397

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

Microsoft Academic Search

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

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

1989-01-01

398

Quantification of DNA by Agarose Gel Electrophoresis and Analysis of the Topoisomers of Plasmid and M13 DNA Following Treatment with a Restriction Endonuclease or DNA Topoisomerase I  

ERIC Educational Resources Information Center

A two-session laboratory exercise for advanced undergraduate students in biochemistry and molecular biology is described. The first session introduces students to DNA quantification by ultraviolet absorbance and agarose gel electrophoresis followed by ethidium bromide staining. The second session involves treatment of various topological forms of…

Tweedie, John W.; Stowell, Kathryn M.

2005-01-01

399

Influence of one- and two-dimensional gel electrophoresis procedure on metal–protein bindings examined by electrospray ionization mass spectrometry, inductively coupled plasma mass spectrometry, and ultrafiltration  

Microsoft Academic Search

Three independent methods, (i) electrospray ionization mass spectrometry (ESI-MS), (ii) carrying out the complete protein preparation procedure required for protein gel electrophoresis (GE) including extraction, precipitation, washing, and desalting with subsequent microwave digestion of the produced protein fractions for metal content quantification, and (iii) ultrafiltration for separating protein-bound and unbound metal fractions, were employed to elucidate the influences of protein

Anne-Christine Schmidt; Bianca Störr; Nicolai-Alexeji Kummer

2011-01-01

400

Two early replicated, developmentally controlled genes of Physarum display different patterns of DNA replication by two-dimensional agarose gel electrophoresis  

Microsoft Academic Search

The nature of replication origins in eukaryotic chromosomes has been examined in some detail only in yeast, Drosophila, and mammalian cells. We have used highly synchronous cultures of plasmodia of the myxomycete Physarum and two-dimensional agarose gel electrophoresis to examine replication of two developmentally controlled, early replicated genes over time in S-phase. A single, discrete origin of replication was found

John D. Diller; Helmut W. Sauer

1993-01-01

401

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

PubMed Central

Double-stranded (ds), as well as denatured, single-stranded (ss) DNA samples can be analyzed on urea–agarose gels. Here we report that after denaturation by heat in the presence of 8 M urea, the two strands of the same ds DNA fragment of ?1–20-kb size migrate differently in 1 M urea containing agarose gels. The two strands are readily distinguished on Southern blots by ss-specific probes. The different migration of the two strands could be attributed to their different, base composition-dependent conformation impinging on the electrophoretic mobility of the ss molecules. This phenomenon can be exploited for the efficient preparation of strand-specific probes and for the separation of the complementary DNA strands for subsequent analysis, offering a new tool for various cell biological research areas.

Hegedus, Eva; Kokai, Endre; Kotlyar, Alexander; Dombradi, Viktor; Szabo, Gabor

2009-01-01

402

Detection of tetracysteine-tagged proteins using a biarsenical fluorescein derivative through dry microplate array gel electrophoresis.  

PubMed

The design of an extended-run 96-well sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system and the development of protein detection technology based upon fluorescein derivatives that bind to peptide epitope tags, allows the creation of a truly high-throughput analysis of protein expression, where less than 20 min are needed to separate proteins and analyze results. We demonstrate the overall capabilities of such a method combination in a complex cell lysate background, while comparing the specific results obtained using a biarsenical fluorescein-derivative and tetracysteine epitope-tagged proteins with total protein staining using a fluorescent gel stain and with Western blotting where an anti-oligohistidine (His) tag antibody has been employed. When applied on purified target proteins without extraneous protein background, the demonstrated sensitivity of the assay on the extended-run 96-array precast SDS-PAGE system allows detection of quantities of tagged protein as low as 1 pmol per band. PMID:15300761

Feldman, Galia; Bogoev, Roumen; Shevirov, Julia; Sartiel, Adam; Margalit, Ilana

2004-08-01

403

[Progress in combination of gel electrophoresis and laser ablation inductively coupled plasma mass spectrometry for trace elements determination in proteins].  

