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1

How to Use 2D Gel Electrophoresis in Plant Proteomics.  

PubMed

Two-dimensional electrophoresis has nurtured the birth of proteomics. It is however no longer the exclusive setup used in proteomics, with the development of shotgun proteomics techniques that appear more fancy and fashionable nowadays.Nevertheless, 2D gel-based proteomics still has valuable features, and sometimes unique ones, which make it often an attractive choice when a proteomics strategy must be selected. These features are detailed in this chapter, as is the rationale for selecting or not 2D gel-based proteomics as a proteomic strategy. PMID:24136513

Rabilloud, Thierry

2014-01-01

2

Comparing 2-D Electrophoresis Gels Across Internet Databases  

Microsoft Academic Search

\\u000a In refs.\\u000a 1-3 and in the previous edition of this book, we described a computerassisted visual method, Flicker, for comparing two two-dimensional\\u000a (2-D) protein gel images across the Internet, http:\\/\\/www.lecb.ncifcrf.gov\\/flicker\\/. This approach may be useful for comparing similar samples created in different laboratories to help putatively identify\\u000a or suggest protein spot identification. Two-dimension gels and associated databases are increasingly appearing

Peter F. Lemkin; Gregory C. Thornwall

3

Independent component analysis of 2-D electrophoresis gels.  

PubMed

We present a novel application of independent component analysis (ICA), an exploratory data analysis technique, to two-dimensional electrophoresis (2-DE) gels, which have been used to analyze differentially expressed proteins across groups. Unlike currently used pixel-wise statistical tests, ICA is a data-driven approach that utilizes the information contained in the entire gel data. We also apply ICA on wavelet-transformed 2-DE gels to address the high dimensionality and noise problems typically found in 2-DE gels. Also, we use an analysis-of-variance (ANOVA) approach as a benchmark for comparison. Using simulated data, we show that ICA detects the group differences accurately in both the spatial and wavelet domains. We also apply these techniques to real 2-DE gels. ICA proves to be much faster than ANOVA, and unlike ANOVA it does not depend on the selection of a threshold. Application of principal component analysis reduces the dimensionality and tends to improve the performance by reducing the noise. PMID:18958894

Safavi, Haleh; Correa, Nicolle; Xiong, Wei; Roy, Anindya; Adali, Tülay; Korostyshevskiy, Valeriy R; Whisnant, Carol C; Seillier-Moiseiwitsch, Françoise

2008-10-01

4

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

2013-05-17

5

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

Microsoft Academic Search

BACKGROUND: Many proteomics initiatives require a seamless bioinformatics integration of a range of analytical steps between sample collection and systems modeling immediately assessable to the participants involved in the process. Proteomics profiling by 2D gel electrophoresis to the putative identification of differentially expressed proteins by comparison of mass spectrometry results with reference databases, includes many components of sample processing, not

Romesh Stanislaus; Liu Hong Jiang; Martha Swartz; John Arthur; Jonas S. Almeida

2004-01-01

6

Differential proteomic profiles from distinct Toxoplasma gondii strains revealed by 2D-difference gel electrophoresis.  

PubMed

Toxoplasma gondii is an obligate intracellular protozoan that infects mammals and birds. Human infection during pregnancy may cause severe damage to the fetus. Reactivation of latent infection in immunocompromised patients can cause life-threatening encephalitis. T. gondii strains are highly diverse but only a few lineages (Type I, II and III) are widely spread. In mouse model, Type I strains are highly virulent, whereas Type II and III strains are intermediately or non virulent. It is not clear how much quantitative difference exists in proteomic profiles among these distinct T. gondii lineages. In the present study, the proteomic profiles of T. gondii tachyzoites from these lineages were investigated by two dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS) technologies. A total of 2321 protein spots were detected. Overall, the GT1 strain of Type I lineage and the strain PTG of Type II lineage have highly similar proteomic profiles and both are different from that of the CTG strain of Type III lineage. Eighty-four protein spots were differentially expressed by greater than 1.5-fold in relative abundance and 10 of them were identified to 7 T. gondii proteins in existing database. Investigation of the quantitative differences in proteomics among distinct T. gondii strains should facilitate our understanding of difference in biological processes and pathogenesis of distinct T. gondii genotypes, which will provide basic information to determine treatment regimen for different manifestation of toxoplasmosis. PMID:23340323

Zhou, Huaiyu; Zhao, Qunli; Das Singla, Lachhman; Min, Juan; He, Shenyi; Cong, Hua; Li, Ying; Su, Chunlei

2013-01-20

7

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

ERIC Educational Resources Information Center

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

Kim, Thomas D.; Craig, Paul A.

2010-01-01

8

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

ERIC Educational Resources Information Center

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

Kim, Thomas D.; Craig, Paul A.

2010-01-01

9

Hsp27-2D-gel electrophoresis is a diagnostic tool to differentiate primary desminopathies from myofibrillar myopathies.  

PubMed

Small heat shock proteins prevent abnormal protein folding and accumulation. We analyzed the expression of hsp27 and alphaB-crystallin in skeletal muscle specimens of patients with desminopathies, plectinopathies, myotilinopathy, and other myofibrillar myopathies by means of differential centrifugation, 2D-gel electrophoresis, Western blotting, and mass spectrometry. Hsp27-P82 and -P15 as well as alphaB-crystallin-P59 and -P45 are the major serine phosphorylation isoforms in normal and diseased human skeletal muscle. 2D-gel-electrophoresis revealed spots of hsp27 in a range of pH 5.3-6.4 in samples of all skeletal muscle specimens, except for the seven desminopathies. They indicated a shift of the main hsp27-spot to alkaline pH degrees, which may help to differentiate primary desminopathies from other myopathies with structural pathology of the desmin cytoskeleton. PMID:15978589

Clemen, Christoph S; Fischer, Dirk; Roth, Udo; Simon, Stéphanie; Vicart, Patrick; Kato, Kanefusa; Kaminska, Anna M; Vorgerd, Matthias; Goldfarb, Lev G; Eymard, Bruno; Romero, Norma B; Goudeau, Bertrand; Eggermann, Thomas; Zerres, Klaus; Noegel, Angelika A; Schröder, Rolf

2005-07-01

10

Hsp27-2D-gel electrophoresis is a diagnostic tool to differentiate primary desminopathies from myofibrillar myopathies  

Microsoft Academic Search

Small heat shock proteins prevent abnormal protein folding and accumulation. We analyzed the expression of hsp27 and ?B-crystallin in skeletal muscle specimens of patients with desminopathies, plectinopathies, myotilinopathy, and other myofibrillar myopathies by means of differential centrifugation, 2D-gel electrophoresis, Western blotting, and mass spectrometry. Hsp27-P82 and -P15 as well as ?B-crystallin-P59 and -P45 are the major serine phosphorylation isoforms in

Christoph S. Clemen; Dirk Fischer; Udo Roth; Stéphanie Simon; Patrick Vicart; Kanefusa Kato; Anna M. Kaminska; Matthias Vorgerd; Lev G. Goldfarb; Bruno Eymard; Norma B. Romero; Bertrand Goudeau; Thomas Eggermann; Klaus Zerres; Angelika A. Noegel; Rolf Schröder

2005-01-01

11

Two-dimensional differential in gel electrophoresis (2D-DIGE) analysis of grape berry proteome during postharvest withering.  

PubMed

The practice of postharvest withering is commonly used to correct quality traits and sugar concentration of high quality wines. To date, changes in the metabolome during the berry maturation process have been well documented; however, the biological events which occur at the protein level have yet to be fully investigated. To gain insight into the postharvest withering process, we studied the protein expression profiles of grape (Corvina variety) berry development focusing on withering utilizing a two-dimensional differential in gel electrophoresis (2D-DIGE) proteomics approach. Comparative analysis revealed changes in the abundance of numerous soluble proteins during the maturation and withering processes. On a total of 870 detected spots, 90 proteins were differentially expressed during berry ripening/withering and 72 were identified by MS/MS analysis. The majority of these proteins were related to stress and defense activity (30%), energy and primary metabolism (25%), cytoskeleton remodelling (7%), and secondary metabolism (5%). Moreover, this study demonstrates an active modulation of metabolic pathways throughout the slow dehydration process, including de novo protein synthesis in response to the stress condition and further evolution of physiological processes originated during ripening. These data represent an important insight into the withering process in terms of both Vitis germplasm characterization and knowledge which can assist quality improvement. PMID:20945943

Di Carli, Mariasole; Zamboni, Anita; Pè, Mario Enrico; Pezzotti, Mario; Lilley, Kathryn S; Benvenuto, Eugenio; Desiderio, Angiola

2010-12-01

12

Separation and identification of hen egg protein isoforms using SDS–PAGE and 2D gel electrophoresis with MALDI-TOF mass spectrometry  

Microsoft Academic Search

Knowledge of the chemical composition and physicochemical properties of native egg white and yolk is necessary to interpret the functional and biological properties attributed to specific egg components. To date, many of the proteins located in this complex biological fluid remain uncharacterised, if not unknown. High-resolution techniques for proteome analysis, including SDS–PAGE and 2-dimensional (2D) gel electrophoresis, combined with mass

Vassilios Raikos; Rasmus Hansen; Lydia Campbell; Stephen R. Euston

2006-01-01

13

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

PubMed Central

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

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

2010-01-01

14

Normalization and expression changes in predefined sets of proteins using 2D gel electrophoresis: A proteomic study of L-DOPA induced dyskinesia in an animal model of Parkinson's disease using DIGE  

Microsoft Academic Search

BACKGROUND: Two-Dimensional Difference In Gel Electrophoresis (2D-DIGE) is a powerful tool for measuring differences in protein expression between samples or conditions. However, to remove systematic variability within and between gels the data has to be normalized. In this study we examined the ability of four existing and four novel normalization methods to remove systematic bias in data produced with 2D-DIGE.

Kim Kultima; Birger Scholz; Henrik Alm; Karl Sköld; Marcus Svensson; Alan R. Crossman; Erwan Bezard; Per E. Andrén; Ingrid Lönnstedt

2006-01-01

15

Capillary HPLC–ICP MS mapping of selenocompounds in spots obtained from the 2-D gel electrophoresis of the water-soluble protein fraction of selenized yeast  

Microsoft Academic Search

A method based on ICP collision-cell MS detection in capillary HPLC was developed to gain an insight into the purity and identity of selenium-containing proteins separated by 1-D and 2-D electrophoresis. The bands and spots obtained after the separation of water-soluble proteins in selenized yeast were digested with trypsin prior to chromatography. Selenium could be detected down to the subpicogram

Laure Tastet; Dirk Schaumlöffel; Brice Bouyssiere; Ryszard Lobinski

2006-01-01

16

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.

17

Difference gel electrophoresis.  

PubMed

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

Timms, John F; Cramer, Rainer

2008-12-01

18

Enhanced Sensitivity Employing Zwitterionic and pI Balancing Dyes (Z-CyDyes) Optimized for 2D-Gel Electrophoresis Based on Side Chain Modifications of CyDye Fluorophores. New Tools For Use in Proteomics and Diagnostics.  

PubMed

The CyDye family of fluorescent dyes is currently the overwhelming choice for applications in proteomic analysis, using two-dimensional difference gel electrophoresis (2D-DIGE). Protein labeling with CyDyes is hampered by protein precipitation and gel smearing when used above minimal labeling. The solubility of labeled protein may be improved by introducing water solubilizing groups on the dye such as cysteic acids. However, addition of a negatively charged functionality will have the undesired effect of shifting the pI in relation to the unlabeled protein. These limitations have been addressed through the synthesis of highly water-soluble and pI balancing zwitterionic CyDye fluorophores (Z-CyDyes). The new dyes feature a cysteic acid motif, a titratable amine functionality and a NHS activated ester group. In side by side 2D-DIGE comparisons of Z-CyDyes and CyDyes, the new dyes significantly enhanced protein spot volume and the number of spots that were detected. Z-CyDyes have the potential to enhance the depth of proteome coverage and provide a general strategy for improving the performance of protein tagging reagents. PMID:23941326

Epstein, Mark G; Reeves, Benjamin D; Maaty, Walid S; Fouchard, David; Dratz, Edward A; Bothner, Brian; Grieco, Paul A

2013-08-13

19

Rotor for Centrifugal Testing of Electrophoresis Gel.  

National Technical Information Service (NTIS)

The patent application describes a rotor for the preparation of identical electrophoresis gels where the liquid is permitted to flow into a rotor bowl under dynamic conditions. The rotor comprises a cylindrical body, a sealable closure, a central core, an...

1974-01-01

20

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

21

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

22

DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.  

SciTech Connect

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

SUTHERLAND,B.M.; BENNETT,P.V.; SUTHERLAND, J.C.

2004-03-24

23

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

24

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

25

Quantitative and reproducible two-dimensional gel analysis using Phoretix 2D Full.  

PubMed

Quantitative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is used to determine changes in individual protein levels in complex protein mixtures. To provide reliable data, the software used for 2-D gel image analysis must provide a linear response over a wide dynamic range of data output. Here, we show that Phoretix 2D Full analysis of 2-D gels stained with colloidal Coomassie Brilliant Blue G-250 can provide a linear measure of changes in protein quantity. We show using a complex mixture of Arabidopsis thaliana proteins, that this is true for essentially all focused proteins, in a data output range greater than three orders of magnitude. An analysis of the factors that affect errors in the results demonstrated that reproducibility of the data is significantly improved by user seeding, whereas it is reduced by use of the background subtraction algorithms. PMID:11465508

Mahon, P; Dupree, P

2001-06-01

26

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

Microsoft Academic Search

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

Sjouke Hoving; Hans Voshol; Jan van Oostrum

27

EXTRACTION METHODS FOR ANALYSIS OF CITRUS LEAF PROTEINS BY TWO-DIMENSIONAL GEL ELECTROPHORESIS  

Technology Transfer Automated Retrieval System (TEKTRAN)

A general procedure for two-dimensional (2-D) gel electrophoresis of Citrus leaves has been developed. We evaluated the resolution of 2-D protein patterns for Tris-HCl and KCl extractions of water soluble proteins and a phenol extraction for extraction of hydrophobic cell membrane proteins from Cha...

28

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

29

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

30

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

31

Zone Electrophoresis of Human Parotid Saliva in Acrylamide Gel.  

National Technical Information Service (NTIS)

The report concerns an examination of acrylamide gel as another medium for the zone electrophoresis of parotid saliva proteins. Preliminary experiments with acrylamide-gel strips yielded protein patterns in which twenty or more fractions could be visually...

T. S. Meyer B. L. Lamberts

1965-01-01

32

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

33

Protein gel electrophoresis in the undergraduate physics laboratory  

Microsoft Academic Search

We describe an undergraduate laboratory experiment in protein gel electrophoresis that uses readily available apparatus and materials. The separation of a mixture of stained proteins by gel electrophoresis was videotaped. Position-time data for the proteins generated from analysis of digitized videotape images allowed for calculation of protein terminal velocities. The dependence of protein terminal velocity on molar mass was determined

Danny G. Miles; David W. Bushman; Zhong-Ying Chen

2005-01-01

34

Processing of DNA and Protein Electrophoresis Gels by Image Analysis  

Microsoft Academic Search

With recent legislation allowing for the registration of new cultivars, the analysis of DNA and protein electrophoresis gels is becoming increasingly important for cultivar identification. DNA fragments or proteins of different molecular weights are separated using electrophoresis, giving a series of bands with positions corresponding to the molecular weight. Image analysis of the gels removes much of the subjectivity of

Donald G. Bailey; C. Bruce Christie

35

Advances in agarose gel electrophoresis of serum lipoproteins  

Microsoft Academic Search

Agarose gel electrophoresis has been extensively employed by researchers to gain a greater understanding of lipoprotein biology and its relationship to cardiovascular disease. Advances in this technique have been made in the visualization and quantitation of separated lipoproteins, in the use of agarose gel electrophoresis for detection and quantitation of apolipoproteins of the separated lipoproteins, and in the detection of

Phillip Greenspan; Fei-wen Mao; Beung-Ho Ryu; Robert L. Gutman

1995-01-01

36

Inexpensive and Safe DNA Gel Electrophoresis Using Household Materials  

ERIC Educational Resources Information Center

|Gel electrophoresis is the single most important molecular biology technique and it is central to life sciences research, but it is often too expensive for the secondary science classroom or homeschoolers. A simple safe low-cost procedure is described here that uses household materials to construct and run DNA gel electrophoresis. Plastic…

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

2012-01-01

37

Inexpensive and Safe DNA Gel Electrophoresis Using Household Materials  

ERIC Educational Resources Information Center

Gel electrophoresis is the single most important molecular biology technique and it is central to life sciences research, but it is often too expensive for the secondary science classroom or homeschoolers. A simple safe low-cost procedure is described here that uses household materials to construct and run DNA gel electrophoresis. Plastic…

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

2012-01-01

38

New urinary EPO drug testing method using two-dimensional gel electrophoresis  

Microsoft Academic Search

We present a two-dimensional electrophoresis (2DE) method for the detection of the drug, recombinant erythropoietin (rHuEPO) in urine and its separation from endogenous erythropoietin (HuEPO). This method involves a one-step acetonitrile precipitation of the proteins in urine, addition of an internal standard, two-dimensional gel electrophoresis (2D PAGE), a single Western blot and chemiluminescent immunodetection.

Alamgir Khan; Jasmine Grinyer; Son T. Truong; Ed J. Breen; Nicolle H. Packer

2005-01-01

39

Nondenaturing electrophoresis of lipoproteins in agarose and polyacrylamide gradient gels  

SciTech Connect

The plasma lipoproteins frequently are classified according to density and/or electrophoretic mobility. The lipoprotein classes differ characteristically also in particle size and apolipoprotein composition. Each class is heterogeneous in size and composition as well. Nondenaturing electrophoresis in agarose gels and polyacrylamide gradient gels are complementary analytical methods for classification of lipoproteins and determining distribution profiles of the major classes. In addition, gradient gel electrophoresis (GGE) has a high resolving capability for subfractionating each class according to particle size. Combination of gel electrophoresis with immunoblotting yields information on heterogeneity in apolipoprotein distribution. 14 refs., 6 figs., 3 tabs.

Shore, V.G.

1989-12-19

40

Guidelines for reporting the use of gel electrophoresis in proteomics  

Microsoft Academic Search

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

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

2009-01-01

41

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

42

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

PubMed

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

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

2008-11-19

43

State-of-the-art two-dimensional gel electrophoresis: a key tool of proteomics research  

Microsoft Academic Search

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the most popular and versatile method of protein separation among a rapidly growing array of proteomics technologies. Based on two distinct procedures, it combines isoelectric focusing (IEF), which separates proteins according to their isoelectric point (pI), and SDS-PAGE, which separates them further according to their molecular mass. At present, 2D-PAGE is capable of simultaneously

Odile Carrette; Pierre R Burkhard; Jean-Charles Sanchez; Denis F Hochstrasser

2006-01-01

44

Plant protein isolation and stabilization for enhanced resolution of two-dimensional polyacrylamide gel electrophoresis  

Microsoft Academic Search

Two-dimensional polyacrylamide gel electrophoresis (2D–PAGE) is the common method of choice for proteomic analysis. By introducing several small changes, a method was developed that not only improved the resolution and reproducibility of 2D–PAGE but also shortened the time of analysis. Precipitation by alkaline phenol and methanol\\/ammonium acetate was the choice for protein extraction. However, instead of precipitating the proteins overnight

Annamraju D. Sarma; Nathan W. Oehrle; David W. Emerich

2008-01-01

45

A systematic study of field inversion gel electrophoresis.  

PubMed Central

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

Heller, C; Pohl, F M

1989-01-01

46

RNA conformational changes analyzed by comparative gel electrophoresis.  

PubMed

The study of biologically relevant native RNA structures is important to understand their cellular function(s). Native gel electrophoresis provides information about such native structures in solution as a function of experimental conditions. The application of native gel electrophoresis in a comparative manner allows to obtain precise information on relative angles subtended between given pair of stems in an RNA molecule. By adapting this approach, it is possible to obtain very specific structural information such as the amplitude of dihedral angles and helical rotation. As an example, we will describe how native gel electrophoresis can be used to study the folding of the S-adenosylmethionine (SAM) sensing riboswitch. PMID:24136609

Eschbach, Sébastien H; Lafontaine, Daniel A

2014-01-01

47

Diced electrophoresis gel assay for screening enzymes with specified activities.  

PubMed

We have established the diced electrophoresis gel (DEG) assay as a proteome-wide screening tool to identify enzymes with activities of interest using turnover-based fluorescent substrates. The method utilizes the combination of native polyacrylamide gel electrophoresis (PAGE) with a multiwell-plate-based fluorometric assay to find protein spots with the specified activity. By developing fluorescent substrates that mimic the structure of neutrophil chemoattractants, we could identify enzymes involved in metabolic inactivation of the chemoattractants. PMID:23581642

Komatsu, Toru; Hanaoka, Kenjiro; Adibekian, Alexander; Yoshioka, Kentaro; Terai, Takuya; Ueno, Tasuku; Kawaguchi, Mitsuyasu; Cravatt, Benjamin F; Nagano, Tetsuo

2013-04-16

48

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

49

Nondenaturing electrophoresis of lipoproteins in agarose and polyacrylamide gradient gels  

Microsoft Academic Search

The plasma lipoproteins frequently are classified according to density and\\/or electrophoretic mobility. The lipoprotein classes differ characteristically also in particle size and apolipoprotein composition. Each class is heterogeneous in size and composition as well. Nondenaturing electrophoresis in agarose gels and polyacrylamide gradient gels are complementary analytical methods for classification of lipoproteins and determining distribution profiles of the major classes. In

1989-01-01

50

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

51

Pulsed-field gel electrophoresis of bacterial chromosomes.  

PubMed

The separation of fragments of DNA by agarose gel electrophoresis is integral to laboratory life. Nevertheless, standard agarose gel electrophoresis cannot resolve fragments bigger than 50 kb. Pulsed-field gel electrophoresis is a technique that has been developed to overcome the limitations of standard agarose gel electrophoresis. Entire linear eukaryotic chromosomes, or large fragments of a chromosome that have been generated by the action of rare-cutting restriction endonucleases, can be separated using this technique. As a result, pulsed-field gel electrophoresis has many applications, from karyotype analysis of microbial genomes, to the analysis of chromosomal strand breaks and their repair intermediates, to the study of DNA replication and the identification of origins of replication. This chapter presents a detailed protocol for the preparation of Escherichia coli chromosomal DNA that has been embedded in agarose plugs, digested with the rare-cutting endonuclease NotI, and separated by contour-clamped homogeneous field electrophoresis. The principles in this protocol can be applied to the separation of all fragments of DNA whose size range is between 40 kb and 1 Mb. PMID:23913293

Mawer, Julia S P; Leach, David R F

2013-01-01

52

Electrophoresis in Polyvinyl-Alcohol Gel.  

National Technical Information Service (NTIS)

It was found that electrophoresis in polyvinyl-alcohol, although not completely equivalent to that in polyacrylamide, is entirely possible and can accordingly be employed as an alternative method in certain cases, e.g. difficulties of procurement of polya...

C. Reich H. Sieber

1967-01-01

53

Gel Electrophoresis of Gold-DNA Nano-Conjugates  

SciTech Connect

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

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

2006-01-10

54

Clustering of Clinical Strains of Helicobacter pylori Analyzed by Two-Dimensional Gel Electrophoresis  

Microsoft Academic Search

Strain variations of Helicobacter pylori have been tested by numerous methods and compared among different patient groups. The aim of this study was to investigate whether H. pylori expresses disease-specific proteins that can be detected by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). H. pylori strains isolated from duodenal ulcer, gastric cancer, and gastritis patients were analyzed. Extensive variation in spot

HELENA ENROTH; T. Akerlund; ANNA SILLEN; LARS ENGSTRAND

2000-01-01

55

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

2012-02-15

56

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

57

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

58

Protein gel electrophoresis in the undergraduate physics laboratory  

NASA Astrophysics Data System (ADS)

We describe an undergraduate laboratory experiment in protein gel electrophoresis that uses readily available apparatus and materials. The separation of a mixture of stained proteins by gel electrophoresis was videotaped. Position-time data for the proteins generated from analysis of digitized videotape images allowed for calculation of protein terminal velocities. The dependence of protein terminal velocity on molar mass was determined and found to agree with predictions made by current theory. We also introduce a model that draws on simple physical concepts to help students place the experimental results in context.

Miles, Danny G.; Bushman, David W.; Chen, Zhong-Ying

2005-12-01

59

Purification of DNA Oligos by Denaturing Polyacrylamide Gel Electrophoresis (PAGE).  

PubMed

After chemical synthesis, the oligonucleotide preparation contains the desired full-length oligonucleotide but also all of the DNA molecules that were aborted during each cycle in the synthesis, and the by-products generated during the chemical reactions. The purification of oligonucleotides is a critical step for demanding applications where the exact length or sequence of the oligonucleotide is important, or for oligonucleotides longer than 50 bases. There are several methods of increasing oligonucleotide purity, the choice of which will depend on modifications of the oligonucleotides and their intended use. Polyacrylamide gel purification (PAGE purification) is the method of choice when the highest percentage of full-length oligonucleotide is desired. This chapter describes a protocol for oligonucleotide purification using denaturing polyacrylamide gel electrophoresis, and includes oligonucleotide preparation, polyacrylamide gel electrophoresis, and purification from the gel slice by two different methods: by diffusion or by electroelution. This chapter also includes recommendations as well as protocol advice. PMID:24011037

Lopez-Gomollon, Sara; Nicolas, Francisco Esteban

2013-01-01

60

Online Tool for Analysis of Denaturing Gradient Gel Electrophoresis Profiles  

PubMed Central

We present an online tool (EquiBands, http://www.univie.ac.at/IECB/limno/equibands/EquiBands.html) that quantifies the matching of two bands considered to be the same in different samples, even when samples are applied to different denaturing gradient gel electrophoresis gels. With an environmental example we demonstrate the procedure for the classification of two bands of different samples with the help of EquiBands.

Huber, Florian; Peduzzi, Peter

2004-01-01

61

Online tool for analysis of denaturing gradient gel electrophoresis profiles.  

PubMed

We present an online tool (EquiBands, http://www.univie.ac.at/IECB/limno/equibands/EquiBands.html) that quantifies the matching of two bands considered to be the same in different samples, even when samples are applied to different denaturing gradient gel electrophoresis gels. With an environmental example we demonstrate the procedure for the classification of two bands of different samples with the help of EquiBands. PMID:15240327

Huber, Florian; Peduzzi, Peter

2004-07-01

62

Analysis of protein glycation using phenylboronate acrylamide gel electrophoresis  

Microsoft Academic Search

The incorporation of the specialized carbohydrate affinity ligand methacrylamido phenylboronic acid in polyacrylamide gels for SDS-PAGE analysis has been successful for the separation of carbohydrates and has here been adapted for the analysis of post-translationally modified proteins. While conventional SDS-PAGE analysis cannot distinguish between glycated and unglycated proteins, methacrylamido phenylboronate acrylamide gel electrophoresis (mP-AGE) in low loading shows dramatic retention

Marta P. Pereira Morais; Julia D. Mackay; Savroop K. Bhamra; J. Grant Buchanan; T. D. James; John S. Fossey; Jean M. H. van den Elsen

2010-01-01

63

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

64

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

NASA Astrophysics Data System (ADS)

Intact protein masses can be measured directly from immobilized pH gradient (IPG) isoelectric focusing (IEF) gels loaded with mammalian and prokaryotic samples, as demonstrated here with murine macrophage and Methanosarcina acetivorans cell lysates. Mass accuracy and resolution is improved by employing instruments which decouple the desorption event from mass measurement; e.g., quadrupole time-of-flight instruments. MALDI in-source dissociation (ISD) is discussed as a means to pursue top-down sequencing for protein identification. Methods have been developed to enzymatically digest all proteins in an IEF gel simultaneously, leaving the polyacrylamide gel attached to its polyester support. By retaining all gel pieces and their placement relative to one another, sample handling and tracking are minimized, and comparison to 2-D gel images is facilitated. MALDI-MS and MS/MS can then be performed directly from dried, matrix-treated IPG strips following whole-gel trypsin digestion, bottom-up methodology. Side-to-side proteomics, highlighting the link between virtual and classical 2-D gel electrophoresis, is introduced to describe a method whereby intact masses are measured from one side (the IEF gel), while proteins are identified based on analyses performed from the other side (the SDS-PAGE gel).

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

2005-02-01

65

Chromosomal localization of structural genes and regulators in wheat by 2D electrophoresis of ditelosomic lines  

Microsoft Academic Search

Among the 782 spots observed in two-dimensional gel electrophoresis of denatured proteins from etiolated wheat shoots, 185 were found to be variable between the euploid and 26 ditelosomic lines of ‘Chinese Spring’. Thirty-five structural genes were located on 17 chromosome arms. Numerous intensity changes showing alterations in protein levels were observed and led to the following statements: 1) regulators are

C. Colas des Francs; H. Thiellement

1985-01-01

66

Statistical analysis of denaturing gel electrophoresis (DGE) fingerprinting patterns  

Microsoft Academic Search

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

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

2002-01-01

67

DNA Electrophoresis in Agarose Gels: Mobility vs. Length Dependence  

Microsoft Academic Search

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

Afshin Beheshti; David van Winkle; Randolph Rill

2001-01-01

68

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

69

Analysis of RNA Folding by Native Polyacrylamide Gel Electrophoresis  

Microsoft Academic Search

Polyacrylamide gel electrophoresis under native conditions (native PAGE) is a well-established and versatile method for probing nucleic acid conformation and nucleic acid–protein interactions. Native PAGE has been used to measure RNA folding equilibria and kinetics under a wide variety of conditions. Advantages of this method are its adaptability, absolute determination of reaction endpoints, and direct analysis of conformational hetereogeneity within

Sarah A. Woodson; Eda Koculi

2009-01-01

70

Pulsed-field gel electrophoresis typing of Staphylococcus aureus isolates  

Technology Transfer Automated Retrieval System (TEKTRAN)

Pulsed-field gel electrophoresis (PFGE) is the most applied and effective genetic typing method for epidemiological studies and investigation of foodborne outbreaks caused by different pathogens, including Staphylococcus aureus. The technique relies on analysis of large DNA fragments generated by th...

71

Two-dimensional gel electrophoresis: vertical isoelectric focusing.  

PubMed

Two-dimensional gel electrophoresis (2-DE) is one of the most powerful tools for separating proteins based on their size and charge. 2-DE is very useful to separate two proteins with identical molecular weights but different charges, which cannot be achieved with just sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Here, a simpler and easier version of 2-DE is presented which is also faster than all the currently available techniques. In this modified version of 2-DE, isoelectric focusing is carried out in the first dimension using a vertical SDS-PAGE apparatus. Following the first-dimensional IEF, each individual lane is excised from the IEF gel and, after a 90° rotation, is inserted into a second-dimensional SDS-PAGE, which can be stained with Coomassie Brilliant Blue for protein analysis or immunoblotted for further analysis. This version of IEF can be run in less than 2 h compared to the overnight run required by O'Farrell's method. Difficult tube gel casting and gel extrusion as well as tube gel distortion are eliminated in our method. This method is simpler, faster, and inexpensive. Both dimensions can be done on the same SDS-PAGE apparatus, and up to ten samples can be run simultaneously using one gel. PMID:22585490

Dorri, Yaser

2012-01-01

72

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

73

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

PubMed Central

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

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

1982-01-01

74

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

75

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

76

Thermally reversible gels in electrophoresis. I: Matrix characterization.  

PubMed

Two series of thermally reversible, hydrogen-bonded gels have been characterized: 5% PVA-4% PEG and 5% PVA-0.04% borate gels. They both have extremely low melting points (16-17 degrees 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's in the PVA polymer should be engaged in H-bond formation. PMID:3154960

Righetti, P G; Snyder, R S

1988-01-01

77

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

PubMed

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

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

2006-10-01

78

Triple-spot proteins in two-dimensional gel electrophoresis.  

PubMed Central

A triple-spot pattern of polypeptides occurring in two-dimensional gel electrophoresis of proteins is described. The presence of a mutant protein, Pc 1 Duarte, which results in a splitting of all three polypeptides, is evidence that they are produced by the same gene. This pattern is seen in about 1% of the proteins from a variety of sources. Typically, about 50% of the protein occurs as a single major spot, the remainder occurring as two polypeptides with an additional negative charge and slightly different molecular weight. The reproducibility of this pattern implies a functional significance which is presently unknown. The implication of this configuration for patterns seen by one-dimensional gel electrophoresis is discussed. Images Fig. 1

Comings, D E; Peters, K E

1979-01-01

79

Pulsed-Field Gel Electrophoresis Typing of Staphylococcus aureus Isolates.  

PubMed

Pulsed-field gel electrophoresis (PFGE) is the most applied and effective genetic typing method for epidemiological studies and investigation of foodborne outbreaks caused by different pathogens, including Staphylococcus aureus. The technique relies on analysis of large DNA fragments generated by the cleavage of intact bacterial chromosomes with a rare cutting restriction enzyme, subsequently resolved by pulsed-field electrophoresis with periodic changes of the orientation of the electrical field across the gel. The high discriminatory power, improved reproducibility due to standardization of experimental protocols and data interpretation guidelines, and establishment of a national PFGE database of S. aureus profiles have made it a valuable means for global tracking of S. aureus infection sources and determination of genetic relatedness of outbreak isolates. PMID:24085692

He, Yiping; Xie, Yanping; Reed, Sue

2014-01-01

80

Polyphenol oxidase activity staining in polyacrylamide electrophoresis gels.  

PubMed

An analytical method allowing the detection of polyphenol oxidase activity on polyacrylamide gel electrophoresis (PAGE) is described. The method is rapid, sensitive and specific and is based on a coupling reaction between 4-tert-butyl-o-benzoquinone and the aromatic amine, 4-amino-N,N-diethylaniline sulphate. Catecholase activity of polyphenol oxidase appears as blue stained bands on a colourless background. PMID:9178091

Rescigno, A; Sollai, F; Rinaldi, A C; Soddu, G; Sanjust, E

1997-03-27

81

On-line combination of capillary isoelectric focusing and capillary non-gel sieving electrophoresis using a hollow-fiber membrane interface: a novel two-dimensional separation system for proteins  

Microsoft Academic Search

A novel two-dimensional (2D) separation system for proteins was reported. In the system, a piece of dialysis hollow-fiber membrane was employed as the interface for on-line combination of capillary isoelectric focusing (CIEF) and capillary non-gel sieving electrophoresis (CNGSE). The system is similar equivalent to two-dimensional polyacrylamide gel electrophoresis (2D PAGE), by transferring the principal of 2D PAGE separation to the

Hechun Liu; Chun Yang; Qing Yang; Weibing Zhang; Yukui Zhang

2005-01-01

82

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

83

Fluorescence two-dimensional difference gel electrophoresis and mass spectrometry based proteomic analysis of Escherichia coli.  

PubMed

Separation and relative quantitation of complex protein mixtures remain two of the most challenging aspects of proteomics. Here an advanced technique called fluorescence difference 2-D gel electrophoresis technology (2D-DIGE) has been applied to a model system study of the Escherichia coli proteome after benzoic acid treatment. The molecular weight and charge matched cyanine dyes enable pre-electrophoretic labelling of control and treated samples which are then mixed and run in the same gel. Pooled control and treated samples labelled with Cy trade mark 3 were used as an internal standard for both Cy5 labelled control and treated E. coli samples. Together with DeCyder trade mark imaging analysis software, more accurate quantitative analysis than conventional two-dimensional polyacrylamide gel electrophoresis was achieved. Using matrix-assisted laser desorption/ionization-time of flight and quadrupole-time of flight mass spectrometry a total of 179 differentially expressed protein spots were identified. These included enzymes, stress related and substrate (e.g. amino acids, maltose, ribose and TRP repressor) binding proteins. Of the spots analysed, 77% contained only one protein species per spot, hence the change in protein expression measured was solely attributed to the identified protein. Many membrane proteins and protein isoforms were identified indicating both adequate solubilization of E. coli samples and potential post-translational modification. The results indicate that the regulatory mechanisms following benzoic acid treatment of E. coli are far more complicated than hitherto expected. PMID:12469338

Yan, Jun X; Devenish, Angelica T; Wait, Robin; Stone, Tim; Lewis, Steve; Fowler, Sue

2002-12-01

84

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

85

Increase in local protein concentration by field-inversion gel electrophoresis  

PubMed Central

Background Proteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and non-specific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein separation efficiency. Enhancement of protein separation efficiency was investigated by implementing pulsed field-inversion gel electrophoresis (FIGE). Results Separation of model protein species and large protein complexes was compared between FIGE and constant field electrophoresis (CFE) in different percentages of PAGs. Band intensities of proteins in FIGE with appropriate ratios of forward and backward pulse times were superior to CFE despite longer running times. These results revealed an increase in band intensity per defined gel volume. A biphasic protein relative mobility shift was observed in percentages of PAGs up to 14%. However, the effect of FIGE on protein separation was stochastic at higher PAG percentage. Rat liver lysates subjected to FIGE in the second-dimension separation of two-dimensional polyarcylamide gel electrophoresis (2D PAGE) showed a 20% increase in the number of discernible spots compared with CFE. Nine common spots from both FIGE and CFE were selected for peptide sequencing by mass spectrometry (MS), which revealed higher final ion scores of all nine protein spots from FIGE. Native protein complexes ranging from 800 kDa to larger than 2000 kDa became apparent using FIGE compared with CFE. Conclusion The present investigation suggests that FIGE under appropriate conditions improves protein separation efficiency during PAGE as a result of increased local protein concentration. FIGE can be implemented with minimal additional instrumentation in any laboratory setting. Despite the tradeoff of longer running times, FIGE can be a powerful protein separation tool.

Tsai, Henghang; Low, Teck Yew; Freeby, Steve; Paulus, Aran; Ramnarayanan, Kalpana; Cheng, Chung-pui Paul; Leung, Hon-chiu Eastwood

2007-01-01

86

Analysis of inorganic polyphosphates by capillary gel electrophoresis.  

PubMed

This paper describes the development of a method that uses capillary gel electrophoresis (CGE) to analyze mixtures of inorganic polyphosphate ((P(i))(n)). Resolution of (P(i))(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 (P(i))(n) with n up to approximately 70; preparation and use of authentic standards enables the identification of peaks for (P(i))(n) with n = 1-10. The main advantages of this method over previously reported methods for analyzing mixtures of (P(i))(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 (P(i))(n) by suppressed conductivity. The method is useful for analyzing (P(i))(n) generated by the dehydration of P(i) at low temperature (125-140 degrees 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). PMID:20704373

Lee, Andrew; Whitesides, George M

2010-08-15

87

Separation of fluorescence-labelled terminal restriction fragment DNA on a two-dimensional gel (T-RFs-2D) - an efficient approach for microbial consortium characterization.  

PubMed

Fingerprinting techniques provide access to understanding the ecology of uncultured microbial consortia. However, the application of current techniques such as terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) has been hindered due to their limitations in characterizing complex microbial communities. This is due to that different populations possibly share the same terminal restriction fragments (T-RFs) and DNA fragments may co-migrate on DGGE gels. To overcome these limitations, a new approach was developed to separate terminal restriction fragments (T-RFs) of 16S rRNA genes on a two-dimensional gel (T-RFs-2D). T-RFs-2D involves restriction digestion of terminal fluorescence-labelled PCR amplified 16S rRNA gene products and their high-resolution separation via a two-dimensional (2D) gel electrophoresis based on the T-RF fragment size (1(st) D) and its sequence composition on the denaturing gradient gel (2(nd) D). The sequence information of interested T-RFs on 2D gels can be obtained through serial poly(A) tailing reaction, PCR amplification and subsequent DNA sequencing. By employing the T-RFs-2D method, bacteria with MspI digested T-RF size of 436 (±1) bp and 514 (±1) bp were identified to be a Lysobacter sp. and a Dehalococcoides sp. in a polychlorinated biphenyl (PCB) dechlorinating culture. With the high resolution of 2D separation, T-RFs-2D separated 63 DNA fragments in a complex river-sediment microbial community, while traditional DGGE detected only 41 DNA fragments in the same sample. In all, T-RFs-2D has its advantage in obtaining sequence information of interested T-RFs and also in characterization of complex microbial communities. PMID:21824243

Wang, Shanquan; He, Jianzhong

2011-08-08

88

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

89

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

90

Identification of fibrosis-relevant proteins using DIGE (difference in gel electrophoresis) in different models of hepatic fibrosis.  

PubMed

Proteomics became a more and more important technique for the large-scale analysis of proteins during the last years. Two-dimensional (2D) electrophoresis as a major tool of proteomics is a powerful method to compare two different biological stages (e. g. healthy and diseased tissue) and to find differences in their protein pattern. One major problem in proteomics is the gel to gel variation of two-dimensional gel electrophoresis, which could cause artefacts in the detection of expression differences. The "difference in gel electrophoresis" (DIGE) technique allows the separation of two proteomes in the same gel. The protein pools were labelled with different fluorescent dyes and equal amounts of protein were separated in the same gel. Another advantage of DIGE is the possibility to separate an internal standard labelled with a third dye in the same gel to allow quantitative expression analysis. We compared proteomes of three different fibrosis models with the appropriate control (tissue inhibitor of metalloproteinases-1 (TIMP-1) overexpressing HepG2 cells in comparison to a HepG2 control, freshly isolated HSC in comparison to activated HSC and healthy mouse liver in comparison to fibrotic mouse liver). Among the differentially expressed proteins several were already found to be relevant for fibrosis but we also detected some proteins like the selenium binding protein 2 which might be relevant for hepatic fibrosis. PMID:15650968

Henkel, C; Roderfeld, M; Weiskirchen, R; Scheibe, B; Matern, S; Roeb, E

2005-01-01

91

Dye removal, catalytic activity and 2D crystallization of chloroplast H(+)-ATP synthase purified by blue native electrophoresis.  

PubMed

The proton-ATP synthase of thylakoid membranes from spinach chloroplasts (CF(O)F(1)) and its subcomplexes CF(O) and CF(1) were isolated by blue native electrophoresis (BN-PAGE) [Neff, D. and Dencher, N.A. (1999) Biochem. Biophys. Res. Commun. 259, 569-575] and subsequently electroeluted from the gel. A method was developed to remove most of the dye Coomassie G-250 (CBG) using gel filtration, a prerequisite for many biophysical investigations. The dye was removed from the electroeluted CF(O)F(1), CF(O) or CF(1) and exchanged with the detergent CHAPS. ATP hydrolysis activity of CF(1) and ATP synthesis activity of reconstituted CF(O)F(1) were determined before and after dye removal. The secondary structure of CF(O) was studied by CD spectroscopy in the presence and the absence of the dye. CBG neither abolishes the catalytic activity of the isolated CF(O)F(1) and CF(1) nor affects the subunit composition and the high alpha-helical content of CF(O). In crystallization attempts, 2D arrays of CF(O)F(1) and of CF(O) before and after dye removal were obtained. In the aggregates of CF(O), circular structures with a mean diameter of 6.7 nm were observed. Our results indicate that the combination of BN-PAGE and dye removal by gel filtration is a suitable approach to obtain catalytically active protein complexes for further functional and structural characterization. PMID:10825454

Poetsch, A; Neff, D; Seelert, H; Schägger, H; Dencher, N A

2000-06-01

92

The Application of Polyacrylamide Gel Electrophoresis to the Characterization of Cholinesterase Enzymes Used in Detector Kits.  

National Technical Information Service (NTIS)

Vertical disc gel electrophoresis on polyacrylamide gel tubes has been applied to the separation and identification of cholinesterases from commercially available sources. Cholinesterase from horse serum, bovine erythrocyte, and electric eel was fractiona...

A. Goodman L. M. McCormack H. Martens

1977-01-01

93

Reconstructed protein arrays from 3D HPLC/tandem mass spectrometry and 2D gels: complementary approaches to Porphyromonas gingivalis protein expression  

PubMed Central

We compare typical qualitative protein identification data from two-dimensional (2D) polyacrylamide gel electrophoresis and reconstructed protein arrays, in the context of measuring protein expression by the Gram-negative periodontal pathogen Porphyromonas gingivalis. The arrays were assembled computationally from genome annotations and tandem mass spectrometry data from an off-line HPLC fractionation combined with 2D capillary HPLC analysis of whole proteome enzymatic digests. The 2D separation was carried out with a standard binary gradient HPLC system, modified only slightly with readily available components. Compared to 2D gels, the number of annotated open reading frames identified using the 3D HPLC approach was typically larger by at least a factor of 30. However, the newer technology is currently limited in its ability to reflect the many protein variants derived from posttranscriptional and posttranslational processing.

Wang, Tiansong; Zhang, Yi; Chen, Weibin; Park, Yoonsuk; Lamont, Richard J.

2009-01-01

94

Detection of protein-protein interactions and a group of immunoglobulin G-associated minor proteins in human plasma by nondenaturing and denaturing two-dimensional gel electrophoresis.  

PubMed

The dissociation of noncovalently associated protein-protein complexes in human plasma was examined by comparing two-dimensional gel electrophoresis (2-DE) patterns obtained in two different electrophoretic conditions. A type I 2-DE pattern was obtained running nondenaturing isoelectric focusing (IEF) followed by nondenaturing gel electrophoresis and a type II 2-DE pattern was nondenaturing IEF followed by sodium dodecyl sulfate gel electrophoresis. Micro-sized gels (internal diameter(id) 1.3 x 35 mm polyacrylamide IEF gels and 38 x 38 x 1 mm polyacryamide slab gels) were used to follow the dissociation processes of major plasma proteins. Larger gel sizes (id 3.4 x 160 mm agarose IEF gels and 160 x 120 x 2.8 mm polyacrylamide slab gels) were used to detect minor plasma proteins dissociated from major proteins. About 110 spots, which have not been detected on type I (nondenaturing) 2-D gels, newly appeared on type II large-sized 2-D gels at molecular masses smaller than 67 kDa. Some of these spots had been analyzed and identified, but about 70 minor spots (isoelectric point 5.5-7.5 and relative molecular mass 8-45 kDa) were detected for the first time by applying large volumes of human plasma samples to the large type II 2-D gels. These minor spots could be concentrated on type II 2-D gels by enriching the immunoglobulin G (IgG) fraction under nondenaturing conditions, and they disappeared when IgG was removed from the fraction. These results strongly suggest that many of the minor spots newly detected were bound to IgG in physiological conditions. PMID:12833506

Manabe, Takashi; Yamaguchi, Nao; Mukai, Jun; Hamada, Osamu; Tani, Osamu

2003-06-01

95

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

Microsoft Academic Search

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

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

96

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

PubMed

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

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

1985-06-01

97

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

SciTech Connect

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

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

1980-08-10

98

Setaria digitata in cattle of Thailand identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis.  

PubMed

Adult Thai Setaria worms collected from cattle which were bred, housed and slaughtered in Thailand were morphologically identified as Setaria digitata. Furthermore, in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) adult Thai S. digitata had the same protein profiles as adult Japanese S. digitata, but did not possess the protein with a molecular size of 69 kDa which was confirmed in adult S. marshalli. In addition, there were no differences in the protein profiles between male and female S. digitata. In point of the distribution pattern of the proteins ranging from 73 to 64 kDa revealed by 2D-PAGE, there were no differences between Thai and Japanese S. digitata, and between male and female worms of the species. PMID:10342300

Subhachalat, P; Shirasaka, S; Nakajima, H; Adachi, Y

1999-04-01

99

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

2011-06-25

100

Differential detergent fractionation of isolated hepatocytes: biochemical, immunochemical and two-dimensional gel electrophoresis characterization of cytoskeletal and noncytoskeletal compartments.  

PubMed

Two-dimensional (2-D) gel electrophoresis is often used in toxicologic and metabolic studies to assess treatment- or stage-specific changes in protein synthesis, degradation or posttranslational modification. When combined with cell fractionation studies the detectability of low abundance proteins is enhanced, and changes in subcellular distribution of proteins can also be monitored. Detergent fractionation is a simpler alternative to differential pelleting, which partitions cellular constituents into functionally distinct populations while preserving cytoskeletal integrity. We defined and characterized a differential detergent fractionation (DDF) protocol to enable protein dynamics in cytoskeletal and noncytoskeletal compartments of isolated hepatocytes to be monitored simultaneously. Rat hepatocytes were maintained in suspension culture and fractionated by sequential extraction with detergent-containing buffers (digitonin/EDTA, Triton/EDTA, Tween/deoxycholate). DDF reproducibly yielded four electrophoretically distinct fractions enriched in cytosolic, membrane-organelle, nuclear membrane and cytoskeletal-matrix markers, respectively. Immunoblotting with over 20 different antibodies corroborated the selectivity of fractionation and was used to characterize the distribution profiles of cytoskeletal (actin, tubulins, cytokeratins, vinculin, myosin, desmoplakins, fodrin, nuclear lamins) and noncytoskeletal proteins (heat-shock 70 proteins, glutathione-S-transferase, calpains, carbamoyl phosphate synthetase, etc.), as well as to identify spots in 2-D gels. Detergent buffers were compatible with equilibrium or nonequilibrium 2-D gel electrophoretic analysis. Extensive 2-D maps of acidic and basic proteins in each fraction were generated along with a tabular listing of M(r) and pI. Thus, DDF reproducibly partitions hepatocytic proteins into functionally distinct cytoskeletal and noncytoskeletal compartments that are readily analyzed by 2-D gel electrophoresis. DDF is simple, applicable to use with other cell types or culture systems and is especially useful when biomaterial is limited (i.e., clinical studies). PMID:8026443

Ramsby, M L; Makowski, G S; Khairallah, E A

1994-02-01

101

Dye removal, catalytic activity and 2D crystallization of chloroplast H +ATP synthase purified by blue native electrophoresis  

Microsoft Academic Search

The proton-ATP synthase of thylakoid membranes from spinach chloroplasts (CFOF1) and its subcomplexes CFO and CF1 were isolated by blue native electrophoresis (BN-PAGE) [Neff, D. and Dencher, N.A. (1999) Biochem. Biophys. Res. Commun. 259, 569–575] and subsequently electroeluted from the gel. A method was developed to remove most of the dye Coomassie G-250 (CBG) using gel filtration, a prerequisite for

Ansgar Poetsch; Dirk Neff; Holger Seelert; Hermann Schägger; Norbert A. Dencher

2000-01-01

102

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

103

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

Microsoft Academic Search

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

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

2001-01-01

104

Comparative analysis of denaturing gradient gel electrophoresis and temporal temperature gradient gel electrophoresis in separating Escherichia coli uidA amplicons differing in single base substitutions.  

PubMed

A set of Escherichia coli freshwater isolates was chosen to compare the effectiveness of denaturing gradient gel electrophoresis (DGGE) vs temporal temperature gradient gel electrophoresis (TTGE) for separating homologous amplicons from the respective uidA region differing in one to seven single base substitutions. Both methods revealed congruent results but DGGE showed a five to eight times higher spatial separation of the uidA amplicons as compared with TTGE, although the experiments were performed at comparable denaturing gradients. In contrast to TTGE, DGGE displayed clear and focused bands. The results strongly indicated a significantly higher discrimination efficiency of the spatial chemical denaturing gradient as compared with the temporal temperature denaturing gradient for separating the uidA amplicons. Denaturing gradient gel electrophoresis proved to be highly efficient in the differentiation of E. coli uidA sequence types. PMID:10849270

Farnleitner, A H; Kreuzinger, N; Kavka, G G; Grillenberger, S; Rath, J; Mach, R L

2000-06-01

105

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

National Technical Information Service (NTIS)

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. Se...

D. A. McGregor

1993-01-01

106

Trapping of Megabase-Sized DNA Molecules during Agarose Gel Electrophoresis  

Microsoft Academic Search

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

Sergio Gurrieri; Steven B. Smith; Carlos Bustamante

1999-01-01

107

Evidence of Agar Gel Electrophoresis Changes of Lipoprotein-X after Phospholipase A and Deoxycholic Acid  

Microsoft Academic Search

The effect of phospholipase A from snake venom and deoxycholic acid on lipoprotein-X (LP-X) recovered from the cathode side of a previous agar gel electrophoresis is described. Adding phospholipase A and deoxycholic acid to the removed cathodal fraction is followed by a marked migration to the anode side on a second electrophoresis procedure. This seems to confirm that phospholipase A

J. C. Frisón; M. R. Ras; J. Rubiés-Prat; S. Masdeu; S. Schwartz

1977-01-01

108

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

Microsoft Academic Search

An optimized multilocus enzyme electrophoresis method, which involves polyacrylamide–agarose gel electrophoresis followed by electrophoretic transfers on nitrocellulose sheets, was developed for the analysis of enzyme polymorphism in several aerobic and anaerobic bacterial species including Staphylococcus aureus, Streptococcus pneumoniae, S. agalactiae, Klebsiella pneumoniae and K. oxytoca, Clostridium bifermentans and C. sordellii, and Prevotella bivia. Serial electrophoretic transfers (during 5–15 min each)

Marie-Laure Combe; Jean-François Lemeland; Martine Pestel-Caron; Jean-Louis Pons

2000-01-01

109

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

PubMed Central

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

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

2013-01-01

110

Analyses of mouse and Drosophila proteins by two-dimensional gel electrophoresis  

Microsoft Academic Search

Two-dimensional gel electrophoresis was employed for the protein analysis of several different mouse tissues and Drosophila. The number of protein spots detected with conventional protein dye staining techniques ranged from 110 in erythrocyte lysate to 320 in liver homogenate. Strain variation of protein spots on the gels was examined in five different tissues from two strains of inbred mice (DBA\\/2J

Chi-Yu Lee; Daniel Charles; Duane Bronson; Michael Griffin; Lawrence Bennett

1979-01-01

111

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

Microsoft Academic Search

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

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

2003-01-01

112

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

113

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

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

2012-01-01

114

DNA detection system using molecularly imprinted polymer as the gel matrix in electrophoresis  

Microsoft Academic Search

To develop a simple and inexpensive method for DNA detection, we prepared a molecularly imprinted polymer (MIP) for recognizing a specific double-stranded DNA (dsDNA) sequence and used it in an electrophoretic gel matrix. The MIP gel has many binding sites that are complementary in size, shape, and arrangement of functional groups of the target dsDNA sequence. During MIP gel electrophoresis

Masayo Ogiso; Norihiko Minoura; Toshio Shinbo; Toshimi Shimizu

2007-01-01

115

High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes.  

PubMed

Clear native electrophoresis and blue native electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native electrophoresis. However, clear native electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during electrophoresis and therefore has been used rarely. To preserve the advantages of both electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during electrophoresis. This improved clear native electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native electrophoresis. We demonstrate the superiority of high resolution clear native electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I-V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native electrophoresis make this technique superior for functional proteomics analyses. PMID:17426019

Wittig, Ilka; Karas, Michael; Schägger, Hermann

2007-04-09

116

Microscope slide electrophoresis of serum lipoproteins in agarose gel  

Microsoft Academic Search

An ultramicro electrophoretic technique for the separation of serum lipoproteins is described using agarose gel on microscope slides.The interaction between the agarose gel and lipoproteins has been reduced and the separation between ?- and pre-?-lipoproteins has been improved over existing methods. A semiquantitative assay of the lipoproteins can be done in a densitometer. It is suggested that the interaction between

M. C. Elphick

1971-01-01

117

High Resolution Clear Native Electrophoresis for In-gel Functional Assays and Fluorescence Studies of Membrane Protein Complexes  

Microsoft Academic Search

Clear native electrophoresis and blue native electro- phoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic ac- tivity assays. This problem can be overcome by omitting the dye in clear native electrophoresis. However, clear native electrophoresis suffers from enhanced protein

Ilka Wittig; Michael Karas; Hermann Schagger

2007-01-01

118

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

PubMed Central

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

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

2007-01-01

119

Molecular heterogeneity of classical and Duarte galactosemia: mutation analysis by denaturing gradient gel electrophoresis.  

PubMed

Classical galactosemia is caused by one common missense mutation (Q188R) and by several rare mutations in the galactose-1-phosphate uridyltransferase (GALT) gene. The most common variant of GALT, the Duarte variant, occurs as two types, Duarte-1 (D-1) and Duarte-2 (D-2), both of which carry the sequence change N314D. D-1 increases, whereas D-2 decreases GALT activity. To study the molecular genetics of classical and Duarte galactosemia, we analyzed the GALT mutations in 30 families with classical galactosemia, in 10 families with the D-2 variant and in 3 individuals carrying the D-1 allele by denaturing gradient gel electrophoresis (DGGE). DGGE detected 59 of the 60 classical galactosemia alleles. Q188R accounted for 60%, K285N accounted for 28% of these alleles. Eight novel candidate galactosemia mutations were found. On all D-2 alleles N314D occurred in cis with two intronic sequence changes, on the D-1 alleles in cis with a neutral mutation in exon 7. We conclude that the mutations causing galactosemia are highly heterogeneous and that K285N is a second common galactosemia mutation in our population. PMID:9222760

Greber-Platzer, S; Guldberg, P; Scheibenreiter, S; Item, C; Schuller, E; Patel, N; Strobl, W

1997-01-01

120

Determination of Human Serum Lipoprotein Patterns by Agarose Gel Electrophoresis.  

National Technical Information Service (NTIS)

An electrophoretic procedure is reported for the separation and detection of lipoproteins in serum. The technique utilizes agarose gel as supporting medium and oil red O dye for staining; it is rapid, very sensitive relatively simple, and inexpensive. In ...

N. M. Papadopoulos J. A. Kintzios

1969-01-01

121

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

122

Two-dimensional phosphate-affinity gel electrophoresis for the analysis of phosphoprotein isotypes.  

PubMed

Herein, we describe three kinds of 2-DE using phosphate-affinity PAGE for the analysis of phosphoprotein isotypes. The first dimension is a urea-PAGE, IEF/NEPHGE, or SDS-PAGE, which are widely used. The second dimension is a phosphate-affinity SDS-PAGE using a phosphate-binding tag molecule, Phos-tag (Mn(2+)-Phos-tag SDS-PAGE). The first 2-D procedure coupling urea-PAGE and Mn(2+)-Phos-tag SDS-PAGE was applied to the separation of beta-casein phosphoisotypes. A typical protein sample containing multiple phosphoisotypes from beta-casein (with five phosphorylation sites) was prepared by partial dephosphorylation with alkaline phosphatase. The second procedure coupling IEF/NEPHGE and Mn(2+)-Phos-tag SDS-PAGE was applied to the separations of phosphoisotypes of caseins and in vitro kinase reaction products of Tau. The third procedure coupling normal SDS-PAGE and Mn(2+)-Phos-tag SDS-PAGE was applied to the separation of A431 cell lysates before and after stimulation with an epidermal growth factor. This procedure followed by immunoblotting with anti-mitogen-activated protein kinase(MAPK) and anti-Shc antibodies demonstrated the detection of phosphoisotypes in each protein isoform of MAPK1/2 (44 and 42 kDa) and Shc (66, 52, and 46 kDa) after the stimulation. By these novel 2-D procedures, the separations of phosphoprotein isotypes should be improved relative to those by current gel electrophoresis methods, including 1-D Mn(2+)-Phos-tag SDS-PAGE. PMID:19156764

Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Matsubara, Mamoru; Aoki, Yuri; Ohie, Shiori; Mouri, Yuka; Koike, Tohru

2009-02-01

123

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

124

Characterization of DNA metabolizing enzymes in situ following polyacrylamide gel electrophoresis  

SciTech Connect

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

Longley, M.J.; Mosbaugh, D.W. (Oregon State Univ., Corvallis (United States))

1991-03-12

125

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

126

Pulsed-field gel electrophoresis for epidemiologic studies of Campylobacter hyointestinalis isolates.  

PubMed Central

Campylobacter hyointestinalis was isolated from five members of the same family who had previously consumed raw milk. Pulsed-field gel electrophoresis of genomic DNAs from the five strains, after digestion with restriction endonuclease SalI, revealed that three strains had identical genome patterns and therefore appeared to be related, whereas the other two had completely different genome patterns and appeared to be unrelated. We report here for the first time the isolation of C. hyointestinalis from family members who had consumed raw milk. Our study also demonstrates the usefulness of pulsed-field gel electrophoresis for epidemiologic studies of this unusual campylobacter. Images

Salama, S M; Tabor, H; Richter, M; Taylor, D E

1992-01-01

127

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

PubMed

Agarose gel electrophoresis of the plasmid deoxyribonucleic acids from 60 gram-negative bacilli recovered during investigations of nosocomial epidemics was used to fingerprint the strains. This method was as specific at differentiating bacterial strains as more conventional phenotyping methods. In all cases, plasmid band fingerprints of epidermic strains isolates were identical whereas coisolate plasmid deoxyribonucleic acid patterns were different. Agarose gel electrophoresis of plasmid deoxyribonucleic acid is proposed as a method which can be used in conventional microbiology laboratories as an adjunct to or, possibly, replacement for other methods of identifying bacterial strains. PMID:6788798

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

1981-06-01

128

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

129

In-gel phosphatase assay using non-denaturing two-dimensional electrophoresis.  