PubMed

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has become a very efficient and sensitive trace, ultratrace, and surface analytical technique for the in situ study of the concentration and distribution of the elements in life sciences with high spatial resolution. It is being used more and more frequently in biological, medical materials and protein research, which will lead to a better understanding of physiology and pathology process in cells and tissues. The present review mainly introduces the strategies of combination of gel electrophoresis (GE) with LA-ICP-MS for the quantification of trace elements in proteins, including the proteins separation, elements detection and calibration methods. The paper emphasizes the basic conditions of the proteins separation, focusing on the stability of proteins during GE and the treatment methods of staining and drying of the gel to enable successful detection of the elements by LA-ICP-MS. In addition, the application of GE-LA-ICP-MS in phosphoproteins, selenoproteins and metal-binding proteins is introduced in detail. The prospects and challenge for this technique are discussed as well for further study. PMID:22497164

Wang, Ying; Guo, Yan-li; Yuan, Hong-lin; Wei, Yong-feng; Yan, Hong-tao; Chen, Hui-hui

2012-01-01

404

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

PubMed

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

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

2013-01-01

405

SDS-polyacrylamide gel electrophoresis of buffalo bulls seminal plasma proteins and their relation with semen freezability.  

PubMed

The objective of this study was to evaluate the protein profiles of seminal plasma in buffalo bulls and to examine their correlation with semen characteristics. Semen of 10 buffalo bulls were collected by a bovine artificial vagina. Semen characteristics (motility, morphology, viability and concentration) were recorded. A part of the semen sample (1 ml) was diluted by tris-egg yolk-glycerol extender, packed in French straws and was frozen in liquid nitrogen. The straws were later thawed and semen characteristics were compared with those of the fresh semen. Seminal plasma was harvested by centrifugation; treated with cold ethanol and then, underwent SDS-polyacrylamide gel electrophoresis (PAGE). Twenty five protein bands were identified on the gel, of which those of <35.5 kDa were prominent (72% of the bands). Of these protein fractions, 24.5 kDa was significantly correlated with sperm progressive motility in fresh and viability in frozen-thawed semen while 45 kDa bands were correlated with abnormal morphology in frozen-thawed semen; 55 kDa protein fractions were correlated with sperm viability of fresh semen. Progressive motility, viability and abnormal sperm morphology of frozen-thawed semen were highly correlated with these parameters in the fresh semen. In conclusion, seminal plasma protein fractions in buffalo bulls are similar to those reported in other animal species and have some correlations with semen characteristics before and after freezing. PMID:17433580

Asadpour, R; Alavi-Shoushtari, S M; Rezaii, S Asri; Ansari, M H Kh

2007-12-01

406

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

2008-01-01

407

Analysis of high density lipoproteins by a modified gradient gel electrophoresis method  

Microsoft Academic Search

A high resolution electrophoretic method has been developed to separate plasma high density lipoprotein (HDL) particles by size using 4-3076 polyacrylamide agarose (PAA) gradient gels, Sudan black B staining, and laser densitometry. Fourteen distinct HDL bands were observed with HDL-1 being designated as the largest particle and HDL-14 as the smallest particle. HDL-1 was similar in size to ferritin (Stokes

Zhengling Li; Judith R. McNamara; Jose M. Ordovas; Ernst J. Schaeferl

408

Multimeric analysis of von Willebrand factor by molecular sieving electrophoresis in sodium dodecyl sulphate agarose gel.  

PubMed

We report the development and optimisation of an agarose gel electrophoretic method for the separation and detection of von Willebrand Factor (vWF) multimers. The method has been specifically developed for use in the clinical evaluation and classification of patients with von Willebrand's Disease (vWD) and clearly shows structural multimer abnormalities associated with the bleeding diathesis of this inherited bleeding disorder. PMID:2084949

Raines, G; Aumann, H; Sykes, S; Street, A

1990-11-01

409

Solubilization of membrane proteins for two-dimensional gel electrophoresis: identification of sarcoplasmic reticulum membrane proteins.  

PubMed

Solubilization of membrane proteins for two-dimensional electrophoresis (2DE) is very difficult. In this study, we report the use of 1,2-diheptanoyl-sn-glycero-3-phosphatdiyl choline (DHPC) as a detergent to solubilize integral membrane proteins for 2DE. Rat ventricular microsomal fractions enriched with sarco(endo)plasmic reticulum (SR) membrane proteins were used as a model system. Compatibility of DHPC with a high concentration of urea increases the solubility of proteins compared with sulphobetaines or ASB-14. Peptide mass analysis assisted in the identification of key SR membrane proteins including SR Ca(2+) ATPase and other membrane proteins, which have not previously been reported on 2DE. These results suggest that DHPC is a better detergent for solubilizing membrane proteins and may be useful in generating proteomic maps for most complex organelles including SR. PMID:14715292

Babu, Gopal J; Wheeler, Debra; Alzate, Oscar; Periasamy, Muthu

2004-02-01

410

Comparison of proteomes in various human plasma preparations by two-dimensional gel electrophoresis.  