PubMed

We developed a method for detecting phosphatase activities in crude tissue extracts after separation of proteins by a novel non-denaturing two-dimensional electrophoresis. In the first dimension, protein samples were separated by a MicroRotofor, a liquid-phase isoelectric focusing, in the presence or absence of urea. In the second dimension, fractionated proteins by the MicroRotofor were resolved by a native polyacrylamide gel electrophoresis in the presence of 20 mM 2-mercaptoethanol. After electrophoresis, the polyacrylamide gel was directly immersed in a reaction mixture containing 4-methylumbelliferyl phosphate (MUP), a fluorogenic substrate, and phosphatase activities were detected as fluorescent bands. In this assay, a variety of phosphatase activities were clearly detected in gel when the tissue extracts were separated by the MicroRotofor in the presence of 1.5 M urea. Furthermore, after detecting phosphatase activities in polyacrylamide gel at neutral pH, its activities at acidic pH could be detected by immersing the gel in sodium citrate buffer (pH 3.0). Therefore, this method is a quite useful technique to analyze various phosphatases by sequential reactions with MUP under different conditions after sample separation by the two-dimensional electrophoresis. PMID:22992841

Baba, Hiromi; Masuda, Yukihiro; Sueyoshi, Noriyuki; Kameshita, Isamu

2012-09-19

130

Gel electrophoresis of linear and star-branched DNA.  

PubMed

The electrophoretic mobility of double-stranded DNA in polyacrylamide gel is investigated using an activated hopping model for the transport of a charged object within a heterogeneous medium. The model is premised upon a representation of the DNA path through the gel matrix as a series of traps with alternating large and small cross sections. Calculations of the trap dimensions from gel data show that the path imposes varying degrees of confinement upon migrating analytes, which retard their forward motion in a size-dependent manner. An expression derived for DNA mobility is shown to provide accurate predictions for the dynamics of linear DNA (67-622 bp) in gels of multiple concentrations. For star-branched DNA, the incorporation within the model of a length scale previously proposed to account for analyte architecture [Yuan et al., Anal. Chem. 78, 6179 (2006)] leads to mobility predictions that compare well with experimental results for a wide range of DNA shapes and molecular weights. PMID:22304125

Lau, Henry W; Archer, Lynden A

2011-12-22

131

Characterization of lymphoid cells by two-dimensional mini gel electrophoresis of proteins  

Microsoft Academic Search

Cytosol proteins from normal lymphocytes, leukemic lymphocytes, and different cultured lymphoid cell lines were separated by two-dimensional mini gel electrophoresis. By staining with Coomassie blue, specific protein patterns were obtained. Very similar gel maps were producted by the cytosol proteins of chronic lymphocytic leukemia cells, hairy cells, and of in vitro grown B cells. Protein 36\\/6.2 (molecular weight\\/isoelectric point), consistently

F. W. Hirsch; C. Bröckl; K. J. Bross; G. Dölken

1983-01-01

132

Isoelectric focusing--polynucleotide/polyacrylamide-gel electrophoresis. A technique to separate and characterize nuclease activities.  

PubMed Central

Individual native nuclease activities from human leucocytes are separated by using two-dimensional gel electrophoresis in an apparatus that allows the simultaneous running of 28 gels. Proteins are separated by isoelectric focusing in a disc gel, followed by electrophoresis into a slab gel containing DNA. Protein denaturants are avoided in the second dimension by the use of a running pH well above the optimal pH for DNAase (deoxyribonuclease) activity. Electrophoresed gels are incubated in appropriate buffers to activate nuclease activity. After staining for intact DNA, the positions of active enzymes, unobscured by the presence of other proteins, are revealed as colourless spots in a reddish-purple field. The technique is easy to use and is sensitive to 50pg of DNAase I. Versatility is provided by the use of either acidic or basic electrophoresis running buffers and by the use of specific gel incubation conditions to reveal different sets of enzyme activities. Two DNAases active at pH 7.4 in the presence of Mg2+ and Ca2+, and sixteen DNAases active at acidic pH and not requiring metals, are detected. Treatment of the human enzymes with specific glycosidases reveals that many of the human DNAases are glycoproteins containing negatively charged moieties and may be derived from modification of parent activities. Images Fig. 3. Fig. 4. Fig. 5.

Karpetsky, T; Brown, G E; McFarland, E; Brady, S T; Roth, W; Rahman, A; Jewett, P

1984-01-01

133

Characterization of bovine collagens using capillary electrophoresis — an alternative to slab gel electrophoresis  

Microsoft Academic Search

A capillary electrophoresis method was developed and characterized for analyzing the spectrum of collagen subspecies in collagen preparations. The Bio-Rad CE-SDS protein kit was used for the dynamic sieving separation of collagen subspecies in this CE method (DSCE). The optimized method utilized a 36 cm (or 24 cm)×50 ?m uncoated capillary, electrophoretic injection at 10 kV for 10 s, a

Paul Chang; Son Kuan; Gert Eberlein; David Burke; Richard Jones

2000-01-01

134

Thermal gradient gel electrophoresis analysis of bioprotection from pollutant shocks in the activated sludge microbial community  

Microsoft Academic Search

The authors used a culture-independent approach, namely, thermal gradient gel electrophoresis (TGGE) analysis of ribosomal sequences amplified directly from community DNA, to determine changes in the structure of the microbial community following phenol shocks in the highly complex activated sludge ecosystem. Parallel experimental model sewage plants were given shock loads of chlorinated and methylated phenols and simultaneously were inoculated (i)

CHRISTINE A. EICHNER; RAINER W. ERB; KENNETH N. TIMMIS; I. Wagner-Doebler

1999-01-01

135

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

Microsoft Academic Search

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

Sergio Gurrieri; D. Beach; C. Bustamante; E. Rizzarelli

1990-01-01

136

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

137

Pulsed-Field Gel Electrophoresis Study of Mycobacterium abscessus Isolates Previously Affected by DNA Degradation  

Microsoft Academic Search

DNA degradation (which results in a smear pattern) occurs with almost 50% of Mycobacterium abscessus strains during pulsed-field gel electrophoresis (PFGE). We assessed the potential benefit of using thiourea- containing buffer with M. abscessus by studying 69 isolates not previously typeable by PFGE (i.e., those with a smear pattern). Random (epidemiologically unrelated) isolates that were typeable (no smear pattern) were

Yansheng Zhang; Mitchell A. Yakrus; Edward A. Graviss; Natalie Williams-Bouyer

138

Comparison of Leuconostoc oenos Strains by Pulsed-Field Gel Electrophoresis  

PubMed Central

Pulsed-field gel electrophoresis of chromosomal DNA digested with NotI or SfiI was used to differentiate individual strains of Leuconostoc oenos. L. oenos isolates with 13 different restriction digest patterns were detected in New Zealand wines undergoing malolactic fermentation. The average genome size was estimated to be 1,800 kb. Images

Kelly, W. J.; Huang, C. M.; Asmundson, R. V.

1993-01-01

139

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

140

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

PubMed Central

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

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

2011-01-01

141

Biologist's perspective on analytical imaging systems as applied to protein gel electrophoresis  

Microsoft Academic Search

High-resolution two-dimensional polyacrylamide gel electrophoresis entails the separation of proteins in the first dimension according to their charge and in the second dimension according to their relative mobility (RF). The technique is capable of simultaneously resolving thousands of polypeptides as a constellation pattern of spots. Ultimately, the level of success in the analysis of such protein patterns depends upon the

Wayne F. Patton

1995-01-01

142

Assay of plant proteins with bicinchoninic acid for high resolution two-dimensional polyacrylamide gel electrophoresis  

Microsoft Academic Search

A method is described for estimating proteins in the same plant tissue sample that is solubilized for separation by two-dimensional polyacrylamide gel electrophoresis. The method uses a modified bicinchoninic acid (BCA) protein assay procedure and a modified standard urea solubilization buffer to estimate microgram values of unknown protein concentration, in the presence of 9 M urea and 4% Nonidet P-40,

Alan R. Orr; Brett A. Wagner; Catherine T. Howard; Orlando A. Schwartz

1988-01-01

143

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

144

DNA damage in exfoliated buccal cells of smokers assessed by the single cell gel electrophoresis assay  

Microsoft Academic Search

The alkaline single-cell gel electrophoresis assay or comet assay is a sensitive and rapid method for DNA strand breaks and detection of alkali labile sites at the single cell level, it further provides information on the presence of damage among individual cells. In this paper we explore the use of this technique utilizing exfoliated buccal mucosa cells from non-smokers (9

E. Rojas; M. Valverde; M. Sordo; P. Ostrosky-Wegman

1996-01-01

145

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

ERIC Educational Resources Information Center

|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…

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

2008-01-01

146

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

147

A Non-Denaturing Gel Electrophoresis System for the Purification of Membrane Bound Proteins  

Microsoft Academic Search

A new method is described for the purification of a membrane bound glycoprotein, the kappa opioid receptor from human placental tissue. The method uses preparative slab-gel electrophoresis in the presence of the non-denaturing detergent CHAPS. A linear relationship between log molecular weight and SDS PAGE electrophoretic mobility of known molecular weight markers, in the presence of CHAPS, is observed. Using

Anna G. Cavinato; Robert M. Macleod; Mahmoud S. Ahmed

1988-01-01

148

Enterotoxin D producing strains of Staphylococcus aureus are typeable by pulsed-field gel electrophoresis (PFGE)  

Microsoft Academic Search

Certain strains of Staphylococcus aureus (S. aureus) are capable of producing enterotoxins. In dairy products, these enterotoxins are the second major cause of foodborne disease in France. The main objective of this study was to type a selection of enterotoxigenic and non-enterotoxigenic strains of S. aureus using a reliable method, pulsed-field gel electrophoresis (PFGE), to explore the genetic relationships between

L. Villard; H. Lamprell; E. Borges; F. Maurin; Y. Noël; E. Beuvier; J. F. Chamba; A. Kodjo

2005-01-01

149

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

PubMed

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

Marcone, Carmine

2013-01-01

150

Two-dimensional acrylamide gel electrophoresis of cancer-patient serum proteins.  

PubMed

Sera from normal volunteers, patients with a variety of non-neoplastic diseases and patients with malignant or benign tumors were examined by two-dimensional acrylamide gel electrophoresis. In this technique the serum is first separated in an acrylamide gel column followed by a second electrophoresis at right angles to the first separation in a continuous concave 2 to 30 percent gradient acrylamide gel slab. The stained two-dimensional gel slab appears as a "fingerprint" pattern or "map" of the separated serum proteins. Both qualitative and quantitative differences in the fingerprint patterns of cancer-patient sera were observed. The quantitative alterations did not appear specifically associated with malignant tumors. However, several qualitative differences were detected, of which some may represent markers of malignancy as they were not observed in the normal, abnormal and benign tumors control sera. At least one of the abnormal protein stained spots, a prealbumin, appears to be restricted and related in some way to cancer of the lymphoreticular system. These data, although limited, support earlier indications that two-dimensional acrylamide gel electrophoresis offers a new and powerful tool to the cancer scientist for the detection of alterations in cancer-patient sera. A discussion of its possible use to examine other biological fluid and tumor extracts from the cancer patient is presented. PMID:4376659

Wright, G L

151

Identification of Frankia Strains by Two-Dimensional Polyacrylamide Gel Electrophoresis  

PubMed Central

Fifteen Frankia strains from five different plant species were analyzed by two-dimensional polyacryl-amide gel electrophoresis to determine their relatedness by comparing the polypeptide patterns obtained. Three major subgroups (A, C, and D) were found in the Alnus-Comptonia-Myrica cross-inoculation group. An isolate from Purshia tridentata had a unique protein pattern and represents a distinct group of frankiae. Members of group A were isolated from root nodules of Alnus incana subsp. rugosa and Alnus viridis subsp. crispa. Group C organisms were from A. incana subsp. rugosa and Comptonia peregrina nodules, and group D organisms were from A. incana subsp. rugosa, A. viridis subsp. cripsa, and Myrica pensylvanica root nodules. Isolates from each gel group were obtained at several widely separated geographical locations. The results indicate that two-dimensional polyacrylamide gel electrophoresis is useful for identifying Frankia isolates. Images

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

1984-01-01

152

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

PubMed

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

Gurrieri, S; Smith, S B; Bustamante, C

1999-01-19

153

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

154

Pinnacle: a fast, automatic and accurate method for detecting and quantifying protein spots in 2-dimensional gel electrophoresis data  

Microsoft Academic Search

Motivation: One of the key limitations for proteomic studies using 2- dimensional gel electrophoresis (2DE) is the lack of rapid, robust, and reproducible methods for detecting, matching, and quantifying protein spots. The most commonly used approaches involve first detecting spots and drawing spot boundaries on individual gels, then matching spots across gels, and finally quantifying each spot by calculating normalized

Jeffrey S. Morris; Brittan N. Clark; Howard B. Gutstein

2008-01-01

155

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

PubMed

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

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

2012-09-10

156

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

PubMed

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

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

2013-04-12

157

An automatic Method to identify and extract information of DNA bands in Gel Electrophoresis images.  

PubMed

This paper presents a system for the automatic processing of Digital Images obtained from Gel Electrophoresis. The system identifies automatically the number and the location of lanes in the digital image, as well as the location of bands on each lane, without any intervention from the user. A reference lane with a know substance is used to compute the molecular weight of the observed (unknown) bands. The system performance was tested using 12 images, obtained from 4 gels with 3 different exposures. A total of 5443 bands were tested in 12 images, 672 reference / observed lane pairs. The average error in the estimation of molecular weight of 9.2%. PMID:19963482

Caridade, C R; Margal, A S; Mendonga, T; Pessoa, A M; Pereira, S

2009-01-01

158

Analysis of Glucose6Phosphate Dehydrogenaseafter Purification usingGel Electrophoresis and Western Blotting  

Microsoft Academic Search

The matrix used in our work was a polyacrylamide gel of 5% concentration of the acrylamide monomer crosslinked to N,N'-methylene bisacrylamide (bisacrylamide). In order to estimate purity and molecular weight in electrophoresis the surfactant sodium dodecyl sulfate (SDS) is used. b-mercaptoethanol (BME) was applied to reduce the disulfide bonds and allow determination of migration by molecular weight only and not

Ivon Brito

2002-01-01

159

Analysis ofSalmonella typhiIsolates from Southeast Asia by Pulsed-Field Gel Electrophoresis  

Microsoft Academic Search

Pulsed-field gel electrophoresis (PFGE) revealed that multiple genetic variants of Salmonella typhi are simultaneously present in Southeast Asia and are associated with sporadic cases of typhoid fever and occa- sional outbreaks. Comparative analysis of PFGE patterns also suggested that considerable genetic diversity exists among S. typhi strains and that some PFGE patterns are shared between isolates obtained from Malaysia,Indonesia,andThailand,implyingmovementofthesestrainswithintheseregionsofSoutheastAsia, where

KWAI-LIN THONG; SAVITHRI PUTHUCHEARY; ROHANI M. YASSIN; PRATIWI SUDARMONO; MARIA PADMIDEWI; EDDY SOEWANDOJO; INDRO HANDOJO

160

Study on nuclear and cytoplasmic genome expression in wheat by two-dimensional gel electrophoresis  

Microsoft Academic Search

In this first analysis the protein patterns obtained by two-dimensional gel electrophoresis of 8 day-old leaves from 18 alloplasmic wheat lines are compared. From 440 spots retained on the basis of their reproducibility, 36 proteins were observed to vary in different cytoplasms, allowing us to distinguish the T. aestivum cytoplasm from 5 Aegilops cytoplasms. Twenty-four of the 36 variable proteins

M. Zivy; H. Thiellement; D. de Vienne; J.-P. Hofmann

1983-01-01

161

Study on nuclear and cytoplasmic genome expression in wheat by two-dimensional gel electrophoresis  

Microsoft Academic Search

Two-dimensional gel electrophoresis of denaturated proteins were performed at five developmental stages or organs (hereafter referred to as stage-organs) on two wheat lines with four different cytoplasms. Five hundred and fifty to 712 reproducible spots were scored depending on the stage-organ. Each stage-organ is unambiguously characterized and several types of control of protein quantity are recorded. Post-translational modifications are hypothetized

M. Zivy; H. Thiellement; D. de Vienne; J. P. Hofmann

1984-01-01

162

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

Microsoft Academic Search

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

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

163

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

Microsoft Academic Search

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

J. C. Auguet; H. Montanié; P. Lebaron

2006-01-01

164

Manual 768 or 384 well microplate gel 'dry' electrophoresis for PCR checking and SNP genotyping  

PubMed Central

Electrophoresis continues to be a mainstay in molecular genetic laboratories for checking, sizing and separating both PCR products, nucleic acids derived from in vivo or in vitro sources and nucleic acid–protein complexes. Many genomic and genetic applications demand high throughput, such as the checking of amplification products from many loci, from many clones, from many cell lines or from many individuals at once. These applications include microarray resource development and expression analysis, genome mapping, library and DNA bank screening, mutagenesis experiments and single nucleotide polymorphism (SNP) genotyping. PCR hardware compatible with industry standard 96 and 384 well microplates is commonplace. We have previously described a simple system for submerged horizontal 96 and 192 well polyacrylamide or agarose microplate array diagonal gel electrophoresis (MADGE) which is microplate compatible and suitable for PCR checking, SNP typing (restriction fragment length polymorphism or amplification refractory mutation system), microsatellite sizing and identification of unknown mutations. By substantial redesign of format and operations, we have derived an efficient ‘dry’ gel system that enables direct 96 pin manual transfer from PCR or other reactions in microplates, into 768 or 384 well gels. Combined with direct electrode contact in clamshell electrophoresis boxes which plug directly to contacts in a powered stacking frame and using 5–10 min electrophoresis times, it would be possible (given a sufficient supply of PCRs for examination) for 1 million gel tracks to be run per day for a minimal hardware investment and at minimal reagent costs. Applications of this system for PCR checking and SNP genotyping are illustrated.

Gaunt, Tom R.; Hinks, Lesley J.; Rassoulian, Hamid; Day, Ian N. M.

2003-01-01

165

Sodium dodecyl sulfate-agarose gel electrophoresis of urinary proteins: application to multiple myeloma  

Microsoft Academic Search

We evaluated a new sodium dodecyl sulfate-agarose gel electrophoresis (SDS-AGE) for urinary protein analysis in patients with multiple myeloma (MM; n 5 47; ages, 62 6 2 years, mean 6 SE). Abnormal proteinuria (mean 5 1872 6 360 mg\\/24 h) was present in 95% of the samples; 75% of the patients had some sign of renal dysfunction (glomerular and\\/or tubular)

Thierry Le Bricon; Danielle Erlich; Djaouida Bengoufa; Michelle Dussaucy; Jean-Pierre Garnier; Bernard Bousquet

166

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

Microsoft Academic Search

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

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

2001-01-01

167

Sodium Dodecyl Sulfate-Polyacrylamide Gel Protein Electrophoresis of Freshwater Photosynthetic Sulfur Bacteria  

Microsoft Academic Search

Sodium dodecyl sulfate-polyacrylamide gel protein electrophoresis (SDS-PAGE) was carried out using different bacterial strains\\u000a of the photosynthetic sulfur bacteria Chlorobium, Thiocapsa, Thiocystis, and Chromatium cultured in the laboratory, and the natural blooms in two karstic lakes (Lake Cisó and Lake Vilar, NE Spain) where planktonic\\u000a photosynthetic bacteria (purple and green sulfur bacteria) massively developed accounting for most of the microbial

M. Begoña Osuna; Emilio O. Casamayor

2011-01-01

168

Rapid Detection of Globin Gene Mutations and Polymorphisms by Temporal Temperature Gradient Gel Electrophoresis  

Microsoft Academic Search

Background: Inherited hemoglobin disorders represent the most common Mendelian disease worldwide. Pre- vention programs based on molecular diagnosis of het- erozygous carriers and\\/or patients require the use of reliable mutation scanning methods in at-risk popula- tions. Methods: We developed a rapid and highly specific mutation-screening test based on temporal temperature gradient gel electrophoresis (TTGE). We analyzed 889 -thalassemia genes from

Ramachandran V. Shaji; Eunice Sindhuvi Edison; Balasubramanian Poonkuzhali; Alok Srivastava; Mammen Chandy

169

Determination of Serum Lipoproteins in Clinically Healthy Iranian Crossbred Cattle by Agarose Gel Electrophoresis  

Microsoft Academic Search

Nazifi, S., Saeb, M. and Rowghani, E. 2003. Determination of serum lipoproteins in clinically healthy Iranian crossbred cattle by agarose gel electrophoresis. J. Appl. Anim. Res., 23: 59–64.To determine the lipoprotein components of the serum, blood samples were collected from the jugular vein of 100 clinically healthy Iranian crossbred cattle according to their age (<1, 1–2, 2–3, 3–4, 4–5 and

S. Nazifi; M. Saeb; E. Rowghani

2003-01-01

170

Comparison of lipoprotein analysis by agarose gel and paper electrophoresis with analytical ultracentrifugation  

Microsoft Academic Search

A comparison has been made of human serum lipoprotein analysis by agarose gel and paper electrophoresis with a standard method\\u000a of analytical ultracentrifugation. Samples were obtained from 28 patients with various disorders of lipoprotein metabolism.\\u000a Correspondence was shown between the following electrophoretic and ultracentrifugal fractions: ? and Sf 0–20; pre-? and Sf 20–400; ?1 and total HDL. The deviations observed

R. P. Noble; F. T. Hatch; J. A. Marzimas; F. T. Lindgren; L. C. Jensen; G. L. Adamson

1969-01-01

171

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

172

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

Microsoft Academic Search

An annual seasonal cycle of composition of a bacterioplankton community in an oligotrophic coastal system was studied by denaturing gradient gel electrophoresis (DGGE) using five different primer sets. Analysis of DGGE fingerprints showed that primer set 357fGC-907rM grouped samples according to seasons. Addition- ally, we used the set of 16S rRNA genes archived in the RDPII database to check the

Olga Sanchez; Josep M. Gasol; Ramon Massana; Jordi Mas; Carlos Pedros-Alio

2007-01-01

173

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

174

Detection of single nucleotide polymorphism in the FUT2 gene by temperature gradient gel electrophoresis  

Microsoft Academic Search

A simple and rapid analysis system for single nucleotide polymorphisms (SNPs) was investigated for the FUT2 gene using the temperature gradient gel electrophoresis (TGGE) method. The 426-bp or 259-bp FUT2 fragments were amplified from heterozygous samples using primers, and the heteroduplex and homoduplex bands were detected by TGGE. The FUT2 fragments amplified from homozygous samples were denatured and re-annealed with

Tomoaki Mitani; Atsushi Akane

2005-01-01

175

Molecularly imprinted polymer as chiral selector for enantioseparation of amino acids by capillary gel electrophoresis  

Microsoft Academic Search

Summary  A capillary electrophoresis method for the enantioseparation of D,L-aromatic amino acids using molecularly imprinted polymer as chiral selector has been developed. Methacrylic acid was used as functional monomer and ethylene glycol dimethacrylate (EDMA) as crosslinking monomer. The molecularly imprinted polymer was packed in capillaries by incorporating with acrylamide gel. The composition of the polymerization mixtures affects the separation factor to

J. M. Lin; T. Nakagama; K. Uchiyama; T. Hobo

1996-01-01

176

Further development of an electroosmotic medium pump system for preparative disk gel electrophoresis  

Microsoft Academic Search

A simple and practical 6.8-cm-diameter (36.30-cm2 cross-sectional-area) preparative disk gel electrophoresis device, based on the design of M. Hayakawa et al. (Anal. Biochem. 288 (2001) 168), in which the elution buffer is driven by an electroosmotic buffer flow through the membrane into the elution chamber from the anode chamber was constructed. We have found that the dialysis membranes employed provide

Mitsuo Hayakawa; Yumiko Hosogi; Hisashi Takiguchi; Teruaki Shiroza; Yasuko Shibata; Koichi Hiratsuka; Michiko Kiyama-Kishikawa; Susumu Hamajima; Yoshimitsu Abiko

2003-01-01

177

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

Microsoft Academic Search

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

Ángel Alegría; Renata González; Mario Díaz; Baltasar Mayo

2011-01-01

178

Capillary blotting of glycosaminoglycans on nitrocellulose membranes after agarose-gel electrophoresis separation.  

PubMed

A method for the blotting and immobilizing of several nonsulfated and sulfated complex polysaccharides on membranes made hydrophilic and positively charged by cationic detergent after their separation by conventional agarose gel electrophoresis is illustrated. This new approach to the study of glycosaminoglycans (GAGs) utilizes the capacity of agarose gel electrophoresis to separate single species of polysaccharides from mixtures and the membrane technology for further preparative and analytical uses.Nitrocellulose membranes are derivatized with the cationic detergent cetylpyridinium chloride and mixtures of GAGs are capillary blotted after their separation in agarose gel electrophoresis. Single purified species of variously sulfated polysaccharides are transferred on derivatized membranes with an efficiency of 100% and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining). This enables a lower amount limit of detection of 0.1 microg. Nonsulfated polyanions, for example hyaluronic acid, may also be transferred to membranes with a limit of detection of approximately 0.1-0.5 microg after irreversible or reversible staining. The membranes may be stained with reversible staining and the same lanes are used for immunological detection or other applications. PMID:19378049

Volpi, Nicola; Maccari, Francesca

2009-01-01

179

Two-dimensional gel electrophoresis analysis of the abundance of virulent exoproteins of group A streptococcus caused by environmental changes.  

PubMed

Group A streptococci regulate the expression of virulence factors in response to environmental change. In order to investigate this mechanism, the growth of group A streptococci and the abundance of virulent exoprotein production in culture supernatant were analyzed by two-dimensional gel electrophoresis (2-D electrophoresis) under several culture conditions. Judging from alterations in their growth, group A streptococci were affected by various environmental stresses. Under high O(2) and low CO(2 )concentrations, streptococcal pyrogenic exotoxin B (SpeB) and streptococcal pyrogenic exotoxin F (SpeF) significantly decreased, and the streptococcal inhibitor of complement (Sic) increased. At 30 degrees C, increases in endo-beta- N-acetylglucosaminidase (EndoS) and alpha-amylase were also detected, while at 41 degrees C EndoS became undetectable and SpeB and SpeF decreased. Sic, SpeF and mitogenic factor 3 (Mf3) decreased when cells were cultured in higher NaCl concentrations, and EndoS disappeared following culture of the cells in high glucose concentration. An increase in acid phosphatase and a decrease in several other proteins were detected when the cells were cultivated in high iron concentrations. These results suggest that group A streptococci have a versatile adaptation system that responds to several environmental stresses by altering the level of exoprotein production. PMID:14673516

Nakamura, Tadahiro; Hasegawa, Tadao; Torii, Keizo; Hasegawa, Yoshinori; Shimokata, Kaoru; Ohta, Michio

2003-12-12

180

Glutamine Synthetase Regulation, Adenylylation State, and Strain Specificity Analyzed by Polyacrylamide Gel Electrophoresis  

PubMed Central

We used polyacrylamide gel electrophoresis to examine the regulation and adenylylation states of glutamine synthetases (GSs) from Escherichia coli (GSE) and Klebsiella aerogenes (GSK). In gels containing sodium dodecyl sulfate (SDS), we found that GSK had a mobility which differed significantly from that of GSE. In addition, for both GSK and GSE, adenylylated subunits (GSK-adenosine 5?-monophosphate [AMP] and GSE-AMP) had lesser mobilities in SDS gels than did the corresponding non-adenylylated subunits. The order of mobilities was GSK-AMP < GSK < GSE-AMP < GSE. We were able to detect these mobility differences with purified and partially purified preparations of GS, crude cell extracts, and whole cell lysates. SDS gel electrophoresis thus provided a means of estimating the adenylylation state and the quantity of GS present independent of enzymatic activity measurements and of determining the strain origin. Using SDS gels, we showed that: (i) the constitutively produced GS in strains carrying the glnA4 allele was mostly adenylylated, (ii) the GS-like polypeptide produced by strains carrying the glnA51 allele was indistinguishable from wild-type GSK, and (iii) strains carrying the glnA10 allele contained no polypeptide having the mobility of GSK or GSK-AMP. Using native polyacrylamide gels, we detected the increased amount of dodecameric GS present in cells grown under nitrogen limitation compared with cells grown under conditions of nitrogen excess. In native gels there was neither a significant difference in the mobilities of adenylylated and non-adenylylated GSs nor a GS-like protein in cells carrying the glnA10 allele. Images

Bender, Robert A.; Streicher, Stanley L.

1979-01-01

181

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

182

Multi-Channel Gel Electrophoresis and Continuous Fraction Collection Apparatus for High Throughput Protein Separation and Characterization.  

National Technical Information Service (NTIS)

To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis col...

J. Jin M. Choi M. D. Biggin R. A. Nordmeyer

2010-01-01

183

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

184

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

PubMed Central

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

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

2011-01-01

185

Analysis of aldosterone-induced differential receptor-independent protein patterns using 2D-electrophoresis and mass spectrometry.  

PubMed

In the human body the mineralocorticoid aldosterone is responsible for maintaining water and electrolyte homeostasis and therefore controlling blood pressure. In addition, aldosterone has recently been associated with severe heart failure. Besides receptor-dependent action, the damaging effects of aldosterone may also be partly mediated through non-genomic mechanisms. The present study focuses on the mineralocorticoid receptor-independent action of aldosterone at the protein level. We chose the fission yeast Schizosaccharomyces pombe as a model organism, since this yeast does not contain nuclear steroid receptors, but many genes and regulatory mechanisms that are close to those of mammals. Using 2D-electrophoresis we identified for the first time protein spots affected by aldosterone in a nuclear receptor-free system. Mass spectrometry analysis using MALDI-TOF MS and nanoLC-MS/MS approaches allowed the unambiguous identification of 11 proteins that showed increased or decreased levels, which may represent newly identified players and pathways of aldosterone-induced action. Two proteins with a connection to osmotic regulation (NAD-dependent malic enzyme and glycerol-3-phosphate-dehydrogenase), as well as two proteins involved in the overall organization of the cytoskeleton, vip1 and glyceraldehyde-3-phosphate dehydrogenase, which was also found to be specifically affected by aldosterone in human HCT116 cells, are discussed. PMID:16913842

Böhmer, Susanne; Carapito, Christine; Wilzewski, Britta; Leize, Emmanuelle; Van Dorsselaer, Alain; Bernhardt, Rita

2006-07-01

186

Screening for serum lipoprotein abnormalities: Comparison of ultracentrifugal, paper and thin-layer starch-gel electrophoresis techniques  

Microsoft Academic Search

Three methods for evaluation of serum lipoprotein abnormalities were compared: paper electrophoresis using buffer containing\\u000a albumin, ultracentrifugation at d 1.21, and thin-layer starch-gel electrophoresis. Analyses by paper electrophoresis and by\\u000a ultracentrifugation of 109 sera of patients with cholesterol levels between 78 and 1150 mg\\/100 ml showed that the electrophoretic\\u000a procedure while not quantitative was an effective procedure for detecting and

Lena A. Lewis

1969-01-01

187

Detection of low-molecular weight allergens resolved on two-dimensional electrophoresis with acid–urea polyacrylamide gel  

Microsoft Academic Search

Two-dimensional electrophoresis with immobilized pH gradient (IPG) followed by acetic acid\\/urea–polyacrylamide gel electrophoresis (AU–PAGE) was developed for the detection of low-molecular weight food allergens. Wheat proteins were used to test the applicability of AU–PAGE for the analysis of food allergens. Isoelectric focusing (IEF) for first dimension was performed with IPG pH 3–10. AU–PAGE was performed as a second-dimensional electrophoresis and

Kazumi Kitta; Mayumi Ohnishi-Kameyama; Tatsuya Moriyama; Tadashi Ogawa; Shinichi Kawamoto

2006-01-01

188

Analysis of radiation damage of DNA by atomic force microscopy in comparison with agarose gel electrophoresis studies  

Microsoft Academic Search

DNA damage induced with ionizing radiation is considered one of the main causes of cell inactivation. Several methods including gel electrophoresis, pulsed-field gel electrophoresis, neutral filter elution method, neutral sedimentation and electron microscopy have been applied to analyze this type of DNA damage. A new method employing an atomic force microscope (AFM) for nanometer-level-structure analysis of DNA damage induced with

Masahiro Murakami; Hideo Hirokawa; Isamu Hayata

2000-01-01

189

Regional differences in luminal fluid polypeptides of the rat testis and epididymis revealed by two-dimensional gel electrophoresis.  

PubMed

Luminal fluid samples were collected by micropuncture of the seminiferous tubule, rete testis, and defined levels of the epididymal tubule. After removal of spermatozoa by centrifugation, the supernatant fluids were analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and an ultrasensitive silver staining procedure to define the sequential change in protein composition along the excurrent duct system. Fluid from each segment displayed a characteristic 2-D PAGE map composed of numerous polypeptides. Seminiferous tubule fluid contained a wide array of polypeptides, with most concentrated in the 45 Kd to 90 Kd range, but, in contrast, rete testis fluid lacked most of these polypeptides. The major complex of rete testis fluid comigrated with serum albumin and was present in all distal segments. Other major rete testis components were not noted distally. Fluid from the caput was characterized by new major components of 30 to 37 Kd, 28 to 30 Kd, 24 Kd, and 23 Kd, each of which consisted of multiple spots of apparent isoelectric variants; all except the 30 to 37 Kd complex were present in the fluid from more distal segments. Proceeding distally, there was a temporal appearance of new polypeptides, especially in the molecular weight range below 30 Kd. Two-dimensional PAGE analysis of detergent extracts of washed spermatozoa indicate that a specific subset of these fluid polypeptides are sperm associated. PMID:3972717

Olson, G E; Hinton, B T

190

Differential in-gel electrophoresis (DIGE) analysis of CHO cells under hyperosmotic pressure: osmoprotective effect of glycine betaine addition.  

PubMed

The use of glycine betaine combined with hyperosmolality is known to be an efficient means for achieving high protein production in recombinant Chinese hamster ovary (rCHO) cells. In order to understand the intracellular events and identify the key factors in rCHO cells cultivated with glycine betaine under hyperosmotic conditions, two-dimensional differential in-gel electrophoresis (2D-DIGE) followed by mass spectrometric analysis was applied. Differentially expressed 19 protein spots were selected and 16 different kinds of proteins were successfully identified. The identified proteins were associated with cellular metabolism (PEPCK, GAPDH, and PK), cellular architecture (?-tubulin and ?-actin), protein folding (GRP78 and OSP94), mRNA processing (Rbm34, ACF, and IPMK), and protein secretion (?-COP). 2D-Western blot analysis of ?-tubulin, GAPDH, Peroxidoxin-1, and GRP78 confirmed the proteomic findings. The proteins identified from this study, which are related to cell growth and antibody production, can be applied to cell engineering for maximizing the efficacy of the use of glycine betaine combined with hyperosmolality in rCHO cells. PMID:22252946

Kim, Jee Yon; Kim, Yeon-Gu; Lee, Gyun Min

2012-01-23

191

Two-dimensional differential in-gel electrophoresis for identification of gastric cancer-specific protein markers.  

PubMed

Gastric cancer is the second most common fatal malignancy in the world. Proteomics studies of clinical tumor samples have led to the identification of specific protein markers of gastric cancer detection and better understanding the carcinogenesis of gastric cancer. Gastric cancer tissue of epithelial origin and adjacent normal mucosa were examined in pair by fluorescence 2-D differential in-gel electrophoresis proteomics analysis utilizing 2-D PAGE protein separation. Intensity changes of 33 spots were detected with statistical significance. Twenty-two out of the 33 spots were identified by MALDI-TOF MS or MS/MS. Of the 9 up-regulated proteins, 7 were identified, including heat shock protein 60 (HSP60), mutant desmin, effector cell proteinase receptor 1 splice form 1b, hypothetical protein, unnamed protein product, and manganese superoxide dismutase (MnSOD), a protein similar to alpha-actin. Of the 20 down-regulated proteins, 16 were identified, including selenium binding protein 1, fibrinogen gamma, HSP27, tubulin alpha 6, zinc finger protein 160, prostaglandin F synthase, and eukaryotic translation elongation factor 1 alpha 1. Our results suggest that MnSOD may be a potential serum marker for molecular diagnosis of gastric carcinoma, and DIGE is a useful technique for screening differentially expressed proteins in cancer tissues. PMID:19424620

Wu, Cheng; Luo, Zhiwen; Chen, Xueyun; Wu, Chaoqun; Yao, Dingkang; Zhao, Peng; Liu, Lijie; Shi, Bin; Zhu, Liang

2009-06-01

192

A plant dye from Lawsonia inermis for protein staining after polyacrylamide gel electrophoresis.  

PubMed

A reddish-brown dye was isolated from the leaves of Lawsonia inermis by extraction with calcium hydroxide (pH 11-12). A 3.6% crude extract in ethanol/water, 1:1 v/v, was used for direct staining, without fixation, of bovine serum albumin, casein and human serum proteins, following polyacrylamide gel electrophoresis in cylindrical gels. After staining for 30 min the gels were destained for 1/2-2 h with 7% acetic acid at 60 degrees C. Protein staining with Amido Black 10B and Coomassie Brilliant Blue R-250, according to standard protocols, required destaining for 24 h and more to obtain a comparably cleared background. Staining with the plant dye and Coomassie Brilliant Blue had a similar overall staining sensitivity but some minor components of human serum showed different staining characteristics with each of the two dyes. Staining with the plant dye excels over standard staining by speed and simplicity. PMID:1692790

Ali, R; Sayeed, S A

1990-04-01

193

Previsible silver staining of protein in electrophoresis gels with mass spectrometry compatibility.  

PubMed

A convenient silver staining method for protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels is described. The method is previsible, sensitive, and mass spectrometry (MS) compatible. Two visible counter ion dyes, ethyl violet (EV) and zincon (ZC), were used in the first staining solution with a detection limit of 2 to 8 ng/band in approximately 1h. The dye-stained gel can be further stained by silver staining, which is based on acidic silver staining employing ZC with sodium thiosulfate as silver ion sensitizers. Especially, ZC has silver ion reducing power by cleavage of the diazo bond of the dye during silver reduction. The second silver staining can be completed in approximately 1h with a detection limit of 0.2 ng/band. PMID:18804088

Jin, Li-Tai; Li, Xiao-Kun; Cong, Wei-Tao; Hwang, Sun-Young; Choi, Jung-Kap

2008-08-27

194

Proteomic Profiling Of Two-Dimensional Gel Electrophoresis Protein Expression Data  

NASA Astrophysics Data System (ADS)

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

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

2008-01-01

195

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

196

DNA detection system using molecularly imprinted polymer as the gel matrix in electrophoresis.  

PubMed

To develop a simple and inexpensive method for DNA detection, we prepared a molecularly imprinted polymer (MIP) for recognizing a specific double-stranded DNA (dsDNA) sequence and used it in an electrophoretic gel matrix. The MIP gel has many binding sites that are complementary in size, shape, and arrangement of functional groups of the target dsDNA sequence. During MIP gel electrophoresis (MIPGE), migration of the target dsDNA should be hindered by the capture effect of the binding sites in the MIP gel. This was confirmed by observation of deviations from the linear relationship between the migration distances of the DNA standard size markers in the polyacrylamide gel and those in the MIP gel. The migration distances of nontarget dsDNA maintained a linear relationship, however. In addition, the sequence selectivity of dsDNA in this method was investigated by using the Ha-ras gene and its point mutants. Except for A.T to T.A base pair substitution, mutant dsDNA (for example, substitution from A.T to C.G and from G.C to T.A) could be distinguished from the target (wild-type) dsDNA. Although some improvement in A.T (T.A) base pair distinction is still needed, this study is the first to demonstrate detection of a specific dsDNA sequence with MIPs and, as such, opens up a new realm for practical applications of MIPs. PMID:16987649

Ogiso, Masayo; Minoura, Norihiko; Shinbo, Toshio; Shimizu, Toshimi

2006-09-20

197

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

198

A review of the CLIP system for the quantitative analysis of two-dimensional electrophoresis gels.  

PubMed

This paper reviews the CLIP image processing system for the complete analysis of two-dimensional electrophoresis images. The analysis problem for two-dimensional gel images can be broken down into three issues: segmentation of individual gel images, alignment and comparison of pairs of gel images, and information storage and retrieval. This paper describes these problems and reviews how the CLIP system handles each of them. Segmentation is the location and isolation of each protein spot on an individual gel image and also the extraction of individual spot data such as position, area and volume. There are three basic stages: background field correction, noise filtering, spot detection and information extraction. Alignment and comparison of gel images involves matching protein spots between two gels. This can be quite difficult because there is not a simple relationship which can transform one gel image onto another. The database issues concern storing all the information which has been obtained from the above operations such that retrieval of this information can be readily performed. The advantage of the CLIP system over others is speed of processing. CLIP series computers use one processor for every pixel of the camera image such that image processing algorithms run in parallel. The main disadvantage is in the cost of these machines. With the declining trend in the cost of parallel processors, these machines will become more and more viable alternatives. This papers reviews the algorithms for the analysis of two-dimensional gels. It is shown that CLIP is flexible enough to perform more than one type of algorithm for a particular operation. PMID:2364927

Potter, D J

1990-05-01

199

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

NSDL National Science Digital Library

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

Hsu, Tien T.

2005-08-17

200

Gel electrophoresis of a charge-regulated, bi-functional particle.  

PubMed

Adopting a Brinkman fluid model, we analyzed the electrophoresis of a charged-regulated, bi-functional particle containing both acidic and basic functional groups in a gel solution. Both the long-range hydrodynamic effect arising from the liquid drag and the short-range steric effect from particle-polymer interaction are considered. The type of particle considered is capable of simulating both biocolloids such as microorganisms and cells, and particles with adsorbed polyelectrolyte or membrane layer. Our model describes successfully the experimental data in the literature. The presence of gel has the effect of reducing the particle mobility and alleviating double-layer polarization so that the particle behavior is less complicated than that in the case where gel is absent. On the other hand, both the quantitative and qualitative behaviors of a particle depend highly on solution pH and background salt concentration, yielding interesting and significant results. These results provide valuable information for both experimental data interpretation and electrophoresis devices design. PMID:23161269

Hsu, Jyh-Ping; Huang, Chih-Hua; Tseng, Shiojenn

2013-01-30

201

The TYCHO system for computer analysis of two-dimensional gel electrophoresis patterns  

SciTech Connect

We describe here a computer system for the analysis of high-resolution two-dimensional gel-electrophoresis patterns, with some initial applications. The system (called TYCHO) comprises programs for image acquisition, background subtraction and smoothing, spot detection, gaussian spot modeling, and pattern matching and comparison. It is based on a conventional minicomputer, but makes extensive use of a high-speed array processor in the image-processing and -modeling steps. Used in concert with the ISO-DALT two-dimensional electrophoresis system (Anal. Biochem. 85:331-354, 1978), TYCHO allows quantitative measurement of hundreds of proteins in complex biological samples, and constitutes the initial data-reduction system required for work towards a Human Protein Index.

Anderson, N.L.; Taylor, J.; Scandora, A.E.; Coulter, B.P.; Anderson, N.G.

1981-11-01

202

The TYCHO system for computer analysis of two-dimensional gel electrophoresis patterns.  

PubMed

We describe here a computer system for the analysis of high-resolution two-dimensional gel-electrophoresis patterns, with some initial applications. The system (called TYCHO) comprises programs for image acquisition, background subtraction and smoothing, spot detection, gaussian spot modeling, and pattern matching and comparison. It is based on a conventional minicomputer, but makes extensive use of a high-speed array processor in the image-processing and -modeling steps. Used in concert with the ISO-DALT two-dimensional electrophoresis system (Anal. Biochem. 85:331-354, 1978), TYCHO allows quantitative measurement of hundreds of proteins in complex biological samples, and constitutes the initial data-reduction system required for work towards a Human Protein Index. PMID:7296832

Anderson, N L; Taylor, J; Scandora, A E; Coulter, B P; Anderson, N G

1981-11-01

203

Detecting single base substitutions, mismatches and bulges in DNA by temperature gradient gel electrophoresis and related methods  

Microsoft Academic Search

Temperature gradient gel electrophoresis (TGGE) and related methods can separate DNA fragments that differ by a single base pair or defect. This article describes the basic features of TGGE, and reviews the theoretical model of DNA unwinding and its ability to predict DNA mobility in a temperature gradient gel. Recent applications of TGGE and related methods that were directed at

Roger M. Wartell; Seyed Hosseini; Sandra Powell; Jian Zhu

1998-01-01

204

Proteomic analysis of the hippocampus in Alzheimer's disease model mice by using two-dimensional fluorescence difference in gel electrophoresis.  

PubMed

We previously identified the E693? mutation in amyloid precursor protein (APP) in patients with Alzheimer's disease (AD) and then generated APP-transgenic mice expressing this mutation. As these mice possessed abundant A? oligomers from 8 months of age but no amyloid plaques even at 24 months of age, they are a good model to study pathological effects of amyloid ? (A?) oligomers. The two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) technology, using a mixed-sample internal standard, is now recognized as an accurate method to determine and quantify proteins. In this study, we examined the proteins for which levels were altered in the hippocampus of 12-month-old APP(E693?)-transgenic mice using 2D-DIGE and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Fourteen proteins were significantly changed in the hippocampus of APP(E693?)-transgenic mice. Actin cytoplasmic 1 (?-actin), heat shock cognate 71kDa, ?-enolase, ATP synthase subunit ?, tubulin ?-2A chain, clathrin light chain B (clathrin) and dynamin-1 were increased. Heat shock-related 70kDa protein 2, neurofilament light polypeptide (NFL), stress-induced-phosphoprotein 2, 60kDa heat shock protein (HSP60), ?-internexin, protein kinase C and casein kinase substrate in neurons protein 1 (Pacsin 1), ?-enolase and ?-actin were decreased. Western blotting also validated the changed levels of HSP60, NFL, clathrin and Pacsin 1 in APP(E693?)-transgenic mice. The identified proteins could be classified as cytoskeleton, chaperons, neurotransmission, energy supply and signal transduction. Thus, proteomics by 2D-DIGE and LC-MS/MS has provided knowledge of the levels of proteins in the early stages of AD brain. PMID:23276639

Takano, Masaoki; Yamashita, Takuya; Nagano, Kazuya; Otani, Mieko; Maekura, Kouji; Kamada, Haruhiko; Tsunoda, Shin-Ichi; Tsutsumi, Yasuo; Tomiyama, Takami; Mori, Hiroshi; Matsuura, Kenji; Matsuyama, Shogo

2012-12-29

205

Anaerobic multiphasic gel electrophoresis of the molybdoproteins in extracts of Clostridium pasteurianum.  

PubMed

Simple procedures using multiphasic buffer systems for anaerobic electrophoresis have been devised to identify oxygen-labile metalloproteins. An anaerobic slab gel apparatus was developed with cooling and design for anaerobic conditions. Included is a procedure to remove sample wells after stacking proteins in a crude extract, to prevent streaking (background) caused by continuous leakage of "nonstacked protein" from the sample wells. Identification of eleven Mo zones in extracts of Clostridium pasteurianum demonstrates the usefulness of the technique in identifying radiolabeled oxygen-labile proteins in cell-free crude lysates. PMID:4014654

Hinton, S M; Mortenson, L E

1985-03-01

206

Analysis of RNA structure by ultraviolet crosslinking and denaturation gel electrophoresis  

SciTech Connect

Electrophoresis in polyacrylamide gels containing both formamide and urea is a high-resolution technique for the analysis of crosslinked RNA species. Combined with a specific crosslinking agent like uv irradiation, it allows a rapid fingerprint of structural differences between RNA forms. The technique reveals significant differences in the pattern of uv crosslinking of free Escherichia coli 16 S ribosomal RNA compared with the RNA in active or inactive 30 S subunits. Ultraviolet photocrosslinks seen only in the 30 S particle are likely to be tertiary structure contacts.

Quarless, S.A.; Cantor, C.R.

1985-06-01

207

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

2013-07-01

208

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

SciTech Connect

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

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

2010-03-20

209

Performing isoelectric focusing and simultaneous fractionation of proteins on a rotary valve followed by sodium dodecyl-polyacrylamide gel electrophoresis.  

PubMed

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 second-D separation) on either a capillary gel electrophoresis instrument or a slab-gel system. The detailed valve configuration is illustrated, and the experimental conditions and operation protocols are discussed. PMID:23819755

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

2013-07-02

210

Disparity between Multilocus Enzyme Electrophoresis, Microsatellite Markers and Pulsed-Field Gel Electrophoresis in epidemiological tracking of Candida albicans.  

PubMed

Various molecular systems are available for epidemiological, genetic, evolutionary, taxonomic and systematic studies of innumerable fungal infections, especially those caused by the opportunistic pathogen C. albicans. A total of 75 independent oral isolates were selected in order to compare Multilocus Enzyme Electrophoresis (MLEE), Electrophoretic Karyotyping (EK) and Microsatellite Markers (Simple Sequence Repeats - SSRs), in their abilities to differentiate and group C. albicans isolates (discriminatory power), and also, to evaluate the concordance and similarity of the groups of strains determined by cluster analysis for each fingerprinting method. Isoenzyme typing was performed using eleven enzyme systems: Adh, Sdh, M1p, Mdh, Idh, Gdh, G6pdh, Asd, Cat, Po, and Lap (data previously published). The EK method consisted of chromosomal DNA separation by pulsed-field gel electrophoresis using a CHEF system. The microsatellite markers were investigated by PCR using three polymorphic loci: EF3, CDC3, and HIS3. Dendrograms were generated by the SAHN method and UPGMA algorithm based on similarity matrices (S(SM)). The discriminatory power of the three methods was over 95%, however a paired analysis among them showed a parity of 19.7-22.4% in the identification of strains. Weak correlation was also observed among the genetic similarity matrices (S(SM)(MLEE)xS(SM)(EK)xS(SM)(SSRs)). Clustering analyses showed a mean of 9+/-12.4 isolates per cluster (3.8+/-8 isolates/taxon) for MLEE, 6.2+/-4.9 isolates per cluster (4+/-4.5 isolates/taxon) for SSRs, and 4.1+/-2.3 isolates per cluster (2.6+/-2.3 isolates/taxon) for EK. A total of 45 (13%), 39 (11.2%), 5 (1.4%) and 3 (0.9%) clusters pairs from 347 showed similarity (S(J)) of 0.1-10%, 10.1-20%, 20.1-30% and 30.1-40%, respectively. Clinical and molecular epidemiological correlation involving the opportunistic pathogen C. albicans may be attributed dependently of each method of genotyping (i.e., MLEE, EK, and SSRs) supplemented with similarity and grouping analysis. Therefore, the use of genotyping systems that give results which offer minimum disparity, or the combination of the results of these systems, can provide greater security and consistency in the determination of strains and their genetic relationships. PMID:20619303

Boriollo, Marcelo Fabiano Gomes; Dias, Ricardo Antunes; Fiorini, João Evangelista; Oliveira, Nelma de Mello Silva; Spolidório, Denise Madalena Palomari; de Souza, Henrique Marques Barbosa; Figueira, Antonio Vargas de Oliveira; Pizzirani-Kleiner, Aline Aparecida

2010-07-07

211

Simplified protocol for pulsed-field gel electrophoresis analysis of Streptococcus pneumoniae.  

PubMed

A variety of pulsed-field gel electrophoresis (PFGE) protocols for the molecular subtyping of Streptococcus pneumoniae have been reported; most are time-consuming and complex. We sought to modify reference PFGE protocols to reduce the time required while creating high-quality gels. Only protocol modifications that resulted in high-quality banding patterns were considered. The following protocol components were modified. Lysis enzymes (lysozyme, mutanolysin, and RNase A) were deleted in a stepwise fashion, and then the lysis buffer was deleted. Lysis and digestion were accomplished in a single step with EDTA and N-lauroyl sarcosine (ES; pH 8.5 to 9.3) incubation at 50 degrees C in the absence of proteinase K. All enzymes except the restriction enzyme were omitted. A minimum incubation time of 6 h was required to achieve high-quality gels. All of the reactions were performed within 9 h, and the total protocol time from lysis to gel completion was reduced from 3 days to only 36 h. Combining lysis and digestion into a single step resulted in a substantial reduction in the time required to perform PFGE for S. pneumoniae. The ES solution may have caused cell lysis by activating N-acetylmuramyl-L-alanine amidase, the pneumococcal autolysin. PMID:10618114

McEllistrem, M C; Stout, J E; Harrison, L H

2000-01-01

212

TOTAL PROTEIN AND 2-D GEL ANALYSIS OF ENTEROCOCCUS FAECALIS PROTEINS EXTRACTED BY FOUR METHODS  

Technology Transfer Automated Retrieval System (TEKTRAN)

Both the identificatio of proteins and changes in levels of protein expression are important in determining the adaptation of Enterococcus faecalis to infection and stress. Two-dimensional electrophoresis is often used to make these determinations. Various chemical and mechanical methods are used ...

213

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

PubMed Central

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

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

2009-01-01

214

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

215

Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms  

SciTech Connect

The authors 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 by hybridization with a nick-translated DNA fragment or more clearly with RNA copies synthesized on each strand of the DNA fragment as probes. As the mobility shift caused by nucleotide substitutions might be due to a conformational change of single-stranded DNAs, we designate the features of single-stranded DNAs as single-strand conformation polymorphisms (SSCPs). Like restriction fragment length polymorphisms (RFLPs), SSCPs were found to be allelic variants of true Mendelian traits, and therefore they should be useful genetic markers. Moreover, SSCP analysis has the advantage over RFLP analysis that it can detect DNA polymorphisms and point mutations at a variety of positions in DNA fragments. Since DNA polymorphisms have been estimated to occur every few hundred nucleotides in the human genome, SSCPs may provide many genetic markers.

Orita, Masato; Iwahana, Hiroyuki; Kanazawa, Hiroshi; Hayashi, Kenshi; Sekiya, Takao (National Cancer Center Research Institute, Tokyo (Japan))

1989-04-01

216

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

PubMed

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

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

2002-01-01

217

Electrophoresis of bacteriophage T7 and T7 capsids in agarose gels.  

PubMed Central

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

Serwer, P; Pichler, M E

1978-01-01

218

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

219

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

PubMed Central

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

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

2013-01-01

220

Functional characterization of reductive dehalogenases by using blue native polyacrylamide gel electrophoresis.  

PubMed

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

Tang, Shuiquan; Chan, Winnie W M; Fletcher, Kelly E; Seifert, Jana; Liang, Xiaoming; Löffler, Frank E; Edwards, Elizabeth A; Adrian, Lorenz

2012-11-30

221

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

PubMed Central

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

Fiala, Gina J.

2011-01-01

222

Target Identification of an Antibacterial Compound from Data Mining of 2-D Gel Images  

Microsoft Academic Search

Drug discovery program is a complex scientific process, with very low successful rate of 1\\/5000 compounds, which might be further developed for therapeutic usage. To accelerate the drug discovery process, new “omics” technology has been used. We have described how to use proteomics technology of two-dimensioal gel to identify the protein target of an antibacterial compound. The digital images of

KuoYuan Hwa; Di-Shuan Chiang; Chen-Wen Yao

2009-01-01

223

Application of temperature-gradient gel electrophoresis in taxonomy of coryneform bacteria.  

PubMed

Strains belonging to the Gram-positive coryneform soil bacteria were screened genotypically by temperature-gradient gel electrophoresis (TGGE). This method allows the sequence-specific separation of amplified fragments of 16S rRNA genes. A total of 115 reference strains representing the majority of the species of the genera Aeromicrobium, Agromyces, Arthrobacter, Aureobacterium, Cellulomonas, Curtobacterium, Nocardioides and Terrabacter were characterized. Depending on the genus investigated, the resolution limit of the technique appeared to be at the species or genus level or intermediate between the two. Aberrant TGGE profiles of strains within particular taxa revealed genomic heterogeneity and generic misclassification of nine strains studied. Beyond that, indications of 16S rRNA gene heterogeneity were found within the genomes of three Curtobacterium strains. The misclassifications revealed by TGGE were confirmed using whole-cell fatty acid methyl ester analysis and subsequent comparison with a database. TGGE has been demonstrated to be a useful tool in bacterial taxonomy. PMID:10028252

Felske, A; Vancanneyt, M; Kersters, K; Akkermans, A D

1999-01-01

224

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

SciTech Connect

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

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

1988-01-01

225

Evaluation of Pulsed-Field Gel Electrophoresis Profiles for Identification of Salmonella Serotypes? †  

PubMed Central

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.

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

2010-01-01

226

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

PubMed Central

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

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

1988-01-01

227

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

2010-07-14

228

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

229

Polyacrylamide gel electrophoresis analysis of ribosomal protein AT-L30 from an actinomycete genus, Streptosporangium.  

PubMed

We analyzed the ribosomal AT-L30 proteins from 11 type strains of species belonging to the genus Streptosporangium. The electrophoretic mobilities of the AT-L30 preparations from these strains, as determined by two-dimensional polyacrylamide gel electrophoresis, revealed that they could be divided into three groups. The first group contained Streptosporangium viridogriseum, S. viridogriseum subsp. kofuense, and S. albidum, while the second group contained S. roseum, S. album, S. vulgare, S. nondiastaticum, S. fragile, S. violaceochromogenes, and S. amethystogenes. S. corrugatum was a member of the third group. These groups were completely consistent with Nonomura's previous classification, which was based on morphological criteria. The results of partial amino acid sequencing of AT-L30 preparations from several representative strains strongly supported the hypothesis that each of the three groups of the genus Streptosporangium merits separate generic status. PMID:1736963

Ochi, K; Miyadoh, S

1992-01-01

230

A mobility shift detection method for DNA methylation analysis using phosphate affinity polyacrylamide gel electrophoresis.  

PubMed

We describe a procedure for DNA methylation analysis using the bisulfite-mediated cytosine-to-uracil conversion of a target DNA followed by methylation-specific polymerase chain reaction (MSP) and phosphate affinity polyacrylamide gel electrophoresis (PAGE). The MSP was performed using a 1:1 mixture of 5'-phosphorylated methylation-specific and 5'-OH non-methylation-specific primers. The PAGE using an immobilized phosphate-binding tag molecule (i.e., a polyacrylamide-bound dizinc(II) complex, Zn(2+)-Phos-tag), which selectively captures the 5'-phosphorylated DNA fragment, enabled the mobility shift detection of the methylation-specific product as a slower migration band. Using this novel procedure, we demonstrated the detection of a methylated cytosine base in a pUC19 plasmid. PMID:18394999

Kinoshita-Kikuta, Emiko; Kinoshita, Eiji; Koike, Tohru

2008-03-16

231

Using Microchip Gel Electrophoresis to Probe DNA-Drug Binding Interactions.  

PubMed

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

Shi, Nan; Ugaz, Victor M

2014-01-01

232

Numerical Simulation of Gel Electrophoresis of DNA Knots in Weak and Strong Electric Fields  

PubMed Central

Gel electrophoresis allows one to separate knotted DNA (nicked circular) of equal length according to the knot type. At low electric fields, complex knots, being more compact, drift faster than simpler knots. Recent experiments have shown that the drift velocity dependence on the knot type is inverted when changing from low to high electric fields. We present a computer simulation on a lattice of a closed, knotted, charged DNA chain drifting in an external electric field in a topologically restricted medium. Using a Monte Carlo algorithm, the dependence of the electrophoretic migration of the DNA molecules on the knot type and on the electric field intensity is investigated. The results are in qualitative and quantitative agreement with electrophoretic experiments done under conditions of low and high electric fields.

Weber, C.; Stasiak, A.; De Los Rios, P.; Dietler, G.

2006-01-01

233

Separation of the Messenger RNAs of Newcastle Disease Virus by Gel Electrophoresis  

PubMed Central

We have separated the 18-22S putative messenger RNA of Newcastle disease virus into seven species ranging in molecular weight from 0.55 to 1.53 × 106 using sodium dodecyl sulfate-acrylamide-gel electrophoresis at relatively high concentrations of acrylamide and for a relatively long time. Studies of the number and molecular weights of the proteins and the 18-22S RNAs of the virus suggests that these RNAs are in the right molecular weight range to code for the known proteins of Newcastle disease virus. In preliminary studies using this separation technique, we have demonstrated that: (a) there is no difference between the 18-22S RNA made during a normal infection and when genome replication is blocked; and (b) there is a strain-specific difference between the RNAs of Newcastle disease virus-AV and Newcastle disease virus-HP.