PubMed

Serum or plasma can be utilized in a variety of studies targeted toward the discovery of disease biomarkers. In this study, the proteome profiles of plasma samples prepared using various anticoagulants (EDTA, heparin or citrate), were compared with those of serum using two-dimensional electrophoresis (2-DE). Proteins which evidenced different levels in the plasma and serum were screened and identified using ESI-Q-TOF MS/MS. The proteins which became detectable after the removal of fibrinogen from serum were identified as pigment epithelial differentiating factor (four spots), fetuin-like protein, and the hemopexin precursor. In particular, three proteins, pre-serum amyloid P component, plasma glutathione peroxidase precursor, and tetranectin, evidenced increased volume intensity only in the plasma samples prepared with EDTA. PMID:17368557

Kim, Hyun-Jung; Kim, Mi-Ryung; So, Eun-Jung; Kim, Chan-Wha

2007-06-10

411

An Economical Electrophoresis Apparatus  

ERIC Educational Resources Information Center

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

Andrews, I. M.

1975-01-01

412

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

PubMed

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

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

2014-01-01

413

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

PubMed Central

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

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

2013-01-01

414

Exogenous expression of human SGLT1 exhibits aggregations in sodium dodecyl sulfate polyacrylamide gel electrophoresis  

PubMed Central

Sodium/glucose co-transporter 1 (SGLT1), which actively and energy-dependently uptakes glucose, plays critical roles in the development of various diseases including diabetes mellitus and cancer, and has been viewed as a promising therapeutic target for these diseases. Protein-protein interaction with EGFR has been shown to regulate the expression and activity of SGLT1. Exogenous expression of SGLT1 is one of the essential approaches to characterize its functions; however, exogenously expressed SGLT1 is not firmly detectable by Western blot at its calculated molecular weight, which creates a hurdle for further understanding the molecular events by which SGLT1 is regulated. In this study, we demonstrated that exogenous SGLT1 functions in glucose-uptake normally but is consistently detected near the interface between stacking gel and running gel rather than at the calculated molecular weight in Western blot analysis, suggesting that the overexpressed SGLT1 forms SDS-resistant aggregates, which cannot be denatured and effectively separated on SDS-PAGE. Co-expression of EGFR enhances both the glucose-uptake activity and protein level of the SGLT1. However, fusion with Flag or HA tag at its carboxy- but not its amino-terminus abolished the glucose-uptake activity of exogenous SGLT1 without affecting its protein level. Furthermore, the solubility of SGLT1 aggregates was not affected by other detergents but was partially improved by inhibition of o-link glycosylation. These findings suggested exogenous overexpression of SGLT1 can function normally but may not be consistently detectable at its formula weight due to its gel-shift behavior by forming the SDS-resistant aggregates.

Huang, Wei-Chien; Hsu, Sheng-Chie; Huang, Shyh-Jer; Chen, Yun-Ju; Hsiao, Yu-Chun; Zhang, Weihua; Fidler, Isaiah J; Hung, Mien-Chie

2013-01-01

415

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

416

Novel application of PhastSystem polyacrylamide gel electrophoresis using restriction fragment length polymorphism--internal transcribed spacer patterns of individuals for molecular identification of entomopathogenic nematodes.  

PubMed

différences! [editorial] [editorial]onomic way of identifying and assigning nematodes to taxons, which had already been determined either by comparative sequence analysis of nuclear rDNA internal transcribed spacer (ITS) region or by other methods of molecular or conventional taxonomy, is provided. Molecular identification of entomopathogenic nematodes (EPN) can be upgraded by basing it on PhastSystem polyacrylamide gel electrophoresis (PAGE) analysis of restriction fragment length polymorphism (RFLP) patterns of polymerase chain reaction (PCR)-amplified DNA derived from single nematodes of Steinernema or Heterorhabditis spp. Although analysis from single worms has previously been made on agarose gel, the resolution on PhastSystem PAGE gel is much higher. The DNA sequences selected for analysis were those constituting the internal transcribed spacer region between the 18S and 26S rDNA genes within the rRNA operon. RFLP analysis was carried out by gel electrophoresis on the PhastSystem (Pharmacia) as detailed elsewhere (Triga et al., Electrophoresis 1999, 20, 1272-1277. The downscaling from conventional agarose to PhastSystem gels resulted in pattern of DNA fragments differing from those obtained with agarose gel electrophoresis under conventional conditions by increasing the number of detected fragments. The approach supported previous species identifications and was able to identify several unclassified isolates, such as those from Hungary and Ireland, and provides a method for identification of previously unclassified strains. We confirmed that Heterorhabditis "Irish Type", represented by two strains of different geographical origin, comprise a species different from H. megidis. We also confirmed that strain IS5 belongs to the species H. indicus rather than to H. bacteriophora, as had been suggested previously. PMID:10380767

Pamjav, H; Triga, D; Buzás, Z; Vellai, T; Lucskai, A; Adams, B; Reid, A P; Burnell, A; Griffin, C; Glazer, I; Klein, M G; Fodor, A

1999-06-01

417

A study of the antigenicity of Rickettsia helvetica proteins using two-dimensional gel electrophoresis.  