Collins, Bonnie Spanier; Bratt, Michael A.

1973-01-01

234

Denaturing gradient gel electrophoresis screening for mutations in the hereditary hyperferritinaemia cataract syndrome.  

PubMed

Hereditary hyperferritinaemia cataract syndrome (HHCS) is characterized by hyperferritinaemia without iron overload. It is essential to differentiate true iron accumulation from HHCS as these patients rapidly develop iron-deficient anaemia when subjected to phlebotomies. The diagnosis of HHCS relies on the identification of point mutations or deletions present in the iron-responsive element of the first exon of the L-ferritin gene. However, many samples referred for diagnosis of putative HHCS are normal. To avoid unnecessary DNA sequencing, we have developed a diagnosis strategy based on the screening of the target DNA region by denaturing gradient gel electrophoresis. This method enabled the accurate identification of 11 different previously known mutations. This strategy will be of interest for family studies or for the screening of large series of patients. PMID:11167783

Giansily, M; Beaumont, C; Desveaux, C; Hetet, G; Schved, J F; Aguilar-Martinez, P

2001-01-01

235

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

236

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

237

Surface Modification of Gel-Free Microchannel Surface Electrophoresis Device for DNA Identification  

NASA Astrophysics Data System (ADS)

A gel-free microchannel electrophoresis device for DNA separation and identification was studied. DNA fragments ranging from 3.5 to 21.2 kbp were effectively separated and identified. The channel’s bottom surface was composed of silicon wafer or glass, and the channel wall was composed of SU-8 photoresist. The channel surface was modified with various solutions or plasmas. The separated DNA on the detection electrode was confirmed by electron spectroscopy for chemical analysis. DNA mobility on the glass substrate was higher than that on the Si substrate. In addition, the increase of the ionic strength of the solution on the device decreased the mobility of DNA. In summary, this new device is applicable to large DNA samples.

Lee, Hyun Ho; Kuo, Yue

2008-04-01

238

Aeromonas Isolates from Human Diarrheic Stool and Groundwater Compared by Pulsed-Field Gel Electrophoresis  

PubMed Central

Gastrointestinal infections of Aeromonas species are generally considered waterborne; for this reason, Aeromonas hydrophila has been placed on the United States Environmental Protection Agency Contaminant Candidate List of emerging pathogens in drinking water. In this study, we compared pulsed-field gel electrophoresis patterns of Aeromonas isolates from stool specimens of patients with diarrhea with Aeromonas isolates from patients’ drinking water. Among 2,565 diarrheic stool specimens submitted to a Wisconsin clinical reference laboratory, 17 (0.66%) tested positive for Aeromonas. Groundwater isolates of Aeromonas were obtained from private wells throughout Wisconsin and the drinking water of Aeromonas-positive patients. The analysis showed that the stool and drinking water isolates were genetically unrelated, suggesting that in this population Aeromonas gastrointestinal infections were not linked with groundwater exposures.

Stemper, Mary E.; Standridge, Jon H.

2003-01-01

239

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

2012-12-28

240

AFLP Analysis of Gel Electrophoresis Images Using JelMarker™ Image Reader and GeneMarker®Fragment Analysis Software  

Microsoft Academic Search

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

Tamela Serensits; Zhang Xuanhe; Ni Shouyong; Jonathan Liu

241

Two-dimensional gel electrophoresis and immunoblotting of Campylobacter outer membrane proteins.  

PubMed Central

We characterized outer membrane proteins (OMPs) from selected Campylobacter jejuni, C. coli, and C. fetus strains by two-dimensional gel electrophoresis (2DGE), using isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and by immunoblotting with immune rabbit serum. The flagellar band with a molecular mass of 63 kilodaltons (kDa) demonstrated previously by one-dimensional SDS-PAGE was shown by 2DGE to consist of one or several charge trains, depending upon the species, strain, and type of preparation studied; each of the individual peptides was found to be antigenic by immunoblotting. In contrast, in all of the strains studied, the major OMP (43 to 44 kDa) of C.jejuni and C. coli consisted of a single isomeric form which was weakly immunogenic. Several minor proteins (29 to 31 kDa) were found to be strongly immunogenic by immunoblotting. C. fetus strains possessed two major OMPs of 45 to 47 kDa, each of which consisted of either a single isomer or a major isomer comprising at least 90% of the major OMP. Serum-resistant strains of C. fetus possessed an acid-labile 100-kDa glycoprotein (pI, 4.1) which was markedly diminished or absent in serum-sensitive strains. These 2DGE analyses provide information that is useful in taxonomic and epidemiologic studies and for the purification of surface antigens for the development of campylobacter vaccines and may also facilitate the identification of specific virulence factors. Images

Dunn, B E; Blaser, M J; Snyder, E L

1987-01-01

242

On-line combination of capillary isoelectric focusing and capillary non-gel sieving electrophoresis using a hollow-fiber membrane interface: a novel two-dimensional separation system for proteins.  

PubMed

A novel two-dimensional (2D) separation system for proteins was reported. In the system, a piece of dialysis hollow-fiber membrane was employed as the interface for on-line combination of capillary isoelectric focusing (CIEF) and capillary non-gel sieving electrophoresis (CNGSE). The system is similar equivalent to two-dimensional polyacrylamide gel electrophoresis (2D PAGE), by transferring the principal of 2D PAGE separation to the capillary format. Proteins were focused and separated in first dimension CIEF based on their differences in isoelectric points (pIs). Focused protein zones was transferred to the dialysis hollow-fiber interface, where proteins hydrophobically complexed with sodium dodecyl sulfate (SDS). The negatively charged proteins were electromigrated and further resolved by their differences in size in the second dimension CNGSE, in which dextran solution, a replaceable sieving matrix instead of cross-linked polyacrylamide gel was employed for size-dependent separation of proteins. The combination of the two techniques was attributed to high efficiency of the dialysis membrane interface. The feasibility and the orthogonality of the combined CIEF-CNGSE separation technique, an important factor for maximizing peak capacity or resolution elements, were demonstrated by examining each technique independently for the separation of hemoglobin and protein mixtures excreting from lung cancer cells of rat. The 2D separation strategy was found to greatly increase the resolving power and overall peak capacity over those obtained for either dimension alone. PMID:15680795

Liu, Hechun; Yang, Chun; Yang, Qing; Zhang, Weibing; Zhang, Yukui

2005-03-01

243

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

PubMed Central

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

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

2013-01-01

244

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

PubMed

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

Osuna, M Begoña; Casamayor, Emilio O

2010-06-04

245

Separation of chromosomal DNA molecules from C.albicans by pulsed field gel electrophoresis.  

PubMed Central

Modifications have been made to standard pulse field gel electrophoresis (PFGE) systems to enable very large DNA molecules to be resolved. The single most important modification was to elevate the temperature of electrophoresis to 35 degrees C. This enabled the largest Saccharomyces cerevisiae chromosome to be reproducibly resolved. More impressively, it enabled the DNA of Candida albicans to be clearly resolved into six bands, a feat which was very difficult at lower temperatures. Even so, optimal resolution could only be obtained by carefully adjusting field voltages and switching times. The DNA from the two largest C. albicans chromosomes, which was estimated to be at least 5-10Mbp in size, ran somewhat anomalously, giving fuzzy bands which did not migrate in the direction of the average electric field. That the highest molecular weight band was a distinct chromosome was demonstrated by specific hybridisation to the C. albicans ADE2 gene probe. With further fine tuning, the PFGE system described here should be capable of resolving DNA from the smallest human chromosomes. Images

Snell, R G; Wilkins, R J

1986-01-01

246

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

247

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

248

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

249

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis  

PubMed Central

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms. Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity. However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE). This technique is finding widespread use for the detection and characterization of amyloid conformational variants. Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins. The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated. Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

Halfmann, Randal; Lindquist, Susan

2008-01-01

250

Analysis of the phospholipase C-?1 pleckstrin homology domain using native polyacrylamide gel electrophoresis.  

PubMed

The phospholipase C (PLC)-?1 pleckstrin homology (PH) domain has a characteristic short ?-helix (?2) from residues 82 to 87. The contributions of the ?2-helix toward the inositol 1,4,5-trisphosphate (IP(3)) binding activity and thermal stability of the PLC-?1 PH domain were investigated using native polyacrylamide gel electrophoresis (PAGE). Native PAGE analyses of gel migration shift induced by IP(3) binding and of protein aggregation induced by heating indicated that disruption of the ?-helical conformation by replacement of Lys86 with proline resulted in reduced affinity for IP(3) and in thermal destabilization of the IP(3)-binding state. Although the mutant protein with replacement of Lys86 with alanine showed a slight reduction in thermal stability, the IP(3)-binding affinity was similar to that of the wild-type protein. Replacement of Phe87 with alanine, but not with tyrosine, also resulted in reduced affinity for IP(3) and in thermal instability. These results indicated that the helical conformation of the ?2-helix and the phenyl ring of Phe87 play important roles in the IP(3)-binding activity and thermal stability of the PLC-?1 PH domain. Based on these results, the biological role of the ?2-helix of the PLC-?1 PH domain is discussed in terms of membrane binding. PMID:22995066

Tanio, Michikazu; Nishimura, Katsuyuki

2012-09-18

251

Characterisation of natural hybrids between Pterostylis alveata Garnet and Pterostylis ophioglossa R. Br. (Orchidaceae) by starch gel electrophoresis  

Microsoft Academic Search

A putative natural inter-specific orchid hybrid between Pterostylis alveata and P. ophioglossa from Fingal Point, New South Wales, Australia, with intermediate morphological characters between the parents was tested using starch gel electrophoresis. Four enzyme systems, glucose phosphate isomerase (GPI), uridine diphosphogluconic pyrophosphatase (UDP), malic enzyme (ME) and leucine amino-peptidase (LAP), exhibited precisely the heterozygote pattern in the hybrid plants demonstrating

I. K. Sharma; D. L. Jones

1999-01-01

252

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

Microsoft Academic Search

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

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

2005-01-01

253

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

254

Application of temperature gradient gel electrophoresis to the study of yeast diversity in the estuary of the Tagus river, Portugal  

Microsoft Academic Search

Temperature gradient gel electrophoresis (TGGE) was employed for the assessment of yeast diversity in the estuary of the Tagus river (Portugal). The molecular detection of yeasts was carried out directly from water samples and, in parallel, a cultivation approach by means of an enrichment step was employed. A nested PCR was employed to obtain a fungal amplicon containing the D2

Mário Gadanho; José Paulo Sampaio

2004-01-01

255

One-step purification of R-phycoerythrin from the red macroalga Palmaria palmata using preparative polyacrylamide gel electrophoresis  

Microsoft Academic Search

Phycoerythrin is a major light-harvesting pigment of red algae and cyanobacteria widely used as a fluorescent probe. In this study, phycoerythrin of the red macroalga Palmaria palmata was extracted by grinding the algal sample in liquid nitrogen, homogenisation in phosphate buffer and centrifugation. Phycoerythrin was then purified from this crude extract using preparative polyacrylamide gel electrophoresis (PAGE) with a continuous

A. V. Galland-Irmouli; L. Pons; M. Luçon; C. Villaume; N. T. Mrabet; J. L. Guéant; J. Fleurence

2000-01-01

256

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

PubMed Central

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

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

1995-01-01

257

An examination of the population genetics of Laminaria and other brown algae in the laminariales using starch gel electrophoresis  

Microsoft Academic Search

While some investigators have attempted to use isozyme electrophoresis to gain information on the genetics of brown algae, most have reported unsatisfactory results. Through exhaustive screening and modification of sample preparation techniques, gel and tray buffers systems, plus staining recipes, we have developed procedures that consistently provide scorable bands for over 20 enzyme systems in several laminarian algae. We have

C. D. Neefus; B. P. Allen; H. P. Baldwin; A. C. Mathieson; R. T. Eckert; C. Yarish; M. A. Miller

1993-01-01

258

The characterization of Nigerian varieties of pepper, Capsicum annuum and Capsicum frutescens by SDS polyacrylamide gel electrophoresis of seed proteins  

Microsoft Academic Search

The possibility of using electrophoresis to characterize varieties of pepper, Capsicum annuum and Capsicum frutescens cultivated in Nigeria was investigated. The SDS- polyacrylamide gel electropherogram of extracted total seed proteins of 10 breeding lines in each of the 6 varieties investigated, revealed a pattern in which 12 polypeptide bands with apparent molecular weight range of 22 to 98 kilodaltons could

P. G. C. Odeigah; B. Oboh; I. O. Aghalokpe

1999-01-01

259

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

260

Genetic Relationship between Blood and Nonblood Isolates from Bacteremic Patients Determined by Pulsed-Field Gel Electrophoresis  

PubMed Central

A total of 148 isolates from 55 bacteremic patients were examined by pulsed-field gel electrophoresis. Genetically different nonblood strains were isolated from 13.9% of patients with bacteremia caused by gram-positive cocci and 42.1% with Pseudomonas aeruginosa bacteremia, indicating that antibiograms of a single nonblood P. aeruginosa isolate are not always informative for treatment of bacteremia.

Matsuda, Junichi; Hirakata, Yoichi; Iori, Fumiaki; Mochida, Chikako; Ozaki, Yumi; Nakano, Michiko; Izumikawa, Kohichi; Yamaguchi, Toshiyuki; Yoshida, Ryoji; Miyazaki, Yoshitsugu; Maesaki, Shigefumi; Tomono, Kazunori; Yamada, Yasuaki; Kohno, Shigeru; Kamihira, Shimeru

1998-01-01

261

Use of Pulsed Field Gel Electrophoresis to Determine the Source of Microbial Contamination of Central Venous Catheters  

Microsoft Academic Search

Microorganisms detected in situ on the distal tip of central venous catheters (CVC) within 90 min of insertion were investigated using pulsed-field gel electrophoresis to analyse genomic fragments obtained with the SmaI restriction enzyme. Thirty patients received a triple lumen CVC, which was inserted directly through the skin using the Seldinger technique. In a further 30 patients a triple lumen

M. A. Livesley; S. E. Tebbs; H. A. Moss; M. H. Faroqui; P. A. Lambert; T. S. J. Elliott

1998-01-01

262

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

263

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

264

A new method combining sequential immunoaffinity depletion and differential in gel electrophoresis to identify autoantibodies as cancer biomarkers.  

PubMed

Easily measurable biomarkers are urgently required to detect early stages of cancer progression. Autoantibodies (aAbs), as a component of the humoral immune response against tumor cells, have such potential of diagnostic markers since they are circulating and stable proteins, produced rapidly and easily amenable to in vitro dosage. The identification of aAbs is based on the characterization of tumor-associated antigens (TAA) against which they are directed. Here, we propose a new method for an unbiased identification of TAA and thereby of aAbs as cancer biomarkers. This method that we called sequential immunoaffinity depletion-differential in gel electrophoresis (SID-DIGE) is based on the immunodepletion of tumor cell lysates with IgG from control and tumor-bearing mice and direct matching of the flow throughs of these immunoaffinity separations on the same 2D format. This strategy reduces the complexity of the samples to be analyzed and maximizes the interest of assessing hundreds of proteins simultaneously. SID-DIGE has also the potential, contrary to existing serological proteome analysis (SERPA) techniques, to detect immunogenic proteins with conformational epitopes, including those resulting from post-translational modifications. Using a model of human colorectal tumors in mice for the proof of principle, we showed that SID-DIGE outperforms the conventional SERPA technique, with the identification of 7 common TAA (validating our approach) and 18 additional aAbs proving the potential of this new method. In particular, the identification of aAbs directed against key enzymes supporting glycolysis gives credential to the role of hypoxia as a major determinant of the tumor proteome and thus as a source of immunogenicity. Overall, the developed methodology allowed efficient screening of sera for the identification of aAbs as potential biomarkers. PMID:23916966

Grandjean, Marie; Dieu, Marc; Raes, Martine; Feron, Olivier

2013-08-02

265

Rapid SDS-GEL capillary electrophoresis for the analysis of recombinant NADP +-dependent formate dehydrogenase during expression in Escherichia coli cells and its purification  

Microsoft Academic Search

The level of expression in Escherichia coli cells and different steps of purification of the recombinant NADP+-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from bacterium Pseudomonas sp.101 was analyzed by rapid SDS-Gel capillary electrophoresis (SDS-Gel CE) and compared with SDS polyacrylamide gel electrophoresis (SDS PAGE). First standard proteins were separated in the short capillary and the calibration curve generated, then fractions

V Klyushnichenko; V Tishkov; M.-R Kula

1997-01-01

266

Identification of 2D-gel proteins: A comparison of MALDI\\/TOF peptide mass mapping to ? LC-ESI tandem mass spectrometry  

Microsoft Academic Search

A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis\\u000a from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and ?LC-MS\\/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding\\u000a open reading frame databases by

Hanjo Lim; Jimmy Eng; John R. Yates; Sandra L. Tollaksen; Carol S. Giometti; James F. Holden; Michael W. W. Adams; Claudia I. Reich; Gary J. Olsen; Lara G. Hays

2003-01-01

267

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

PubMed Central

Background: Lung cancer remains the leading cause of cancer-related mortality worldwide. Early detection of lung cancer is problematic due to the lack of a marker with high diagnosis sensitivity and specificity. The purpose of this study was to develop techniques to identify the differential expression protein profiles between tumor and tumor free of lung cancer tissues. Methods: 2-dimensional differential ingel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used to analyze four samples of lung cancer tissue (3 replicates each). Results: From optimized 2DE image, A total of 2561 spots were detected and 427 spots of these were differentially expressed (p<0.01). 40 spots were subjected to mass spectrometry including over expressed proteins and under expressed proteins. Some proteins were the products of oncogenes and others were involved in the regulation of cell cycle and signal transduction such as Annexin III, Selenium binding protein, glyceraldehydes-3-phosphate dehydrogenase, cathepsin D and catalase. Conclusion: These data are valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis, establishing human lung cancer proteome database and screening molecular marker to further study human lung cancer. Using the DIGE approach, we were able to find many proteins that were expressed differently due to the disease state (tumor and tumor-free).

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

2011-01-01

268

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

269

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

PubMed

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

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

2011-05-23

270

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

SciTech Connect

The inclusion of 1% casein or bovine serum albumin in buffer used to reactivate enzymes subjected to sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis resulted in accelerated removal of SDS and restoration of nuclease and beta-galactosidase enzyme activities. Nuclease and beta-galactosidase activities which are absent from gels after longer wash procedures are detectable with this technique. Enzyme activity in gels prepared with SDS which contained inhibitory contaminants was partially restored by the casein wash procedure. The threshold of detection of two-dimensionally separated deoxyribonuclease I using the casein wash procedure was 1 picogram.

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

1990-08-15

271

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

272

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

PubMed

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

Alegría, Angel; González, Renata; Díaz, Mario; Mayo, Baltasar

2010-11-04

273

Investigation of endogenous soybean food allergens by using a 2-dimensional gel electrophoresis approach.  

PubMed

As part of the safety assessment of genetically modified (GM) soybean, 2-dimensional gel electrophoresis analyses were performed with the isoxaflutole and glyphosate tolerant soybean FG72, its non-GM near-isogenic counterpart (Jack) and three commercial non-GM soybean lines. The objective was to compare the known endogenous human food allergens in seeds in the five different soybean lines in order to evaluate any potential unintended effect(s) of the genetic modification. In total, 37 protein spots representing five well known soybean food allergen groups were quantified in each genotype. Qualitatively, all the allergenic proteins were detected in the different genetic backgrounds. Quantitatively, among 37 protein spots, the levels of accumulation of three allergens were slightly lower in the GM soybean than in the non-GM counterparts. Specifically, while the levels of two of these three allergens fell within the normal range of variation observed in the four non-GM varieties, the level of the third allergen was slightly below the normal range. Overall, there was no significant increase in the level of allergens in FG72 soybean seeds. Therefore, the FG72 soybean can be considered as safe as its non-GM counterpart with regards to endogenous allergenicity. Additional research is needed to evaluate the biological variability in the levels of endogenous soybean allergens and the correlation between level of allergens and allergenic potential in order to improve the interpretation of these data in the safety assessment of GM soybean context. PMID:20932868

Rouquié, David; Capt, Annabelle; Eby, William H; Sekar, Vaithilingam; Hérouet-Guicheney, Corinne

2010-10-13

274

Characterization of United Kingdom Isolates of Corynebacterium pseudotuberculosis Using Pulsed-Field Gel Electrophoresis  

PubMed Central

Caseous lymphadenitis is a chronic suppurative disease caused by Corynebacterium pseudotuberculosis and is responsible for serious economic losses to the sheep and goat industry. Caseous lymphadenitis was first reported for goats in the United Kingdom in 1990 and for sheep in 1991. Recent evidence suggests that the prevalence of the disease within the national flock is increasing. Fifty isolates of C. pseudotuberculosis from the United Kingdom comprising sheep and horse isolates, the original goat outbreak strain, and the type strain were characterized by biotyping, antimicrobial susceptibility, production of phospholipase D, and genotyping by pulsed-field gel electrophoresis using SfiI and SmaI. All of the isolates were confirmed as C. pseudotuberculosis, and all produced phospholipase D but none reduced nitrate. Restriction with SfiI generated 16 to 18 bands between 48.5 and 290 kb and differentiated six pulsotypes. We conclude that 80% of the strains tested were epidemiologically related to the outbreak strain and that the equine profile was distinct both phenotypically and genotypically.

Connor, Kathleen M.; Quirie, Malcolm M.; Baird, Graham; Donachie, William

2000-01-01

275

Application and evaluation of denaturing gradient gel electrophoresis to analyse the yeast ecology of wine grapes.  

PubMed

The performance of denaturing gradient gel electrophoresis (DGGE) for analysing yeasts associated with wine grapes was compared with cultural isolation on malt extract agar (MEA). After optimisation of PCR and electrophoretic conditions, the lower limit of yeast detection by PCR-DGGE was 10(2) cfuml(-1), although this value was affected by culture age and the relative populations of the species in mixed culture. In mixed yeast populations, PCR-DGGE detected species present at 10-100-fold less than other species but not when the ratio exceeded 100-fold. Aureobasidium pullulans was the main species isolated from immature, mature, and both damaged and undamaged grapes. It was not detected by PCR-DGGE when present at populations less than 10(3) cfug(-1). When approaching maturity, damaged grapes gave a predominance of Metschnikowia and Hanseniaspora species (10(5)-10(7) cfug(-1)), all detectable using PCR-DGGE. However, various species of Rhodotorula, Rhodosporidium and Cryptococcus were not detected by this method, even when populations were as high as 10(4) cfug(-1). PCR -DGGE was less sensitive than culture on MEA for determining the yeast ecology of grapes and could not reliably detect species present at populations less than 10(4) cfug(-1). However, this method detected a greater diversity of species than agar plating. PMID:15450194

Prakitchaiwattana, Cheunjit J; Fleet, Graham H; Heard, Gillian M

2004-09-01

276

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

277

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

PubMed Central

Microscopic agglutination (MA) analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to determine strain and species similarities and dissimilarities among three species of oral anaerobic spirochetes, Treponema denticola, Treponema pectinovorum, and Treponema vincentii. The MA analysis revealed a diversity of serologic reactivity or sharing of common antigens within each species. However, there was no cross-reactivity or sharing of common antigens among the three species. Distinct SDS-PAGE whole-cell electrophoretograms for each species were obtained. The banding patterns for 16 T. denticola strains revealed 30 distinct proteins, while the banding patterns for 5 strains of T. pectinovorum and 2 strains of T. vincentii revealed 26 and 35 distinct proteins, respectively. Analysis of the electrophoretograms showed that their respective banding patterns could be used to distinguish the three species from one another. In addition, strain differences within each species could be detected. There was a correlation between MA analysis and SDS-PAGE analysis. It is thus suggested that both MA and SDS-PAGE analysis be included in classification schemes for the identification of oral spirochetes. Images

Tall, B D; Nauman, R K

1986-01-01

278

Membranes of the adrenal medulla. Behaviour of insoluble proteins of chromaffin granules on gel electrophoresis  

PubMed Central

Washed membranes of bovine adrenal chromaffin granules contained most of the cholesterol and phospholipids of the particle and 22% of the total protein. The protein/lipid ratio was about 0.45 (w/w). Dopamine(3,4-dihydroxyphenethylamine)?-hydroxylase, Mg2+-activated nucleoside triphosphatase and cytochrome b-559 activities were present in the membrane. ATP was the best substrate for the nucleoside triphosphatase, whose pH optimum was 6.4, Km 7×10?4m and Vmax. 1.8?mol/h per mg of protein. Treatment of the membranes with various detergents caused a preferential solubilization of protein compared with lipids. Membranes dissolved in sodium dodecyl sulphate or phenol–acetic acid–urea were subjected to polyacrylamide-gel electrophoresis at alkaline and acid pH respectively. The electrophoretic patterns given by the proteins of the chromaffin granule membrane were distinct from those given by the chromogranins, and from those given by mitochondrial and microsomal membrane proteins. ImagesPLATE 1

Winkler, H.; Hortnagl, Heide; Hortnagl, H.; Smith, A. D.

1970-01-01

279

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

280

Analysis of aminoacyl- and peptidyl-tRNAs by gel electrophoresis  

PubMed Central

During protein synthesis, the ribosome translates the genetic information encoded within messenger RNAs into defined amino acid sequences. Transfer RNAs (tRNAs) are crucial adaptor molecules in this process, delivering amino acid residues to the ribosome and holding the nascent peptide chain as it is assembled. Here, we present methods for the analysis of aminoacyl- and peptidyl-tRNA species isolated from Escherichia coli. These approaches utilize denaturing gel electrophoresis at acidic pH to preserve the labile ester bonds that link amino acids to tRNA. Specific aminoacyl- and peptidyl-tRNAs are detected by Northern blot hybridization using probes for tRNA isoacceptors. Small peptidyl-tRNAs can be differentiated from aminoacyl-tRNA through selective deacylation of the latter with copper sulfate. Additionally, peptidyl-tRNAs can be detected through metabolic labeling of the nascent peptide. This latter approach is amenable to pulse-chase analysis to examine peptidyl-tRNA turnover in vivo. We have applied these methods to study programmed translational arrests and the kinetics of paused ribosome turnover.

Janssen, Brian D.; Diner, Elie J.; Hayes, Christopher S.

2013-01-01

281

Protein degradation in human pure pancreatic juice analyzed by two-dimensional gel electrophoresis.  

PubMed

Two-dimensional gel electrophoresis (2-DE) was used to study protein degradation in human pure pancreatic juice (PPJ) which was collected at 5 min intervals for 20 min by selective endoscopic cannulation of the main pancreatic duct. In PPJ collected from healthy subjects no significant degradation was observed by incubating PPJ at 37 degrees C up to 6 h. By further incubation for 24 h, glycoprotein-1, procarboxypeptidase A-1 and lipase were nearly completely degraded, while alpha-amylase and procarboxypeptidase B-1 were not degraded under these conditions; alpha-amylase became labile in the presence of 1 mM ethylene diaminetetraacetic acid (EDTA) or 10 mM phenyl methyl sulfonyl fluoride (PMSF). Protein degradation was observed by 2-DE of an initial fraction of PPJ collected from patients with chronic calcific pancreatitis (CCP). The 2-DE patterns of subsequent fractions resembled those of PPJ from healthy subjects. The mixture of the last fraction with the initial fraction showed significant protein degradation, inhibited by adding aprotinin. Furthermore, the extent of protein degradation correlated with the dilatation of the main pancreatic duct as a consequence of intraductal stagnation of pancreatic juice. These findings demonstrate that protein degradation in PPJ is accelerated by intraductal activation of serine proteases in the case of patients with CCP. 2-DE of PPJ from patients with CCP provides useful information for the evaluation of intraductal activation of zymogens and the progress of chronic pancreatitis. PMID:8738347

Furui, T; Ikeda, M; Chao-Ming, L; Okita, K; Nakamura, K

1996-04-01

282

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

2012-02-29

283

Characterization of Protistan Assemblages in the Ross Sea, Antarctica, by Denaturing Gradient Gel Electrophoresis  

PubMed Central

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

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

2004-01-01

284

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

285

Fidelity of Thermococcus litoralis DNA polymerase (Vent) in PCR determined by denaturing gradient gel electrophoresis.  

PubMed Central

DNA synthesis fidelities of two thermostable DNA polymerases, Thermus aquaticus (Taq) and Thermococcus litoralis (Tli, also known as Vent), and a non-thermostable enzyme, a modified T7 DNA polymerase (Sequenase), were determined by analyzing polymerase chain reaction (PCR) products using denaturing gradient gel electrophoresis (DGGE). The error rates were 4.4, 8.9, and 2.4 x 10(-5) errors/bp for modified T7, Taq, and Tli polymerase, respectively. Reducing the nucleotide triphosphate concentration for Tli polymerase during PCR did not alter the fidelity. The ability of DGGE to detect a mutant present at several percent in a wild type population is related to the polymerase fidelity. To examine the sensitivity of mutant detection, human genomic DNA containing a 1% fraction of a known base pair substitution mutant was PCR-amplified with the three enzymes using primers that flank the mutant sequence. The PCR products were analyzed by DGGE. The signal from the mutant present at 1% was visible in the samples amplified with modified T7 and Tli polymerase, but the higher error rate of Taq polymerase did not permit visualization of the signal in DNA amplified with Taq polymerase. Images

Cariello, N F; Swenberg, J A; Skopek, T R

1991-01-01

286

Control of buffer pH during agarose gel electrophoresis of glyoxylated RNA.  

PubMed

There is a shift in buffer pH routinely encountered during electrophoresis. If one is running glyoxylated RNA on agarose gels, this pH shift can have potentially detrimental effects on experimental objectives if the shift is allowed to proceed unabated; this is due to the fact that the RNA will deglyoxylate if the pH is allowed to rise above 8.0. In order to counteract the shift, the buffer may be continuously recirculated or totally replaced at predetermined intervals. Because constant recirculation may be technically bothersome to achieve and because repeated total buffer replacement may require large amounts of highly purified water, we have studied (a) the time course of the pH change encountered during the electrophoretic process and (b) whether or not simple manual mixing of the buffer system at specific intervals was sufficient to maintain pH within acceptable bounds. We found that, under the given conditions, the cathode pH had nearly reached 8.0 at 25 min and had soared to 10.5 at 40 min. We further found that manual mixing at 20-min intervals not only prevented the cathode pH from rising above 7.8, but also restored the buffer to its initial pH. PMID:1712208

O'Conner, J L; Wade, M F; Zhou, Y

1991-03-01

287

Proteins of the kidney microvillus membrane. Identification of subunits after sodium dodecylsullphate/polyacrylamide-gel electrophoresis.  

PubMed Central

The proteins of microvilli prepared from pig kidney were analysed by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The typical pattern stained for protein revealed five major bands, four of which also stained for carbohydrate, and about 15 minor bands. For descriptive purposes the bands were designated numerically by their apparent molecular weights (X10(-3). Well-characterized proteins were identified with four of the five major bands. Dipeptidyl peptidase IV, a serine proteinase that may be specifically labelled with di-isopropyl [32P]phosphorofluoridate, was assigned to band 130. Aminopeptidase M was assigned to band 160, though when released from the membrane by a proteinase, this protein comprises three polypeptides each of lower apparent molecular weight than the native enzyme. Neutral endopeptidase can be assigned to band 95 and actin to band 42. The fifth major band (180) is an extrinsic glycoprotein that has not been identified with any microvillus enzyme activity. These four proteins contribute 21% of the microvillus-membrane protein. Kidney microvillus actin was characterized by a variety of properties and was similar to muscle actin. A computer analysis of the gel pattern indicates that it comprises 9.0% of the microvillus protein. Myosin is not present in the microvillus, but another protein associated with band 95, with properties that distinguish it from neutral endopeptidase, was tentatively identified as alpha-actinin. Alkaline phosphatase was identified as a monomeric polypeptide with an apparent molecular weight of 80000; it is a minor protein of the microvillus and is not discernible as a discrete band in the gel pattern. These and other results permit a model of the organization of the microvillus protein to be suggested. The computer program used has been deposited as Supplementary Publication SUP 50070 (12 pages) at the British Library Lending Division, Boston Spa. Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1976) 153,5. Images PLATE 1 PLATE 2

Booth, A G; Kenny, A J

1976-01-01

288

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

289

Mutations of the G sup s. alpha. -subunit gene in Albright hereditary osteodystrophy detected by denaturing gradient gel electrophoresis  

SciTech Connect

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

Weinstein, L.S.; Friedman, E.; Collins, R.M.; Spiegel, A.M.; Gejman, P.V.; Kadowaki, Takashi; Gershon, E.S. (National Inst. of Health, Bethesda, MD (United States))

1990-11-01

290

Simple, Time-Saving Dye Staining of Proteins for Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis Using Coomassie Blue  

Microsoft Academic Search

A fixation-free and fast protein-staining method for sodium dodecyl sulfate–polyacrylamide gel electrophoresis using Coomassie blue is described. The protocol comprises staining and quick washing steps, which can be completed in 0.5 h. It has a sensitivity of 10 ng, comparable with that of conventional Coomassie Brilliant Blue G staining with phosphoric acid in the staining solution. In addition, the dye

Wei-Hua Dong; Tian-Yun Wang; Fang Wang; Jun-He Zhang

2011-01-01

291

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

292

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

293

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

Microsoft Academic Search

One dominating strain of serotype 1\\/2b was found when serotyping and pulsed-field gel electrophoresis (PFGE) patterns were used for the characterization of 41 Listeria monocytogenes isolates originating from an ice cream plant. Samples were taken from the production environment, equipment and ice cream during the years 1990–1997. Serotyping divided the isolates into two serovars, 1\\/2b and 4b. Three rare-cutting enzymes

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

1999-01-01

294

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

Microsoft Academic Search

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

Lewis M. Graves; Bala Swaminathan

2001-01-01

295

Pulsed-field gel electrophoresis pattern similarities between Listeria monocytogenes isolated from human patients and poultry in Chile  

Microsoft Academic Search

Listeria monocytogenes is the causal agent of listeriosis, a highly lethal food-borne disease. We identified isolates of L.monocytogenes present in poultry products bought at local retailers. The bacterial DNA was isolated and patterns were compared by pulsed-field gel electrophoresis (PFGE). Food isolates were also compared against three strains isolated from patients with listeriosis. Results showed 13 PFGE groups (>90% similarity).

Claudia Foerster; Gisela Gonzalez-Hein; Miriam Troncoso; Guillermo Figueroa

2012-01-01

296

Evaluation of the denaturing gradient gel electrophoresis-apparatus as a parameter influencing soil microbial community fingerprinting  

Microsoft Academic Search

We compared two denaturing gradient gel electrophoresis (DGGE) systems—DCode (Biorad, Hercules, CA, USA) and PhorU (Ingeny,\\u000a Leiden, NL), performing community level 16S and 18S rRNA gene fragment-PCR-DGGE with total DNA extracted from upland pasture\\u000a soil used for outdoor cattle husbandry. The methodological evaluation of the DGGE apparatus as parameter influencing DGGE\\u000a fingerprinting, based on cluster analysis of soil bacterial and

J. AscherM; M. T. Ceccherini; A. Chro?áková; J. Jirout; F. Borgogni; D. Elhottová; M. Šimek; G. Pietramellara

2010-01-01

297

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

298

Typing of Salmonella enterica Serotype Paratyphi C Isolates from Various Countries by Plasmid Profiles and Pulsed-Field Gel Electrophoresis  

Microsoft Academic Search

Pulsed-field gel electrophoresis (PFGE) of 61 Salmonella enterica serotype Paratyphi C isolates from six coun- tries gave five distinct clusters. Twenty-four isolates from five countries were susceptible to 10 antimicrobials tested and gave similar restriction endonuclease digest patterns of the 38-MDa plasmid. In contrast, plasmid and PFGE profiles of 37 multidrug-resistant isolates from Zaire were different from those from other

S. KARIUKI; J. CHEESBROUGH; A. K. MAVRIDIS; C. A. HART

1999-01-01

299

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

PubMed

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

Gough, E J; Gough, N M

1984-01-11

300

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

PubMed Central

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

Gough, E J; Gough, N M

1984-01-01

301

Quantification of Fetal and Total Circulatory DNA in Maternal Plasma Samples Before and After Size Fractionation by Agarose Gel Electrophoresis  

Microsoft Academic Search

Fetal extracellular DNA is mainly derived from apoptotic bodies of trophoblast. Recent studies have shown size differences between fetal and maternal extracellular DNA. We have examined the quantification of fetal (SRY gene) and total (GLO gene) extracellular DNA in maternal plasma in different fractions (100-300, 300-500, 500-700, 700-900, and ? 900 bp) after size fractionation by agarose gel electrophoresis. DNA

I. Hromadnikova; L. Zejskova; J. Doucha; D. Codl

2006-01-01

302

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

303

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

Microsoft Academic Search

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

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

1996-01-01

304

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

SciTech Connect

A typing method for Clostridium difficile based on the incorporation of (/sup 35/S)methionine into cellular proteins, their separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their visualization by autoradiography is described. On analysis of the radiolabeled-protein profiles, nine distinct groups were observed (A to E and W to Z). The method, which is simple, reproducible, and readily expandable, has been applied in epidemiological studies to demonstrate cross-infection and hospital acquisition of C. difficile.

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

1986-01-01

305

Glucose-induced changes of multiple mouse islet proteins analysed by two-dimensional gel electrophoresis and mass spectrometry  

Microsoft Academic Search

Aims\\/hypothesis  The aim of this study was to investigate molecular mechanisms of glucose-induced changes in islets of Langerhans by analysing global changes in protein patterns of islets exposed to elevated glucose concentrations.Methods  Islets were isolated from C57BL\\/6J mice and used either directly or after exposure to 11 mmol\\/l glucose for 24 h. Islet protein profiles were obtained by two-dimensional gel electrophoresis, and protein spots

M. Ahmed; P. Bergsten

2005-01-01

306

Use of pulsed-field gel electrophoresis for investigation of an outbreak of Clostridium difficile infection among geriatric patients  

Microsoft Academic Search

A six-month outbreak ofClostridium difficile infection among elderly residents of a middle-term-care facility was investigated. Pulsed-field gel electrophoresis was used to genotype 22 outbreak strains and 30 epidemiologically unrelated strains. A prospective case-control study was conducted to identify risk factors for epidemicClostridium difficile-associated diarrhea. All epidemiologically unrelatedClostridium difficile strains of the same serogroup could be differentiated by their DNA patterns

D. Talon; P. Bailly; M. Delmée; M. Thouverez; B. Mulin; M. Iehl-Robert; V. Cailleaux; Y. Michel-Briand I

1995-01-01

307

Analysis of brain proteins in Alzheimer’s disease using high-resolution two-dimensional gel electrophoresis  

Microsoft Academic Search

Two-dimensional gel electrophoresis (2-DE), a method which can be used to analyze the expression of many proteins, is a promising and powerful approach which we have begun to use in the characterization of the complex pathologic processes in Alzheimer’s disease (AD). In the present study, a reliable 2-DE database of human brain proteins was created by improving the reproducibility of

T. Tsuji; S. Shimohama; S. Kamiya; T. Sazuka; O. Ohara

1999-01-01

308

Quantification of HIV1 proviral DNA and analysis of genomic diversity by polymerase chain reaction and temperature gradient gel electrophoresis  

Microsoft Academic Search

A competitive polymerase chain reaction\\/temperature gradient gel electrophoresis (PCR\\/TGGE) protocol was developed for exact quantification of HIV-1 proviral DNA copy numbers in clinical samples. An internal standard (ST) that differs from wildtype-sequences only by a single base exchange was used as a competitor in PCR. Quantification of HIV-1 target sequences was achieved by coamplification of defined copy numbers of ST

Ulrike Wieland; Helge Suhr; Bernd Salzberger; Hans J. Eggers; Rüdiger W. Braun; Joachim E. Kühn

1996-01-01

309

Determination of biotin on a protein by quantitative sodium dodecyl sulfate–capillary gel electrophoresis of monomeric avidin  

Microsoft Academic Search

Sodium dodecyl sulfate–capillary gel electrophoresis (SDS–CGE) is performed to quantify monomeric avidin and biotin on a protein. Under non-reducing SDS–CGE conditions, avidin migrates as monomers exhibiting apparent molecular mass 17?000. In the presence of a biotin–protein conjugate, monomeric avidin binds the conjugate and forms a larger complex that migrates later in the separation. The difference between the remaining monomeric avidin

Huey G. Lee; Edward Fritsche

2003-01-01

310

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

311

Assessment of allozyme variation among New Zealand populations of Gracilaria chilensis (Gracialiares, Rhodophyta) using starch-gel electrophoresis  

Microsoft Academic Search

New Zealand populations of Gracilaria chilensis are uniform in anatomical reproductive characteristics but vary morphologically and have been found to separate into two distinct groups with respect to agar methylation level, namely low (24–30%) and high (43–47%). To investigate the genetic variation within New Zealand populations of this species, 14 isozyme loci detected by starch-gel electrophoresis were examined in 17

Sompop Intasuwan; Margaret E. Gordon; Charles H. Daugherty; Graeme C. Lindsay

1993-01-01

312

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

Microsoft Academic Search

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

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

313

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

Microsoft Academic Search

A fundamental question in microbial oceanography concerns the relationship between prokaryote diversity and biogeochemical function in an ecosystem context. We combined bromodeoxyuridine (BrdU) magnetic bead immu- nocapture and PCR-denaturing gradient gel electrophoresis (BUMP-DGGE) to examine phylotype-specific growth in natural marine assemblages. We also examined a broad range of marine bacterial isolates to determine their abilities to incorporate BrdU in order

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

2007-01-01

314

The isolation of high molecular weight DNA from wheat, barley and rye for analysis by pulse-field gel electrophoresis  

Microsoft Academic Search

A method is presented for the preparation of large DNA molecules from protoplasts embedded in agarose blocks of three different cereals-hexaploid bread wheat (Triticum aestivum), barley (Hordeum vulgare) and rye (Secale cereale). Pulse-field gel electrophoresis (PFGE) analysis of these DNA preparations using a contour-clamped homogeneous field (CHEF) apparatus indicated that the size of the DNA molecules was greater than 6

Wing Y. Cheung; Michael D. Gale

1990-01-01

315

Genetic Relationship between Blood and Nonblood Isolates from Bacteremic Patients Determined by Pulsed-Field Gel Electrophoresis  

Microsoft Academic Search

A total of 148 isolates from 55 bacteremic patients were examined by pulsed-field gel electrophoresis. Genetically different nonblood strains were isolated from 13.9% of patients with bacteremia caused by gram- positive cocci and 42.1% with Pseudomonas aeruginosa bacteremia, indicating that antibiograms of a single nonblood P. aeruginosa isolate are not always informative for treatment of bacteremia. Bacteremia arises from preexisting

JUNICHI MATSUDA; YOICHI HIRAKATA; FUMIAKI IORI; CHIKAKO MOCHIDA; YUMI OZAKI; MICHIKO NAKANO; KOHICHI IZUMIKAWA; TOSHIYUKI YAMAGUCHI; RYOJI YOSHIDA; YOSHITSUGU MIYAZAKI; SHIGEFUMI MAESAKI; KAZUNORI TOMONO; YASUAKI YAMADA; SHIGERU KOHNO; SHIMERU KAMIHIRA

316

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

Microsoft Academic Search

Sixteen Salmonella strains isolated from a variety of foods during 2000 and 2003 by the Florida State Department of Agriculture were characterized by various genotypic and phenotypic tests. Among 16 isolates, 15 different serotypes were identified. Pulse-field gel electrophoresis (PFGE) fingerprinting profiles obtained using restriction endonucleases XbaI and BlnI revealed that 16 Salmonella isolates were genetically diverse with 16 unique

Xiaodong Xia; Shaohua Zhao; Allen Smith; James McEvoy; Jianghong Meng; Arvind A. Bhagwat

2009-01-01

317

Alkaline single cell gel electrophoresis of DNA fragments in biomonitoring for genotoxicity: an introductory study on healthy human volunteers  

Microsoft Academic Search

Alkaline single cell gel electrophoresis (also known as the ‘comet assay’) is a rapid method for detecting DNA strand breaks\\u000a in individual cells. Before the assay is used for biomonitoring in human populations the test conditions must be accurately\\u000a characterised. Five healthy male volunteers donating capillary blood over a period of 20 weeks showed a fairly stable level\\u000a of DNA

B. Hellman; Hamid Vaghef; Lennart Friis; Christer Edling

1997-01-01

318

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

319

Allergens in hymenoptera venoms. X. Vespid venoms versus venom sac extracts: comparison by two-dimensional polyacrylamide gel electrophoresis.  

PubMed

Vespid venoms were compared to venom sac extracts by two-dimensional polyacrylamide gel electrophoresis using non-equilibrium pH gradient electrophoresis in the first dimension and sodium dodecyl sulfate electrophoresis in the second. The gels were stained with silver. Fresh venoms from four species, Vespula maculifrons, Polistes fuscatus fuscatus, P. metricus and P. exclamans, were compared with commercially available venom sac extracts from the same species. In each case the venom sac extract contained all of the proteins detected in the fresh venom plus numerous additional proteins which are probably sac components. Yellow jacket and bee (Apis mellifera) proteins were extracted from the gels and tested for IgE binding activity using pooled sera from RAST-positive individuals. Significant IgE binding activity was found for the five known bee allergens and for the major yellow jacket venom proteins. Fresh pure vespid venoms contain a relatively small number of major protein subunits. Venom sac extracts contain the same components plus many other proteins not found in the pure venoms. PMID:6625227

Wood, C L; Timmons, B E; Hoffman, D R

1983-10-01

320

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

2006-05-30

321

Pulsed-field gel electrophoresis diversity of human and bovine clinical Salmonella isolates.  

PubMed

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

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

2010-06-01

322

C4 allotyping by prolonged gel electrophoresis and immunoblotting using monoclonal and polyclonal antibodies.  

PubMed

Out of the 136 individual samples mostly from HLA-typed family members which were submitted to the VIth Complement Genetics Workshop 1989, Mainz, FRG, for C4 allotyping, 129 were analyzed by prolonged gel electrophoresis followed by immunoblotting. The blotting was performed using either a Rodger (Rg):1,2 specific monoclonal, 99H7 (provided by G. Mauff, Cologne, FRG), and/or a Chido (Ch):1 specific monoclonal antibody (MoAb), 2B12 (provided by G.J. O'Neill, Miami, USA), and a polyclonal antihuman C4 antibody. In addition, 29 individuals from the workshop panel were tested with a third antibody, 1217, and 20 individuals with a fourth antibody, 1228 (both provided by D. Bitter-Suermann, Hannover, FRG). Due to the improved typing technique a total of 13 C4A and 16 C4B variants could be discriminated, of which some have not yet been described. Considering as a rule that all allotypes with great hemolytic activity are named C4B, the Ch:1 specific antibody 2B12 is reactive with all B allotypes except C4*B11, C4*B12, C4*B13, some C4*B3, C4*B4, one C4*B5, and weakly with one C4*B6 variant. The Rg:1,2 specific antibody 99H7 recognizes all C4A allotypes except for C4*A91, one C4*A1, and some C4*A12 variants. Therefore all these allotypes mentioned may, like C4*A91, express reversed antigenicity. The two additionally tested MoAbs 1217 and 1228 reacted similar to 2B12 with one exception for each. Correlations of HLA haplotypes with C4 allotypes especially with those of reversed antigenicity are discussed. PMID:2088665

Doxiadis, G; Grosse-Wilde, H

1990-01-01

323

Meta-Analysis of Pulsed-Field Gel Electrophoresis Fingerprints Based on a Constructed Salmonella Database  

PubMed Central

A database was constructed consisting of 45,923 Salmonella pulsed-field gel electrophoresis (PFGE) patterns. The patterns, randomly selected from all submissions to CDC PulseNet during 2005 to 2010, included the 20 most frequent serotypes and 12 less frequent serotypes. Meta-analysis was applied to all of the PFGE patterns in the database. In the range of 20 to 1100 kb, serotype Enteritidis averaged the fewest bands at 12 bands and Paratyphi A the most with 19, with most serotypes in the 13?15 range among the 32 serptypes. The 10 most frequent bands for each of the 32 serotypes were sorted and distinguished, and the results were in concordance with those from distance matrix and two-way hierarchical cluster analyses of the patterns in the database. The hierarchical cluster analysis divided the 32 serotypes into three major groups according to dissimilarity measures, and revealed for the first time the similarities among the PFGE patterns of serotype Saintpaul to serotypes Typhimurium, Typhimurium var. 5-, and I 4,[5],12:i:-; of serotype Hadar to serotype Infantis; and of serotype Muenchen to serotype Newport. The results of the meta-analysis indicated that the pattern similarities/dissimilarities determined the serotype discrimination of PFGE method, and that the possible PFGE markers may have utility for serotype identification. The presence of distinct, serotype specific patterns may provide useful information to aid in the distribution of serotypes in the population and potentially reduce the need for laborious analyses, such as traditional serotyping.

Zou, Wen; Chen, Hung-Chia; Hise, Kelley B.; Tang, Hailin; Foley, Steven L.; Meehan, Joe; Lin, Wei-Jiun; Nayak, Rajesh; Xu, Joshua; Fang, Hong; Chen, James J.

2013-01-01

324

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

325

Meta-analysis of pulsed-field gel electrophoresis fingerprints based on a constructed Salmonella database.  

PubMed

A database was constructed consisting of 45,923 Salmonella pulsed-field gel electrophoresis (PFGE) patterns. The patterns, randomly selected from all submissions to CDC PulseNet during 2005 to 2010, included the 20 most frequent serotypes and 12 less frequent serotypes. Meta-analysis was applied to all of the PFGE patterns in the database. In the range of 20 to 1100 kb, serotype Enteritidis averaged the fewest bands at 12 bands and Paratyphi A the most with 19, with most serotypes in the 13-15 range among the 32 serptypes. The 10 most frequent bands for each of the 32 serotypes were sorted and distinguished, and the results were in concordance with those from distance matrix and two-way hierarchical cluster analyses of the patterns in the database. The hierarchical cluster analysis divided the 32 serotypes into three major groups according to dissimilarity measures, and revealed for the first time the similarities among the PFGE patterns of serotype Saintpaul to serotypes Typhimurium, Typhimurium var. 5-, and I 4,[5],12:i:-; of serotype Hadar to serotype Infantis; and of serotype Muenchen to serotype Newport. The results of the meta-analysis indicated that the pattern similarities/dissimilarities determined the serotype discrimination of PFGE method, and that the possible PFGE markers may have utility for serotype identification. The presence of distinct, serotype specific patterns may provide useful information to aid in the distribution of serotypes in the population and potentially reduce the need for laborious analyses, such as traditional serotyping. PMID:23516614

Zou, Wen; Chen, Hung-Chia; Hise, Kelley B; Tang, Hailin; Foley, Steven L; Meehan, Joe; Lin, Wei-Jiun; Nayak, Rajesh; Xu, Joshua; Fang, Hong; Chen, James J

2013-03-14

326

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

327

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

PubMed Central

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

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

1989-01-01

328

Use of denaturing gradient gel electrophoresis for the identification of mixed oral yeasts in human saliva.  

PubMed

A PCR-denaturing gradient gel electrophoresis (DGGE) method was established for the simultaneous presumptive identification of multiple yeast species commonly present in the oral cavity. Published primer sets targeting different regions of the Saccharomyces cerevisiae 26-28S rRNA gene (denoted primer sets N and U) and the 18S rRNA gene (primer set E) were evaluated with ten Candida and four non-Candida yeast species, and twenty Candida albicans isolates. Optimized PCR-DGGE conditions using primer set N were applied to presumptively identify, by band matching, yeasts in the saliva of 25 individuals. Identities were confirmed by DNA sequencing and compared with those using CHROMagar Candida culture. All primer sets yielded detectable DGGE bands for all species tested. Primer set N yielded mainly single bands and could distinguish all species examined, including differentiating Candida dubliniensis from C. albicans. Primer set U was less discriminatory among species but yielded multiple bands that distinguished subspecies groups within C. albicans. Primer set E gave poor yeast discrimination. DGGE analysis identified yeasts in 17 of the 25 saliva samples. Six saliva samples contained two yeast species: three contained C. albicans and three C. dubliniensis. C. dubliniensis was present alone in one saliva sample (total prevalence 16?%). CHROMagar culture detected yeasts in 16 of the yeast-containing saliva samples and did not enable identification of 7 yeast species identified by DGGE. In conclusion, DGGE identification of oral yeast species with primer set N is a relatively fast and reliable method for the simultaneous presumptive identification of mixed yeasts in oral saliva samples. PMID:23065546

Weerasekera, Manjula M; Sissons, Chris H; Wong, Lisa; Anderson, Sally; Holmes, Ann R; Cannon, Richard D

2012-10-11

329

Fluorescence amplified fragment length polymorphism compared to pulsed field gel electrophoresis for Listeria monocytogenes subtyping  

PubMed Central

Background Listeriosis is a severe infection which mainly affects pregnant women, neonates and immuno-compromised adults. ANSES’s Laboratory for Food safety has been the European Union Reference Laboratory (EURL) for L. monocytogenes in the food chain since 2006. Pulsed Field Gel Electrophoresis (PFGE) is routinely used in the EURL for the surveillance of L. monocytogenes isolated from foods, animals and the environment. One of the main EURL activities is to evaluate alternative molecular subtyping methods to PFGE, and integrate their use within the National Reference Laboratories (NRL) network. Since 2008, the United Kingdom (UK)-NRL for L. monocytogenes at the Health Protection Agency (HPA), London, has used fluorescent Amplified Fragment Length Polymorphism (fAFLP) for the routine surveillance of L. monocytogenes isolated from human clinical cases, food and food processing environments in the UK. This study compares fAFLP with PFGE for subtyping L. monocytogenes. Results A panel of 109 L. monocytogenes isolates from either human cases of listeriosis, foods, food processing environments and animals were used for the comparative evaluation. Among these, 2 strains were tested from duplicate culture by both methods. The panel also included field isolates, isolates associated with outbreaks or sporadic cases and reference strains. The two strains tested in duplicate displayed the same fAFLP and PFGE types. Strains known to be epidemiologically associated with one another were found to have unique PFGE and fAFLP types. FAFLP and PFGE divided the strains into 76 and 82 distinct profiles, or types, respectively. The discriminatory index calculated was 0.993 and 0.996 for fAFLP and PFGE, respectively. Conclusions The discriminatory ability of fAFLP was similar to that of PFGE for the subtyping of L. monocytogenes isolates. As a less labour intensive technique fAFLP may be a better method to use than PFGE in investigating outbreaks of human listeriosis and tracking the source of contamination in food processing facilities in real time.

2013-01-01

330

Cationic electrophoresis.  

PubMed

Denaturing, discontinuous electrophoresis in the presence of SDS has become a standard method for the protein scientist. However, there are situations where this method produces suboptimal results. In these cases, electrophoresis in the presence of positively charged detergents such as cetyltrimethylammonium bromide (CTAB) may work considerably better. Methods for electrophoresis and staining of such gels are presented. PMID:22585477

Buxbaum, Engelbert

2012-01-01

331

Disaggregation of adenylate cyclase during polyacrylamide-gel electrophoresis in mixtures of ionic and non-ionic detergents  

PubMed Central

1. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] solubilized from the rat liver plasma membrane with 1% Lubrol PX and partially purified by gel filtration in buffer containing 0.01% Lubrol PX was physically characterized by polyacrylamide-gel electrophoresis. 2. The molecular radius determined for the partially purified enzyme was 4.9nm, compared with the value of 3.9nm obtained for the enzyme before gel filtration. 3. This difference, representing an approximate doubling of the molecular volume of the enzyme, implied that aggregation with itself or other proteins had occurred during partial purification. 4. Aggregation was not reversed by electrophoresis in the presence of high Lubrol concentrations. 5. Substitution of deoxycholate or N-dodecylsarcosinate for Lubrol PX either for solubilization or during electrophoresis led to poorer resolution of membrane proteins at concentrations giving greater than 70% loss of enzyme activity. 6. Partially purified adenylate cyclase was electrophoresed in the presence of mixed micelles of Lubrol PX and deoxycholate or Lubrol PX and N-dodecylsarcosinate. Different mixtures were examined simultaneously in a suitable apparatus. 7. Electrophoresis in the presence of 0.1% Lubrol plus 0.03% deoxycholate decreased the molecular radius of the cyclase to 4.0nm, with greater than 90% recovery of enzymic activity. The net charge of the enzyme was also increased, indicating ionic detergent binding. 8. With 0.1% Lubrol plus 0.03% N-dodecylsarcosinate the molecular radius was 4.3nm, recovery approx. 50% and net charge similar to that seen in Lubrol plus deoxycholate. 9. The resolution of cyclase from bulk protein, on an analytical scale, was improved in the presence of detergent mixtures, as compared with resolution in Lubrol alone. 10. The results demonstrate the usefulness of polyacrylamide-gel electrophoresis to detect and overcome aggregation problems with membrane proteins and suggest that detergent mixtures in specific ratios may be useful in the purification of adenylate cyclase and other intrinsic membrane proteins. ImagesFig. 3.

Newby, Andrew C.; Chrambach, Andreas

1979-01-01

332

Increase in local protein concentration by field-inversion gel electrophoresis  

Microsoft Academic Search

BACKGROUND: Proteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and non-specific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein separation efficiency. Enhancement of protein separation efficiency was investigated by implementing

Henghang Tsai; Teck Yew Low; Steve Freeby; Aran Paulus; Kalpana Ramnarayanan; Chung-pui Paul Cheng; Hon-chiu Eastwood Leung

2007-01-01

333

Purification and staining of intact yeast DNA chromosomes and real-time observation of their migration during gel electrophoresis.  

PubMed

In the past few years, fluorescence microscopy has been used successfully to characterize the motion of intermediate-size DNA molecules (50-500 kbp) during steady- and pulsed-field gel electrophoresis. However, experimental difficulties had prevented the application of this technique to the direct observation of longer DNA chromosomes (1-2 Mbp). In the present study a particular procedure was followed for the purification and staining of chromosomal yeast DNA to protect it from shear forces. Also, a new highly fluorescent DNA-labelling dye, YOYO-1, was employed to improve brightness and contrast. Finally, the motion of such long DNA molecules (1-2 Mbp) was characterized under steady-field electrophoresis conditions. An accurate description of the molecular mechanisms of motion of such long molecules should provide the basis for a detailed analysis of the mechanisms responsible for DNA trapping. PMID:9337860

Gurrieri, S; Bustamante, C

1997-08-15

334

The use of ASB-14 in combination with CHAPS is the best for solubilization of human brain proteins for two-dimensional gel electrophoresis  

Microsoft Academic Search

Protein extraction is the most important step to reveal a proteome by Two-Dimensional Gel Electrophoresis. Usually, the urea\\/thiourea based standard protein extraction buffer (SB) is combined with detergents with the aim of achieving better resolution and solubilization of different classes of proteins. In order to produce better gels and achieve the greatest spot resolution of Human Brain Proteins, comparisons using

Daniel Martins; Bruno Menezes de Oliveira; Santos Farias; Ricardo Shiniti; Oka Horiuchi; Cleyton Crepaldi Domingues; Eneida de Paula; Sergio Marangoni; Wagner Farid Gattaz; Emmanuel Dias-Neto

2007-01-01

335

Disc Gel Electrophoresis of Blood Sera and Muscle Extracts from Some Catostomid Fishes.  

National Technical Information Service (NTIS)

Disc electrophoresis was used to examine blood sera and low ionic strength muscle extracts from specimens of Ictiobus cyprinellus, I. bubalus, I. niger, Carpiodes carpio, C. velifer, C. cyprinus, Erimyzon oblongus, Minytrema melanops, Moxostoma erythrurum...

G. R. Huntsman

1970-01-01

336

Development of a capillary gel electrophoresis method for monitoring disulfide isomer heterogeneity in IgG2 antibodies.  