PubMed

Rickettsia helvetica is an obligate intracellular Gram-negative microorganism found in Ixodes ricinus ticks. When R. helvetica was first discovered in 1979, little was known about its physiology and it fell into oblivion until it recently was suspected of being pathogenic to humans. However, all efforts to isolate R. helvetica from patients have been unsuccessful, although serological responses against R. helvetica can be demonstrated. The aim of our study was to investigate the protein profile of R. helvetica and study the antigenicity of its proteins using two-dimensional (2D) immunoblot in order to characterize the immunological response against R. helvetica infection. Our results show that in addition to the known PS120 and OmpB antigenic R. helvetica proteins, three other antigens exist: a 60 kDa GroEL protein, a 10 kDa GroES protein and a hitherto unknown 35 kDa hypothetical protein that has similarities with ORF-RC0799 of Rickettsia conorii. Furthermore, the lipopolysaccharide showed strong antigenicity. In this study, we present the first proteome map and the first 2D immunoblot profile of R. helvetica and finally we present the 35 kDa R. helvetica as an additional antigen to the previously known rickettsial antigens. PMID:19338513

Hajem, Nedaa; Weintraub, Andrej; Nimtz, Manfred; Römling, Ute; Påhlson, Carl

2009-04-01

418

[Molecular epidemiological analysis of an outbreak of Campylobacter jejuni using restriction enzyme double-digestion technique for genotyping of isolates by pulsed-field gel electrophoresis].  

PubMed

In an outbreak of gastroenteritis in elementary school students and their families in Chiba Prefecture, Japan, Campylobacter jejuni was isolated from the stools of 14 patients who developed diarrheal illness after a one-day bus trip. C. jejuni was also isolated from the stools of 3 patients not going on the bus trip. Pulsed-field gel electrophoresis (PFGE) analysis was done on 17 isolates of C. jejuni to study genetic relationships among them. PFGE profiles of isolates treated with restriction enzymes Sma I, Ksp I and Kpn I were separated into 9, 10, and 10 types, but the relationship between PFGE profiles and epidemiological profiles was unclear. Dendrograms of PFGE of isolates double-digested with both Sma I and Ksp I were typed into D1, D2, D3 and D4, and profiles compared to profiles of serotyping and flagellin typing of isolates and epidemiological profiles to evaluate genetical and epidemiological relationships. Thirteen isolates of PFGE type D1 possessed serotype G and flagellin type Al and were isolated from patients going on the bus trip. Type D2 isolated from a student going on the bus trip and type D3 isolates from two students not going on the bus trip had serotype B and flagellin type A2. C. jejuni of PFGE type D4, serotype UT, and flagellin type A3 was also isolated from a student not going on the trip. Our results show that at least two outbreaks of C. jejuni occurred simultaneously in people related to the school. Restriction enzyme double-digestion PFGE was thus useful in the molecular epidemiological analysis of the C. jejuni outbreak. PMID:17176857

Yoda, Kiyoe; Yokoyama, Eiji; Uchimura, Masako

2006-11-01

419

High-throughput gender determination using automated denaturant gel capillary electrophoresis.  

PubMed

Sex determination of anonymous samples is a requirement before analysis of DNA variation on X or Y chromosomes. Based on this, we designed a method for screening samples on different DNA capillary sequencing instruments with a sensitivity that is able to quantify sex chromosome abnormalities. The two different amelogenin alleles sited on the X and Y chromosomes were polymerase chain reaction amplified with the same set of primers and separated by denaturant capillary electrophoresis (DCE). Sex chromosome ratios could be reproducibly determined with a relative standard deviation of 8.7%, which is sufficient to distinguish a normal XY karyotype from an XYY karyotype associated with Klinefelter syndrome. Reconstruction experiments demonstrated sensitivity down to a simulated Y:X allelic ratio of 1:127 in all three instruments, enabling the prediction of sex chromosomal aneuploidies. When tested on anonymous pooled and single samples, DCE gave a good prediction of the m