PubMed

A CGE method for monitoring the disulfide isomer distribution characteristic of IgG2 MAbs is presented. Disulfide heterogeneity of MAbs has been studied using various chromatographic and electrophoretic methods. Although CGE operates using a different selectivity mechanism from that of sorption chromatographic techniques, similar trends are present in the data, which allow the CGE method to be used as a complementary method for studying disulfide isomer distribution. This article focuses on the optimization of a capillary-based gel electrophoresis method that can be used to support antibody development including bioprocess optimization, antibody characterization, release, and formulation stability assessment. PMID:20119952

Lacher, Nathan A; Wang, Qian; Roberts, Rachel K; Holovics, Heidi J; Aykent, Serdar; Schlittler, Michael R; Thompson, Melissa R; Demarest, Charles W

2010-01-01

337

Size analysis of residual host cell DNA in cell culture-produced vaccines by capillary gel electrophoresis.  

PubMed

Residual host cell DNA poses potential safety concerns for cell culture-derived vaccines or other biological products. In addition to the quantity of residual DNA, the size distribution is an important measure for determination of its associated risk factor. We have developed a new method for residual DNA size analysis, based on capillary gel electrophoresis (CGE) technology with sensitive laser induced fluorescence detection (LIF). The performance of this method was optimized through empirical selection of appropriate testing conditions and optimized conditions are presented. Examples are given to demonstrate the successful employment of this method for residual DNA size analysis of cell culture-produced vaccine samples. PMID:23313102

Shen, Xuan; Chen, Xiaomu; Tabor, David E; Liu, Yi; Albarghouthi, Methal; Zhang, Yi-Fan; Galinski, Mark S

2013-01-10

338

Simple, time-saving dye staining of proteins for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using Coomassie blue.  

PubMed

A fixation-free and fast protein-staining method for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using Coomassie blue is described. The protocol comprises staining and quick washing steps, which can be completed in 0.5 h. It has a sensitivity of 10 ng, comparable with that of conventional Coomassie Brilliant Blue G staining with phosphoric acid in the staining solution. In addition, the dye stain does not contain any amount of acid and methanol, such as phosphoric acid. Considering the speed, simplicity, and low cost, the dye stain may be of more practical value than other dye-based protein stains in routine proteomic research. PMID:21850222

Dong, Wei-Hua; Wang, Tian-Yun; Wang, Fang; Zhang, Jun-He

2011-08-05

339

Improved pulsed-field gel electrophoresis procedure for the analysis of Flavobacterium columnare isolates previously affected by DNA degradation.  

PubMed

Flavobacterium columnare is a fresh water bacterium that causes columnaris diseases in over 36 fish species. Intra-species typing of F. columnare can be performed by pulsed-field gel electrophoresis (PFGE). However, this method is hampered by the degradation of chromosomal DNA in about 10% of strains. In the current study, DNA degradation problems caused by extracellular DNases were overcome by fixation of cells with formaldehyde prior to isolation. The results substantiate that after problems due to DNases are overcome, PFGE analysis is a reproducible highly discriminating epidemiological method for studying F. columnare isolates regardless of fish host. PMID:18023300

Soto, Esteban; Mauel, Michael; Lawrence, Mark

2007-10-10

340

Pulsed-field gel electrophoresis as an epidemiologic tool in the investigation of laboratory acquired Salmonella typhi infection.  

PubMed

Strains of Salmonella typhi implicated in two separate cases of laboratory acquired infection from patients and the medical laboratory technologists who processed the patients' samples were analysed by pulsed-field gel electrophoresis. Although all four isolates were of bacteriophage type E1, PFGE was able to demonstrate that the strains responsible for the two laboratory acquired cases were not genetically related. The PFGE patterns of the isolates from the MLTs were found to be identical to those of the corresponding patients after digestion with restriction enzyme AvrII. This provided genetic as well as epidemiological evidence for the source of the laboratory acquired infections. PMID:9322288

Koay, A S; Jegathesan, M; Rohani, M Y; Cheong, Y M

1997-03-01

341

A pulsed-field gel electrophoresis map in the ataxia-telangiectasia region of chromosome 11q22. 3  

SciTech Connect

The authors interest in isolating the gene(s) for ataxia-telangiectasia has prompted construction of a physical map of chromosome 11q22.3 using markers localized to this region by linkage analysis and/or hybrid cell panels. Twenty-two markers have been analyzed by pulsed-field gel electrophoresis. Nine of these markers form an [approximately]2-Mb long-range contiguous map. An average distance of 200 kb between probes in this map should facilitate the isolation of new cDNAs, anonymous probes, and YACs in an orderly way. 15 refs., 2 figs.

Uhrhammer, N.; Huo, Y.; Gatti, R.A. (Univ. of California, Los Angeles, CA (United States)); Concannon, P. (Virginia Mason Research Center, Seattle, WA (United States)); Nakamura, Yusuke (Cancer Institute, Tokyo (Japan))

1994-03-15

342

Detection of metalloproteins in human liver cytosol by synchrotron radiation X-ray fluorescence after sodium dodecyl sulphate polyacrylamide gel electrophoresis  

Microsoft Academic Search

An improved method of analysis of metals in protein bands with synchrotron radiation X-ray fluorescence (SRXRF) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation is introduced and applied to human liver cytosol. Through a step of drying the gel before SRXRF determination, the continuous background resulting mainly from the Compton-scattering of X-rays by the gel matrix was substantially reduced, and

Yuxi Gao; Chunying Chen; Peiqun Zhang; Zhifang Chai; Wei He; Yuying Huang

2003-01-01

343

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

Microsoft Academic Search

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

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

1997-01-01

344

Effect of heat and sodium dodecyl sulfate on solubilization of proteins before two-dimensional polyacrylamide gel electrophoresis.  

PubMed

To solubilize biological samples, sodium dodecyl sulfate (SDS) frequently is added and the mixture heated at 70-100 degrees C. However, two-dimensional polyacrylamide gel electrophoresis of a single protein after SDS treatment has not been reported. When rabbit-muscle creatine kinase was so run, we saw considerable difference in the gel staining pattern for the heated and nonheated enzyme dissolved in the SDS solution. After heating for 10 min at 95 degrees C the number of silver-stained spots apparent increased, and staining of several spots intensified. After 60 min, most of the discrete spots disappeared. Evidently the peptide backbone had been hydrolyzed. When the enzyme was simply left at room temperature for four days, the effects were similar. Appearance of new spots and loss of spots apparently are caused by heating alone but are intensified by SDS. Experiments with human serum albumin yielded similar results. PMID:6499173

Hodges, S C; Hirata, A A

1984-12-01

345

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

NASA Astrophysics Data System (ADS)

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

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

2012-06-01

346

Apolipoprotein distribution in human lipoproteins separated by polyacrylamide gradient gel electrophoresis.  

PubMed

The heterogeneity of serum lipoproteins (excluding very low density (VLDL) and intermediate density (IDL) lipoproteins) and that of lipoproteins secreted by HepG2 cells has been studied by immunoblot analysis of the apolipoprotein composition of the particles separated by polyacrylamide gradient gel electrophoresis (GGE) under nondenaturing conditions. The reactions of antibodies to apoA-I, apoA-II, apoE, apoB, apoD, and apoA-IV have revealed discrete bands of particles which differ widely in size and apolipoprotein composition. GGE of native serum lipoproteins demonstrated that apoA-II is present in lipoproteins of limited size heterogeneity (apparent molecular mass 345,000 to 305,000) and that apoB is present in low density lipoproteins (LDL) and absent from all smaller or denser lipoproteins. In contrast, serum apoA-I, E, D, and A-IV are present in very heterogeneous particles. Serum apoA-I is present mainly in particles of 305 to 130 kDa where it is associated with apoA-II, and in decreasing order of immunoreactivity in particles of 130-90 kDa, 56 kDa, 815-345 kDa, and finally within the size range of LDL, all regions where there is little detectable apoA-II. Serum apoE is present in three defined fractions, one within the size range of LDL, one containing heterogeneous particles between 640 and 345 kDa, and one defined fraction at 96 kDa. Serum apoD is also present in three defined fractions, one comigrating with LDL, one containing heterogeneous particles between 390 and 150 kDa, and one band on the migration front. Most of serum apoA-IV is contained in a band comigrating with albumin. GGE of centrifugally prepared LDL shows the presence of apoB, apoE, and apoD, but not that of apoA-I. However, the particles containing apoA-I, which, in serum, migrated within the LDL size range and as bands of 815 to 345 kDa, were recovered upon centrifugation in the d greater than 1.21 g/ml fraction. GGE of high density lipoproteins (HDL) indicated that most of apoA-I, A-II, and A-IV were present in lipoproteins of the same apparent molecular mass (390-152 kDa). ApoD tended to be associated with large HDL, and this was also significant for HDL apoE, which is present in lipoproteins ranging from 640 to 275 kDa. GGE of very high density lipoproteins (VHDL) presented some striking features, one of which was the occurrence of apolipoproteins in very discrete bands of different molecular mass. ApoA-II was bimodally distributed at 250-175 kDa and 175-136 kDa, the latter fraction also containing apoA-I.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:3411236

Vézina, C A; Milne, R W; Weech, P K; Marcel, Y L

1988-05-01

347

Identification of actinomycete communities in Antarctic soil from Barrientos Island using PCR-denaturing gradient gel electrophoresis.  

PubMed

The diversity of specific bacteria taxa, such as the actinomycetes, has not been reported from the Antarctic island of Barrientos. The diversity of actinomycetes was estimated with two different strategies that use PCR-denaturing gradient gel electrophoresis. First, a PCR was applied, using a group-specific primer that allows selective amplification of actinomycete sequences. Second, a nested-PCR approach was used that allows the estimation of the relative abundance of actinomycetes within the bacterial community. Molecular identification, which was based on 16S rDNA sequence analysis, revealed eight genera of actinomycetes, Actinobacterium, Actinomyces, an uncultured Actinomycete, Streptomyces, Leifsonia, Frankineae, Rhodococcus, and Mycobacterium. The uncultured Actinomyces sp and Rhodococcus sp appear to be the prominent genera of actinomycetes in Barrientos Island soil. PCR-denaturing gradient gel electrophoresis patterns were used to look for correlations between actinomycete abundance and environmental characteristics, such as type of rookery and vegetation. There was a significant positive correlation between type of rookery and abundance of actinomycetes; soil samples collected from active chinstrap penguin rookeries had the highest actinomycete abundance. Vegetation type, such as moss, which could provide a microhabitat for bacteria, did not correlate significantly with actinomycete abundance. PMID:22370930

Learn-Han, L; Yoke-Kqueen, C; Shiran, M S; Vui-Ling, C M W; Nurul-Syakima, A M; Son, R; Andrade, H M

2012-02-08

348

Analysis of steric partition behavior of molecules in membranes using statistical physics. Application to gel chromatography and electrophoresis.  

PubMed Central

The principles of statistical physics are used to formulate general expressions for the steric partition behavior of molecules in both random and ordered membrane structures that may be applied to any shape of the solute and/or the volume-excluding element of the membrane. These expressions fully define partitioning in terms of the volume excluded to point molecules and to finite-sized molecules. The mean effective exclusion volume for a molecule is calculated as a function of a global interaction energy, which varies with position, conformation, and orientation of the molecule. It allows consideration of electrostatic and other nonsteric factors. To test the model, specific partition functions are derived for several simple geometries describing the membrane and solute. Frequently, the derived expressions agree with past analyses; however, a new expression describing partitioning within an random network of fibers is derived. It agrees with past results only in the limit of low exclusion volumes. With greater volume exclusions, past results greatly overestimate the partition function. It is applied to gel electrophoresis and chromatography and survives testing with available experimental data. Unlike past analyses, it predicts nonlinear Ferguson plots for agarose gel electrophoresis. In addition, an analytical expression predicting the minimum radius of a sphere excluded from a random fiber matrix is derived, tested, and found to agree with experimental data.

Schnitzer, J E

1988-01-01

349

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

PubMed

Neutral/neutral two-dimensional (2D) agarose gelelectrophoresis was used to investigate populations of the different topological conformations that pBR322 can adopt in vivo in bacterial cells as well as in Xenopus egg extracts. To help in interpretation and identification of all the different signals, undigested as well as DNA samples pretreated with DNase I, topoisomerase I and topoisomerase II were analyzed. The second dimension of the 2D gel system was run with or without ethidium bromide to account for any possible changes in the migration behavior of DNA molecules caused by intercalation of this planar agent. Finally, DNA samples were isolated from a recA-strain of Escherichia coli , as well as after direct labeling of the replication intermediates in extracts of Xenopus laevis eggs. Altogether, the results obtained demonstrated that 2D gels can be readily used to identify most of the complex topological populations that circular molecules can adopt in vivo in both bacteria and eukaryotic cells. PMID:9649629

Martín-Parras, L; Lucas, I; Martínez-Robles, M L; Hernández, P; Krimer, D B; Hyrien, O; Schvartzman, J B

1998-07-15

350

Characterization of ribosomal proteins from different tissues and species of animals by electrophoresis on polyacrylamide gel  

Microsoft Academic Search

Summary Proteins from ribosomes of different tissues and animals were characterized by polyacrylamide disc electrophoresis. The proteins from ribosomes of different tissues from the same animal are qualitatively similar. The results of the experiments with ribosomes from the livers of different species of animals exhibit clear differences, in the electrophoretic patterns of the proteins.

H. Bielka; H. Welfle

1968-01-01

351

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

Microsoft Academic Search

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

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

1993-01-01

352

Preparation of Plant Protein Samples for 2-D PAGE  

Microsoft Academic Search

\\u000a A critical step in the application of O’Farrell’s (1) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) in plant biology research is the preparation of plant protein\\u000a samples free of artefactual protein modifications and without adversely affecting gel resolving power and reproducibility.\\u000a However, it is important to note that many problems could be encountered in this step. A large part of the

David W. M. Leung

353

Enhanced analysis of human breast cancer proteomes using micro-scale solution isoelectrofocusing combined with high resolution 1-D and 2-D gels.  

PubMed

Current methods for quantitatively comparing proteomes (protein profiling) have inadequate resolution and dynamic range for complex proteomes such as those from mammalian cells or tissues. More extensive profiling of complex proteomes would be obtained if the proteomes could be reproducibly divided into a moderate number of well-separated pools. But the utility of any prefractionation is dependent upon the resolution obtained because extensive cross contamination of many proteins among different pools would make quantitative comparisons impractical. The current study used a recently developed microscale solution isoelectrofocusing (musol-IEF) method to separate human breast cancer cell extracts into seven well-resolved pools. High resolution fractionation could be achieved in a series of small volume tandem chambers separated by thin acrylamide partitions containing covalently bound immobilines that establish discrete pH zones to separate proteins based upon their pIs. In contrast to analytical 2-D gels, this prefractionation method was capable of separating very large proteins (up to about 500 kDa) that could be subsequently profiled and quantitated using large-pore 1-D SDS gels. The pH 4.5-6.5 region was divided into four 0.5 pH unit ranges because this region had the greatest number of proteins. By using very narrow pH range fractions, sample amounts applied to narrow pH range 2-D gels could be increased to detect lower abundance proteins. Although 1.0 pH range 2-D gels were used in these experiments, further protein resolution should be feasible by using 2-D gels with pH ranges that are only slightly wider than the pH ranges of the musol-IEF fractions. By combining musol-IEF prefractionation with subsequent large pore 1-D SDS-PAGE (>100 kDa) and narrow range 2-D gels (<100 kDa), large proteins can be reliably quantitated, many more proteins can be resolved, and lower abundance proteins can be detected. PMID:12458011

Zuo, Xun; Hembach, Peter; Echan, Lynn; Speicher, David W

2002-12-25

354

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

Microsoft Academic Search

Double-stranded (ds), as well as denatured, single- stranded (ss) DNA samples can be analyzed on urea-agarose gels. Here we report that after dena- turation by heat in the presence of 8M urea, the two strands of the same ds DNA fragment of ~1-20-kb size migrate differently in 1M urea containing aga- rose gels. The two strands are readily distinguished on

Eva Hegedus; Endre Kokai; Alexander Kotlyar; Viktor Dombradi; Gabor Szabo

2009-01-01

355

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

356

Microchip electrophoresis of oligosaccharides using lectin-immobilized preconcentrator gels fabricated by in situ photopolymerization.  

PubMed

A lectin-impregnated gel was fabricated at the channel crossing point in a microfluidic chip made from polymethyl methacrylate (PMMA). The acrylamide containing lectin was photopolymerized to form a round gel (radius 60 ?m) by irradiation with an argon laser, which was also used for fluorometric detection. This gel was applied to specific concentration, elution, and electrophoretic separation of fluorescent-labeled oligosaccharides. Because the lectin in the polyacrylamide gel was mechanically immobilized, it maintained its activity. The lectin was used to trap up to a few tens of femtomoles of specific oligosaccharides labeled with 8-aminopyrene-1,3,6-trisulfonic acid with 2 min by a factor >800, and the amount trapped corresponded to ca. 70% of lectin in the gel. The trapped oligosaccharides were released from the gel by lowering the pH with an acidic background electrolyte. The oligosaccharides that eluted as a broad band were concentrated by transient isotachophoresis stacking using concentrated sodium borate buffer (pH 11.0). The stacked sample components were then separated and fluorometrically detected at the end of the separation channel. Under the optimized conditions, resolution of the saccharides was good, and was similar to that obtained by pinched injection. The method was applied to preconcentration and analysis of oligosaccharides derived from some glycoproteins. PMID:22433972

Yamamoto, Sachio; Suzuki, Sho; Suzuki, Shigeo

2012-03-21

357

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

Microsoft Academic Search

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

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

1998-01-01

358

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

Microsoft Academic Search

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

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

1999-01-01

359

Allelic variation of the HMW glutenin subunits in Aegilops tauschii accessions detected by sodium dodecyl sulphate (SDS-PAGE), acid polyacrylamide gel (A-PAGE) and capillary electrophoresis  

Microsoft Academic Search

Summary Variability of high molecular weight glutenin subunits (HMW-GS) was studied in 198 accessions of Ae. tauschii (2n=2x=14, DD) by sodium dodecyl sulphate (SDS-PAGE) and acid polyacrylamide gel electrophoresis (A-PAGE) and capillary electrophoresis (CE). A high allelic variation of HMW-GS, including some novel x- and y-type subunits and variable subunit combinations were observed. One accession (TD159) showed a x-type null

Yueming Yan; S. L. K. Hsam; Jianzhong Yu; F. J. Zeller

2003-01-01

360

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

Microsoft Academic Search

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

Philippe Delepelaire; Nam-Hai Chua

1979-01-01

361

Investigation of a 2D two-point maximum entropy regularization method for signal-to-noise ratio enhancement: application to CT polymer gel dosimetry  

NASA Astrophysics Data System (ADS)

This study presents a new method of image signal-to-noise ratio (SNR) enhancement by utilizing a newly developed 2D two-point maximum entropy regularization method (TPMEM). When utilized as an image filter, it is shown that 2D TPMEM offers unsurpassed flexibility in its ability to balance the complementary requirements of image smoothness and fidelity. The technique is evaluated for use in the enhancement of x-ray computed tomography (CT) images of irradiated polymer gels used in radiation dosimetry. We utilize a range of statistical parameters (e.g. root-mean square error, correlation coefficient, error histograms, Fourier data) to characterize the performance of TPMEM applied to a series of synthetic images of varying initial SNR. These images are designed to mimic a range of dose intensity patterns that would occur in x-ray CT polymer gel radiation dosimetry. Analysis is extended to a CT image of a polymer gel dosimeter irradiated with a stereotactic radiation therapy dose distribution. Results indicate that TPMEM performs strikingly well on radiation dosimetry data, significantly enhancing the SNR of noise-corrupted images (SNR enhancement factors >15 are possible) while minimally distorting the original image detail (as shown by the error histograms and Fourier data). It is also noted that application of this new TPMEM filter is not restricted exclusively to x-ray CT polymer gel dosimetry image data but can in future be extended to a wide range of radiation dosimetry data.

Jirasek, A.; Matthews, Q.; Hilts, M.; Schulze, G.; Blades, M. W.; Turner, R. F. B.

2006-05-01

362

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

363

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

364

Thin-layer chromatography and polyacrylamide gel electrophoresis-based assays for sialyltransferases using tetramethylrhodamine-labeled acceptors.  

PubMed

Two novel assay systems for the determination of sialyltransferase activity using a tetramethylrhodamine-labeled disaccharide Galbeta1-4GlcNAc (2) as the acceptor are described. The TMR-labeled disaccharide 2 was synthesized by directly coupling Galbeta1-4GlcNAc-O-(CH(2))(6)NH(2) (1) with 5-tetramethylrhodamine N-hydroxysuccinimide ester. The K(m) value for compound 2 obtained with alpha-2,6-sialyltransferase from rat liver (EC 2.4.99.1) was 160 +/- 20 microM. After incubation of compound 2 with sialyltransferase the product and the unreacted acceptor substrate were separated either by thin-layer chromatography (TLC) on C-18 silica gel plates or by polyacrylamide gel electrophoresis (PAGE). The density of the spots on the TLC plates and the fluorescence of the bands on the gel were quantified. The assay conditions were optimized using crude bovine colostrum extract and also alpha-2, 6-sialyltransferase from rat liver. The detection limits for the TLC and PAGE assays were 1 and 0.4 microU of the rat liver enzyme, respectively. Either assay allows the parallel investigation of several samples at a time and is useful for the testing of fractions during enzyme purification. PMID:10998267

Hubl, U; Slim, G C; Zubkova, O V

2000-10-01

365

Development of a gel monolithic column polydimethylsiloxane microfluidic device for rapid electrophoresis separation.  

PubMed

A beta-cyclodextrin (beta-CD)-bonded gel monolithic column polydimethylsiloxane (PDMS) microfluidic device was developed in a simple and feasible way. Before preparation of gel monolithic column in PDMS microchannel, PDMS surface was activated by UV light to create silanol groups, which is an active molecule to covalently bond 3-(trimethoxysilyl)-propyl methacrylate (Bind-Silane) and seal microfluidic device. By the way, Bind-Silane is a bifunctional molecule to link polyacrylamide (PAA) gel and inner wall of PDMS microchannel covalently. Allyl-beta-CD was used not only as a multifunctional crosslinker in PAA gel to control the size of the pores, but also as a chiral selector for the enantioseparation. The stability, transferring heat and optical characteristic of the microfluidic device were examined. The separation capability of the gel monolithic column was confirmed by the successful separation of fluorescein isothiocyanate (FITC)-labeled arginine (Arg), glutamine acid (Glu), tryptophan (Try), cysteine (Cysteine) and phenylalanine (Phe) in the PDMS microfluidic device less than 100 s at 36 mm effective separation length. A maximum of 2.06 x 10(5) theoretical plates was obtained by the potential strength of 490 V/cm. A pair of FITC-labeled dansyl-D,L-threonine (Dns-Thr) was separated absolutely. PMID:18970558

Zeng, Hu-Lie; Li, Hai-Fang; Wang, Xu; Lin, Jin-Ming

2005-10-20

366

Detection of Salmonellae in Chicken Feces by a Combination of Tetrathionate Broth Enrichment, Capillary PCR, and Capillary Gel Electrophoresis  

PubMed Central

This report describes a rapid detection procedure for salmonellae from chicken feces by the combination of tetrathionate primary enrichment (preenrichment [PE])-bacterial lysis-capillary PCR and capillary gel electrophoresis. Pure Salmonella enterica serovar Enteritidis 64K was reisolated and detected by capillary PCR after buffered peptone water and nutrient broth, tetrathionate broth base Hajna (TTBH), and tetrathionate broth (TTB) preenrichments. When the same culture was mixed with intestinal homogenate, bacteriological reisolation and capillary PCR detection was achieved only by TTBH and TTB preenrichments. Capillary gel electrophoresis revealed that a Salmonella genus-specific 281-bp PCR product was detected when Salmonella strains but not non-Salmonella strains were tested. The detection limit of capillary PCR with whole-cell DNA extracted from pure Salmonella enterica serovars Enteritidis 64K, Typhimurium LT2-CIP60-62, and Gallinarum 64K was 3, 3, and 9 CFU ml?1, respectively. The detection limit of capillary PCR from whole-cell DNA extracted from intestinal homogenate artificially contaminated with the same three strains was 3, 3, and 7 CFU ml?1, respectively. We compared the results of the capillary PCR and bacteriological examination from the natural samples. Thirty-five of 53 naturally contaminated samples produced a specific PCR product. In 9 of the 35 PCR-positive samples, Salmonella could not be detected bacteriologically either by PE or a primary and delayed secondary enrichment (DSE) combination. In the 18 PCR-negative samples, 4 samples were found to harbor Salmonella by both PE and DSE and 14 samples were positive after DSE. Fifty-three additional intestinal homogenate samples, which were negative by their PE and DSE in bacteriological examination, were found to be also negative by their PCRs. The total time required to detect Salmonella with the capillary PCR method we used was approximately 20 h. If samples are from clinically diseased birds, the total time for PCR and detection is reduced to 2 h since the 18-h PE is not required. These results indicate that TTB enrichment, bacterial lysis, and genus-specific capillary PCR combined with capillary gel electrophoresis constitute a sensitive and selective procedure which has the potential to rapidly identify Salmonella-infected flocks.

Carli, K. Tayfun; Unal, Can Bora; Caner, Vildan; Eyigor, Aysegul

2001-01-01

367

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

Microsoft Academic Search

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

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

2010-01-01

368

LaneRuler: Automated Lane Tracking for DNA Electrophoresis Gel Images  

Microsoft Academic Search

We present a novel method for correctly identifying and straightening one dimensional agarose electrophoretic lanes. Our method has been shown to yield comparable accuracy with manual lane tracking results, and to successfully process 98% of DNA fingerprinting gels with no human intervention.

Richard T. F. Wong; Stephane Flibotte; Richard Corbett; Parvaneh Saeedi; Steven J. M. Jones; Marco A. Marra; Jacqueline E. Schein; Inanç Birol

2010-01-01

369

A fluorescent natural product for ultra sensitive detection of proteins in one-dimensional and two-dimensional gel electrophoresis.  

PubMed

Lightning Fast is a sensitive fluorescence-based stain for detecting proteins in one-dimensional and two-dimensional polyacrylamide electrophoresis gels. It contains the fluorophore epicocconone from the fungus Epicoccum nigrum that interacts noncovalently with sodium dodecyl sulfate and protein. Stained proteins can be excited optimally by near-ultraviolet light of about 395 nm or with visible light of about 520 nm. The stain can be excited using a range of sources used in image analysis systems including UVA (ca. 365 nm) and UVB (ca. 302 nm) transilluminators; Xenon-arc lamps; 488 nm and 457 nm Argon-ion lasers; 473 nm and 532 nm neodymium: yttrium aluminum garnet (Nd:YAG) solid-state lasers; 543 nm helium-neon lasers, and emerging violet, blue and green diode lasers. Maximum fluorescence emission of the dye is at approximately 610 nm. The limit of detection in one-dimensional gels stained with Lightning Fast protein gel stain is less than 100 pg of protein, rivaling the current limits of matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Lightning Fast was found to be considerably more sensitive than SYPRO Ruby, SYPRO Orange, silver and Coomassie Brilliant Blue G-250 in matched experiments. Staining takes as little as 3.5 h and stained proteins displayed quantitative linearity over more than four orders of magnitude, thereby allowing visualization of entire proteomes. Lightning Fast protein gel staining is compatible with subsequent peptide mass fingerprinting using MALDI-MS and Edman-based sequencing chemistry. PMID:14673778

Mackintosh, James A; Choi, Hung-Yoon; Bae, Soo-Han; Veal, Duncan A; Bell, Philip J; Ferrari, Belinda C; Van Dyk, Derek D; Verrills, Nicole M; Paik, Young-Ki; Karuso, Peter

2003-12-01

370

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  

PubMed Central

Strains of Salmonella enterica (n = 212) of different serovars and phage types were used to establish a library typing computerized system for serovar Enteritidis on the basis of PCR fingerprinting, infrequent-restriction-site PCR (IRS-PCR), or pulsed-field gel electrophoresis (PFGE). The rate of PCR fingerprinting interassay and intercenter reproducibility was low and was only increased when DNA samples were extracted at the same time and amplified with the same reaction mixtures. Reproducibility of IRS-PCR technique reached 100%, but discrimination was low (D = 0.52). The PFGE procedure showed an intercenter reproducibility value of 93.3%. The high reproducibility of PFGE combined with the previously determined high discrimination directed its use for library typing. The use of PFGE with enzymes XbaI, BlnI, and SpeI for library typing of serovar Enteritidis was assessed with GelCompar 4.0 software. Three computer libraries of PFGE DNA profiles were constructed, and their ability to recognize new DNA profiles was analyzed. The results obtained pointed out that the combination of PFGE with computerized analysis could be suitable in long-term epidemiological comparison and surveillance of Salmonella serovar Enteritidis, specially if the prevalence of genetic events that could be responsible for changes in PFGE profiles in this serovar was low.

Garaizar, Javier; Lopez-Molina, Nuria; Laconcha, Idoia; Lau Baggesen, Dorte; Rementeria, Aitor; Vivanco, Ana; Audicana, Ana; Perales, Ildefonso

2000-01-01

371

Initial proteome analysis of caffeine-induced proteins in Aspergillus tamarii using two-dimensional fluorescence difference gel electrophoresis.  

PubMed

Caffeine is toxic to most microorganisms. However, some filamentous fungi, such as Aspergillus tamarii, are able to metabolize this alkaloid when fed caffeine as the sole nitrogen source. The aim of the present work was to identify intracellular A. tamarii proteins, regulated by caffeine, using fluorescence difference two-dimensional gel electrophoresis. Specific proteins from two culture media of A. tamarii grown either on ammonium sulfate or caffeine as the sole nitrogen source were analysed by mass spectrometry. Thirteen out of a total of 85 differentially expressed spots were identified after database search. Identified up-regulated proteins include phosphoglycerate kinase, malate dehydrogenase, dyp-type peroxidase family protein, heat shock protein, Cu, Zn superoxidase dismutase and xanthine dehydrogenase. Some of the proteins identified in this study are involved in the caffeine degradation pathway as well as in stress response, suggesting that stress proteins could be involved in caffeine metabolism in filamentous fungi. PMID:22391696

Gutiérrez-Sánchez, Gerardo; Atwood, James; Kolli, V S Kumar; Roussos, Sévastianos; Augur, Christopher

2012-03-07

372

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

373

Allozyme comparison of three Trypanosoma species (Kinetoplastida: Trypanosomatidae) of toads and frogs by starch-gel electrophoresis.  

PubMed

Six metabolic enzymes, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, isocitrate dehydrogenase, malate dehydrogenase, phosphoglucomutase, and purine nucleoside phosphorylase, from clonal isolates of 3 presumptive species of Trypanosoma (T. fallisi, T. ranarum, and T. rotatorium) from 3 anuran hosts (Bufo americanus, Rana clamitans, and Rana catesbeiana) were compared using starch-gel electrophoresis. Although bands were shared among the different zymodemes of isolates of the same host genus, low genetic polymorphism of the enzyme loci was observed with few apparent shared bands between samples isolated from frogs and toads. A distance value calculated between toad and frog trypanosome isolates suggests the likelihood of long-time separation of species. Cluster analysis based on overall similarity distinguished the trypanosomes of toads and frogs as separate taxa, suggesting that host specificity and observed morphological differences are consistent with heritable allozyme differences. PMID:1556645

Martin, D S; Desser, S S; Hong, H

1992-04-01

374

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

2011-10-27

375

Epidemiologic application of pulsed-field gel electrophoresis to an outbreak of Campylobacter jejuni in an Austrian youth centre.  

PubMed Central

We report the first documented Campylobacter jejuni outbreak in an Austrian youth centre. Sixty-four children were involved of which 38 showed classical signs of campylobacter gastroenteritis. Since unpasteurized milk distributed by a local dairy was suspected to be the source of infection, stool samples were collected from 20 cows providing the milk. Five of the cows tested positive for C. jejuni. These isolates together with 37 clinical samples were compared by pulsed-field-gel electrophoresis (PFGE). The PFGE patterns, using the restriction endonucleases SmaI and SalI, were identical for the human and bovine isolates.This finding confirmed that the outbreak was caused by the consumption of unpasteurized milk contaminated with C. jejuni.

Lehner, A.; Schneck, C.; Feierl, G.; Pless, P.; Deutz, A.; Brandl, E.; Wagner, M.

2000-01-01

376

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

Microsoft Academic Search

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

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

1993-01-01

377

Studies of blood cholinesterase activity in liver desease (2nd report) Change of esterase zymogram by thin layer agar gel electrophoresis  

Microsoft Academic Search

The present report was performed for purpose to represent the change of serum esterase zymogram in the liver desease and the effect of carbon tetrachloride poisoning upon that of plasma and liver homogenete of the rat. Agar gel electrophoresis carried out under conditions described by Wieme and Sibata. Component of agar gel was as follows; pH6.8 phosphate buffer (~:0.025), 0.75%

H. Saito; Y. Sugawara; K. Komatubara; T. Niimi; T. Adachi; H. Kano; K. Miyata

1967-01-01

378

Structural organization of the human glucocorticoid receptor determined by one- and two-dimensional gel electrophoresis of proteolytic receptor fragments  

SciTech Connect

The structural organization of the steroid-binding protein of the IM-9 cell glucocorticoid receptor was investigated by using one- and two-dimensional gel electrophoresis of proteolytic receptor fragments. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of receptor fragments isolated after trypsin digestion of immunopurified (/sup 3/H)dexamethasone 21-mesylate ((/sup 3/H)DM-) labeled receptor revealed the presence of a stable 26.5-kilodalton (kDa) steroid-containing non-DNA-binding fragment, derived from a larger, less stable, 29-kDa fragment. The 26.5-kDa tryptic fragment appeared to be completely contained within a 41-kDa, steroid-containing, DNA-binding species isolated after chymotrypsin digestion of the intact protein. Two-dimensional electrophoretic analysis of the (/sup 3/H)DM-labeled tryptic fragments resolved two 26.5-kDa and two 29-kDa components. This was the same number of isoforms seen in the intact protein, indicating that the charge heterogeneity of the steroid-binding protein is the result of modification within the steroid-containing, non-DNA-binding, 26.5-kDa tryptic fragment. Two-dimensional analysis of the 41-kDa (/sup 3/H)DM-labeled chymotryptic species revealed a pattern of isoforms more complex than that seen either in the intact protein or in the steroid-containing tryptic fragments. These results suggest that the 41-kDa (/sup 3/H)DM-labeled species resolved by one-dimensional SDS-PAGE after chymotrypsin digestion may be composed of several distinct proteolytic fragments.

Smith, A.C.; Harmon, J.M.

1987-01-27

379

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

PubMed

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

Clements, C; Ralph, S; Petras, M

1997-01-01

380

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

381

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

382

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

NASA Astrophysics Data System (ADS)

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

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

1997-12-01

383

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

384

A modified coomassie brilliant blue G 250 staining method for the detection of chitinase activity and molecular weight after polyacrylamide gel electrophoresis.  

PubMed

A modified Coomassie Brilliant Blue G 250 staining method for detecting chitinolytic enzymes in chitin-containing polyacrylamide gel electrophoresis (PAGE) is presented. The staining formed achromatic zones at the locations of the migrated enzyme. Using Streptomyces griseus chitinase, we have demonstrated that our method is more sensitive and less complicated than the conventional Calcofluor white M2R staining. PMID:18691542

Liau, Chun Yi; Lin, Chung-Sheng

2008-07-01

385

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

Microsoft Academic Search

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

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

1990-01-01

386

Microbial Community Analysis During Composting of Digested Sludge and Sawdust by Using Quinone Profile and Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis  

Microsoft Academic Search

Microbial community structure during the composting of digested sewage sludge and sawdust was analyzed by a combination of quinone profile and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The change in total quinine content (TQ) indicated that microbial biomass reached a peak followed by a decrease, whereas the divergence of quinine (DQ) showed that the microbial community diversity increased continuously

Lijuan Yang; Mengchun Gao; Fangyuan Liang; Chao Qin

2009-01-01

387

The Optimization of a Rapid Pulsed-Field Gel Electrophoresis Protocol for the Typing of Acinetobacter baumannii, Escherichia coli and Klebsiella spp  

Microsoft Academic Search

SUMMARY: Pulsed-field gel electrophoresis (PFGE) is the most common genotyping method used for the typing of a number of bacterial species. Generally, investigators use their own custom-developed protocol, but a standardized PFGE protocol would allow the comparison of typing results between laboratories and the tracing of strains around the country. In the present study, we optimized a PFGE protocol for

Riza Durmaz; Baris Otlu; Fatih Koksal; Salih Hosoglu; Recep Ozturk; Yasemin Ersoy; Elif Aktas; Nafia Canan Gursoy; Ahmet Caliskan

388

Study of microbial community of brewery-treating granular sludge by denaturing gradient gel electrophoresis of 16S rRNA gene  

Microsoft Academic Search

The microbial community structure of granular sludge from an upflow anaerobic sludge blanket (UASB) reactor treating brewery effluent was studied by denaturing gradient gel electrophoresis (DGGE). Twelve major bands were observed in the DGGE fingerprint for the Bacteria domain and four bands for the Archaea domain. Of the bacterial bands observed, six were successfully purified and sequenced. Among them, three

O. C. Chan; W. T. Liu; H. H. P. Fang

2001-01-01

389

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

ERIC Educational Resources Information Center

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

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

2012-01-01

390

Proteomics Analysis by Two-Dimensional Differential Gel Electrophoresis Reveals the Lack of a Broad Response of Neisseria meningitidis to In Vitro-Produced AI-2  

PubMed Central

To investigate the effect of the autoinducer AI-2 on protein expression in Neisseria meningitidis, a luxS mutant of strain MC58 was grown in the presence and absence of in vitro-produced AI-2, and differential protein expression was assessed by two-dimensional differential gel electrophoresis. N. meningitidis did not show a global response to AI-2 signaling activity.

Schauder, Stephan; Penna, Lucia; Ritton, Adeline; Manin, Catherine; Parker, Fabienne; Renauld-Mongenie, Genevieve

2005-01-01

391

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

392

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

Microsoft Academic Search

The bacterial DNA structures at different depths in the Puruogangri (Tibetan Plateau) ice core (83.45 m) were investigated by the denaturing gradient gel electrophoresis (DGGE) DNA fingerprinting technique. DGGE profiles indicated that the bacterial species diversity in glacial ice is high, and indigenous species represented by common bands in all samples may grow on the glacial surface. Bacterial diversity, as

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

2006-01-01

393

Characterization of Triglyceride Rich Lipoproteins with Very Light Density by Ultracentrifugation and Agarose Gel Electrophoresis using Triglyceride and Cholesterol-Staining  

Microsoft Academic Search

Hypertriglyceridemia is an independent risk factor for atherosclerosis. This risk is most likely due to accumulation of circulating triglyceride rich lipoproteins with heterogeneous particles. The identification and characterization of these triglyceride rich lipoproteins is important to detect abnormality of triglyceride metabolism. In the present study, we developed a new method that combines ultra- centrifugation and agarose gel electrophoresis with triglyceride-

Hiroya Hidaka; Minoru Tozuka; Barbara Meyer; Kazuyoshi Yamauchi; Mitsutoshi Sugano; Tetsuo Nakabayashi; Tsutomu Katsuyama

394

Use of single cell gel electrophoresis assays for the detection of DNA-protective effects of dietary factors in humans: Recent results and trends  

Microsoft Academic Search

This article summarises the results of human dietary intervention trials employing the comet assay (single cell gel electrophoresis, SCGE), which have been published in the last few years (i.e., between 2005 and 2008) and describes new trends and developments as well as current problems concerning the design of intervention trials and the interpretation of the results. Most new studies were

Christine Hoelzl; Siegfried Knasmüller; Miroslav Mišík; Andrew Collins; Maria Dušinská; Armen Nersesyan

2009-01-01

395

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

Microsoft Academic Search

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

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

2001-01-01

396

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

397

Role of Real-Time Molecular Typing in the Surveillance of Campylobacter Enteritis and Comparison of Pulsed-Field Gel Electrophoresis Profiles from Chicken and Human Isolates  

Microsoft Academic Search

The goal of the present study was to assess the contribution of real-time molecular typing, used alone or with clinical surveillance, to the prompt identification of clusters of Campylobacter enteritis. Potential poultry sources were sought by comparing the pulsed-field gel electrophoresis genotypes of human and fresh whole retail chicken isolates collected during the same study period. Among 183 human isolates,

Sophie Michaud; Suzanne Menard; Robert D. Arbeit

2005-01-01

398

Comparison of Multiple-Locus Variable-Number Tandem-Repeat Analysis with Pulsed-Field Gel Electrophoresis Typing of Acinetobacter baumannii in China  

PubMed Central

A panel of seven variable-number tandem-repeat (VNTR) markers was selected for Acinetobacter baumannii typing analysis (MLVA-7). Compared with pulsed-field gel electrophoresis (PFGE), MLVA-7 provided greater discrimination. We modified the criteria for MLVA complex assignments proposed previously, and a remarkable congruence between MLVA-7- and PFGE-based strain clustering was observed.

Hu, Yuan; Li, Boqing; Jin, Dazhi; Cui, Zhigang; Tao, Xiaoxia; Zhang, Binghua

2013-01-01

399

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

400

Comparison of multiple-locus variable-number tandem-repeat analysis with pulsed-field gel electrophoresis typing of Acinetobacter baumannii in China.  

PubMed

A panel of seven variable-number tandem-repeat (VNTR) markers was selected for Acinetobacter baumannii typing analysis (MLVA-7). Compared with pulsed-field gel electrophoresis (PFGE), MLVA-7 provided greater discrimination. We modified the criteria for MLVA complex assignments proposed previously, and a remarkable congruence between MLVA-7- and PFGE-based strain clustering was observed. PMID:23345295

Hu, Yuan; Li, Boqing; Jin, Dazhi; Cui, Zhigang; Tao, Xiaoxia; Zhang, Binghua; Zhang, Jianzhong

2013-01-23

401

Measuring the Quantity and Activity of Mitochondrial Electron Transport Chain Complexes in Tissues of Central Nervous System Using Blue Native Polyacrylamide Gel Electrophoresis  

Microsoft Academic Search

Mitochondrial dysfunction and degeneration are associated with many neurodegenerative disorders. A dysfunctional mitochondrial electron transport chain (ETC) impairs ATP production and accelerates the generation of free radicals. To evaluate mitochondrial function, reliable methods are needed. Conventional spectrophotometric assays may not eliminate interference from nonspecific enzyme activities and do not measure quantities of specific ETC complexes. Blue native polyacrylamide gel electrophoresis

Cheolwha Jung; Cynthia M. J. Higgins; Zuoshang Xu

2000-01-01

402

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

403

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

Microsoft Academic Search

Amplified fragment length polymorphism (AFLP) is a recently developed, PCR-based high resolution fingerprinting method that is able to generate complex banding patterns which can be used to delineate intraspecific genetic relationships among bacteria. In the present study, AFLP was evaluated for its usefulness in the molecular typing of Salmonella typhi in comparison to ribotyping and pulsed-field gel electrophoresis (PFGE). Six

Satheesh Nair; Edgar Schreiber; Kwai-Lin Thong; Tikki Pang; Martin Altwegg

2000-01-01

404

Comparison of Multilocus Sequence Typing, Pulsed-Field Gel Electrophoresis, and Antimicrobial Susceptibility Typing for Characterization of Salmonella enterica Serotype Newport Isolates  

Microsoft Academic Search

In the United States, multidrug-resistant phenotypes of Salmonella enterica serotype Newport (commonly referred to as MDR-AmpC) have emerged in animals and humans and have become a major public health problem. Although pulsed-field gel electrophoresis (PFGE) is the current \\

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

2006-01-01

405

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

PubMed Central

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

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

2003-01-01

406

Evaluation of three-dimensional gel electrophoresis to improve quantitative profiling of complex proteomes.  

PubMed

Two-dimensional remains one of the main experimental approaches in proteome analysis. However, comigration of protein leads to several limitations: lack of accuracy in protein identification, impaired comparative quantification, and PTM detection. We have optimized a third additional step of in-gel separation to alleviate comigration associated drawbacks. Spot resolution is strikingly improved following this simple and rapid method and the positive impact on protein and peptide identification from MS/MS data, on the analysis of relative changes in protein abundance, and on the detection of PTM is described. PMID:23592440

Colignon, Bertrand; Raes, Martine; Dieu, Marc; Delaive, Edouard; Mauro, Sergio

2013-06-20

407

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

Microsoft Academic Search

Intact protein masses can be measured directly from immobilized pH gradient (IPG) isoelectric focusing (IEF) gels loaded with mammalian and prokaryotic samples, as demonstrated here with murine macrophage and Methanosarcina acetivorans cell lysates. Mass accuracy and resolution is improved by employing instruments which decouple the desorption event from mass measurement; e.g., quadrupole time-of-flight instruments. MALDI in-source dissociation (ISD) is discussed

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

2005-01-01

408

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

PubMed Central

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

2010-01-01

409

Tracking of antibody reduction fragments by capillary gel electrophoresis during the coupling to microparticles surface.  

PubMed

Due to their high specificity and efficiency, antibodies are ideal ligands for target-specific ultrasound contrast agents. The present study focuses on the chemical stability of antibodies during functionalisation with sulfosuccinimidyl-pyridyldithiopropionamidohexanoate (SPDP), a heterobifunctional linker, which exposes free thiol groups upon treatment with a reducing agent. Thiolated antibodies can then react with thiol-reactive group, such as maleimide present on the microbubble surface to form stable covalent complexes. The immunoglobulin structure relies on several intra- and inter-chain disulfide bridges which might be affected by reducing agents. A capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) method with UV detection was applied to address the effect of the functionalisation process on the structural integrity of the antibodies and revealed that antibody disulfide bonds are prone to reduction as function of the reducing agents. Depending on the coupling conditions, various IgG fragments were identified reflecting different combinations between the light and heavy chains. Furthermore, two commonly used reducing agents, namely triscarboxyethylphosphine (TCEP) and 1,4-dithiothreitol (DTT) were compared under various preparation conditions. Results showed that reduction conditions based on DTT as a reducing agent under acidic pH were more appropriate to preserve intra- and inter-disulfide bridges of SPDP-modified antibodies. PMID:20193997

Cherkaoui, S; Bettinger, T; Hauwel, M; Navetat, S; Allémann, E; Schneider, M

2010-02-01

410

Assessing factors for reliable quantitative proteomics based on two-dimensional gel electrophoresis.  

PubMed

We statistically analysed various factors to get accurate estimates of protein quantities from two-dimensional gels. Yeast proteins were labelled with (35)S or stained with Coomassie Brilliant Blue G-250, and spots were automatically quantified with software packages Kepler, ImageQuaNT, Melanie 3.0 and Progenesis. The different software packages proved to have very similar performances. With (35)S-labelled actin spot as a reference, we studied the staining efficiency of colloidal Coomassie blue as a function of amino acid composition of the protein, and derived an equation to estimate the number of molecules per cell from blue-stained proteins. Absolute quantification of most glycolytic enzymes was carried out in two yeast strains. PMID:15221754

Fiévet, Julie; Dillmann, Christine; Lagniel, Gilles; Davanture, Marlène; Negroni, Luc; Labarre, Jean; de Vienne, Dominique

2004-07-01

411

Assessment of the red cell proteome of young patients with unexplained hemolytic anemia by two-dimensional differential in-gel electrophoresis (DIGE).  

PubMed

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

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

2012-04-03

412

Identification of stable plant cystatin/nematode proteinase complexes using mildly denaturing gelatin/polyacrylamide gel electrophoresis.  

PubMed

The biochemical interactions between two cystatins from rice seeds, oryzacystatin I (OCI) and oryzacystatin II (OCII), and the cysteine proteinases from three plant parasitic nematodes, Meloidogyne hapla, M. incognita and M. javanica, were assessed using standard protease assays and mildly denaturing gelatin/polyacrylamide gel electrophoresis (gelatin/PAGE). Activity detected in extracts of preparasitic second-stage larvae (J2) from M. hapla was optimal at pH 5.5 and was inhibited in vitro by the cysteine proteinase inhibitors trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane, hen egg cystatin, OCI, and OCII. As demonstrated by class-specific activity staining, all the activity measured between pH 3.5 and pH 7.5 was accounted for by a major proteinase form, Mhp1, and two minor forms, Mhp2 and Mhp3. Mhps were also detected in extracts and excretions of parasitic J2 and adult females, indicating their continuous expression throughout development of M. hapla, and their possible involvement in the extracellular degradation of proteins. Interestingly, the two plant cysteine proteinase inhibitors OCI and OCII showed different degrees of affinity for the major proteinase form, Mhp1. Both inhibitors almost completely inactivated this proteinase in native conditions but, unlike OCII, OCI conserved a high affinity for Mhp1 during mildly denaturing gelatin/PAGE, showing the differential stabilities of the OCI/Mhp1 and OCII/Mhp1 complexes. In contrast to Mhp1, the major cysteine proteinases detected in the two closely related species M. incognita and M. javanica were strongly inhibited by OCII, while the inhibition of OCI was partly prevented during electrophoresis. This species-related efficiency of plant cystatins against nematode cysteine proteinases could have practical implications when planning their use to control nematodes of the genus Meloidogyne. PMID:8874065

Michaud, D; Cantin, L; Bonadé-Bottino, M; Jouanin, L; Vrain, T C

1996-08-01

413

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

PubMed Central

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

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

2008-01-01

414

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

PubMed

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

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

2008-12-18

415

Three Isoforms of Cyclophilin A Associated with Human Immunodeficiency Virus Type 1 Were Found by Proteomics by Using Two-Dimensional Gel Electrophoresis and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry  

PubMed Central

Human immunodeficiency virus type 1 (HIV-1) strain LAV-1 (HIV-1LAV-1) particles were collected by ultracentrifugation, treated with subtilisin, and then purified by Sepharose CL-4B column chromatography to remove microvesicles. The lysate of the purified HIV-1LAV-1 particles was subjected to two-dimensional (2D) gel electrophoresis and stained. The 2D gel electrophoresis image suggested that 24 proteins can be identified inside the virion. Furthermore, the stained protein spots were excised and digested with trypsin. The resulting peptide fragments were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Peptide mass fingerprinting data suggested that two isoforms of cyclophilin A (CyPA), one with an isoelectric point (pI) of 6.40 and one with a pI of 6.53, are inside the viral membrane; that another isoform, with a pI of 6.88, is outside the viral membrane; and that the CyPA isoform with a pI of 6.53 is N acetylated. The mechanisms that permit the redistribution of CyPA on the viral surface have not yet been clarified, but it is surmised that the CyPA isoform with a pI of 6.88 may play a critical role in the attachment of virions to the surface of target cells and that both CyPA isoforms with pIs of 6.40 and 6.53 may regulate the conformation of the HIV-1 capsid protein.

Misumi, Shogo; Fuchigami, Takashi; Takamune, Nobutoki; Takahashi, Ichiro; Takama, Michiho; Shoji, Shozo

2002-01-01

416

Genome fingerprinting of Salmonella typhi by pulsed-field gel electrophoresis for subtyping common phage types.  

PubMed Central

The genomic DNA of 39 strains of Salmonella typhi isolated from local residents and patients who had visited countries in the Asian region was analysed for restriction fragment length polymorphisms (RFLP). Pulsed-field gel electrophoretic (PFGE) analysis of Xba I- and Spe I-generated genomic restriction fragments established 22 PFGE types whereas phage typing differentiated the 39 isolates into 9 distinct phage types. This study showed that PFGE is more discriminatory than phage typing as it is capable of subtyping S. typhi strains of the same phage types. Genetic relatedness among the isolates was determined. Seven major clusters were identified at SABs of > 0.80 and the remaining 13 isolates were distributed into minor clusters which were related at SABs of less than 0.80. In conclusion, PFGE analysis in conjunction with distance matrix analysis served as a useful tool for delineating common S. typhi phage types of diverse origins from different geographical locales and separated in time. Images Fig. 1 Fig. 2 Fig. 3

Nair, S.; Poh, C. L.; Lim, Y. S.; Tay, L.; Goh, K. T.

1994-01-01

417

Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis.  

PubMed Central

Sequence heterogeneities in 16S rRNA genes from individual strains of Paenibacillus polymyxa were detected by sequence-dependent separation of PCR products by temperature gradient gel electrophoresis (TGGE). A fragment of the 16S rRNA genes, comprising variable regions V6 to V8, was used as a target sequence for amplifications. PCR products from P. polymyxa (type strain) emerged as a well-defined pattern of bands in the gradient gel. Six plasmids with different inserts, individually demonstrating the migration characteristics of single bands of the pattern, were obtained by cloning the PCR products. Their sequences were analyzed as a representative sample of the total heterogeneity. An amount of 10 variant nucleotide positions in the fragment of 347 bp was observed, with all substitutions conserving the relevant secondary structures of the V6 and V8 regions in the RNA molecules. Hybridizations with specifically designed probes demonstrated different chromosomal locations of the respective rRNA genes. Amplifications of reverse-transcribed rRNA from ribosome preparations, as well as whole-cell hybridizations, revealed a predominant representation of particular sequences in ribosomes of exponentially growing laboratory cultures. Different strains of P. polymyxa showed not only remarkably differing patterns of PCR products in TGGE analysis but also discriminative whole-cell labeling with the designed oligonucleotide probes, indicating the different representation of individual sequences in active ribosomes. Our results demonstrate the usefulness of TGGE for the structural analysis of heterogeneous rRNA genes together with their expression, stress problems of the generation of meaningful data for 16S rRNA sequences and probe designs, and might have consequences for evolutionary concepts.

Nubel, U; Engelen, B; Felske, A; Snaidr, J; Wieshuber, A; Amann, R I; Ludwig, W; Backhaus, H

1996-01-01

418

An improved plant leaf protein extraction method for high resolution two-dimensional polyacrylamide gel electrophoresis and comparative proteomics.  

PubMed

We report here a simple and universally applicable protocol for extracting high quality proteins from plant leaf tissues. The protocol provides improved resolution and reproducibility of two-dimensional polyacrylamide gel electrophoresis (2-DE) and reduces the time required to analyze samples. Partitioning rubisco by polyethylene glycol (PEG) fractionation provides clearer detection of low-abundance proteins. Co-extraction of interfering substances increases the sample conductivity, which results in poor electrophoretic separation. Re-extraction of PEG-fractionated samples with phenol effectively eliminated interfering substances, which results in optimal conductivity during separation in the first dimension of the isoelectric focusing. Smooth focusing reduces analysis time and provides superior resolution in 2-DE gels. Incubating the samples at -80° C instead of -20° C reduced protein precipitation time to 2-3 h. Removal of nonprotein contaminants and the use of sonication increased protein solubility without additional reagents. These changes enabled loading and separation of maximum amounts of proteins, which permitted improved protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). An immunological approach revealed that little or no ribulose-1, 5-bisphosphte bisphosphate carboxylase oxygenase was present in the PEG supernatant. In addition, low-abundance proteins, such as myelocytomatosis transcription factor (MYC) and alpha subunit of heterotrimeric guanine nucleotide-binding protein complex (G?), were detected only in the modified PEG supernatant and not in the total protein. These results suggest that our protocol produced high quality proteins and made many low-abundant proteins available for proteomic analysis. The successful application of this protocol for analyzing the leaf proteomes of soybean, Miscanthus sinensis, barley, Chinese cabbage, peanut and tea (Camellia sinensis) suggests that it could be used for comparative proteomic analysis of a wide range of plant leaves. PMID:23072551

Alam, I; Sharmin, Sa; Kim, K-H; Kim, Y-G; Lee, Jj; Lee, B-H

2012-10-17

419

Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis.  

PubMed

Sequence heterogeneities in 16S rRNA genes from individual strains of Paenibacillus polymyxa were detected by sequence-dependent separation of PCR products by temperature gradient gel electrophoresis (TGGE). A fragment of the 16S rRNA genes, comprising variable regions V6 to V8, was used as a target sequence for amplifications. PCR products from P. polymyxa (type strain) emerged as a well-defined pattern of bands in the gradient gel. Six plasmids with different inserts, individually demonstrating the migration characteristics of single bands of the pattern, were obtained by cloning the PCR products. Their sequences were analyzed as a representative sample of the total heterogeneity. An amount of 10 variant nucleotide positions in the fragment of 347 bp was observed, with all substitutions conserving the relevant secondary structures of the V6 and V8 regions in the RNA molecules. Hybridizations with specifically designed probes demonstrated different chromosomal locations of the respective rRNA genes. Amplifications of reverse-transcribed rRNA from ribosome preparations, as well as whole-cell hybridizations, revealed a predominant representation of particular sequences in ribosomes of exponentially growing laboratory cultures. Different strains of P. polymyxa showed not only remarkably differing patterns of PCR products in TGGE analysis but also discriminative whole-cell labeling with the designed oligonucleotide probes, indicating the different representation of individual sequences in active ribosomes. Our results demonstrate the usefulness of TGGE for the structural analysis of heterogeneous rRNA genes together with their expression, stress problems of the generation of meaningful data for 16S rRNA sequences and probe designs, and might have consequences for evolutionary concepts. PMID:8824607

Nübel, U; Engelen, B; Felske, A; Snaidr, J; Wieshuber, A; Amann, R I; Ludwig, W; Backhaus, H

1996-10-01

420

Allelic variation of the HMW glutenin subunits in Aegilops tauschii accessions detected by sodium dodecyl sulphate (SDS-PAGE), acid polyacrylamide gel (A-PAGE) and capillary electrophoresis  

Microsoft Academic Search

Variability of high molecular weight glutenin subunits (HMW-GS) was studied in198 accessions of Ae. Tauschii (2n=2x=14, DD) by sodium dodecyl sulphate(SDS-PAGE) and acid polyacrylamide gel electrophoresis (A-PAGE) and capillary electrophoresis\\u000a (CE). A high allelic variation of HMW-GS, including some novel x- and y-type subunits and variable subunit combinations were\\u000a observed. One accession(TD159) showed a x-type null form. The results by

Yueming Yan; S. L. K. Hsam; Jianzhong Yu; Yi Jiang; F. J. Zeller

2003-01-01

421

Comparative proteome analysis of Helicobacter pylori clinical strains by two-dimensional gel electrophoresis*  

PubMed Central

Objective: To investigate the pathogenic properties of Helicobacter pylori by comparing the proteome map of H. pylori clinical strains. Methods: Two wild-type H. pylori strains, YN8 (isolated from biopsy tissue of a gastric cancer patient) and YN14 (isolated from biopsy tissue of a gastritis and duodenal ulcer patient), were used. Proteomic analysis, using a pH range of 3–10 and 5–8, was performed. The individual proteins were identified by quadrupole time-of-flight (Q-TOF) mass spectrometer and protein database search. Results: Variation in spot patterns directed towards differential protein expression levels was observed between the strains. The gel revealed prominent proteins with several protein “families”. The comparison of protein expressions of the two strains reveals a high variability. Differentially present or absent spots were observed. Nine differentially expressed protein spots identified by Q-TOF included adenosine triphosphate (ATP)-binding protein, disulfide oxidoreductase B (DsbB)-like protein, N utilization substance A (NusA), ATP-dependent protease binding subunit/heat shock protein, hydantoin utilization protein A, seryl-tRNA synthetase, molybdenum ABC transporter ModD, and hypothetical proteins. Conclusions: This study suggests that H. pylori strains express/repress protein variation, not only in terms of the virulence proteins, but also in terms of physiological proteins, when they infect a human host. The difference of protein expression levels between H. pylori strains isolated from gastric cancer and gastritis may be the initiator of inflammation, and result in the different clinical presentation. In this preliminary study, we report seven differential proteins between strains, with molecule weights from approximately 10 kDa to approximately 40 kDa. Further studies are needed to investigate those proteins and their function associated with H. pylori colonization and adaptation to host environment stress.

Zhang, Ya-nan; Ding, Shi-gang; Huang, Liu-huan; Zhang, Jing; Shi, Yan-yan; Zhong, Li-jun

2011-01-01

422

Analysis of microbial diversity on deli slicers using polymerase chain reaction and denaturing gradient gel electrophoresis technologies.  

PubMed

Cross-contamination of pathogenic and spoilage bacteria from food-contact surfaces to food products is a serious public health issue. Bacteria may survive and attach to food-contact surfaces by residual food components and/or background bacteria which may subsequently transfer to other food products. Deli slicers, generally used for slicing ready-to-eat products, can serve as potential sources for considerable bacterial transfer. The objective of this study was to assess the extent and distribution of microbial diversity of deli slicers by identification of pathogenic and background bacteria. Slicer-swab samples were collected from restaurants in Arkansas and Texas in the United States. Ten surface areas for each slicer were swabbed using sterile sponges. Denaturing gradient gel electrophoresis (DGGE) was applied to investigate the fingerprint of samples, and each band was further identified by sequence analysis. Pseudomonads were identified as the dominant bacteria followed by Enterobacteriaceae family, and lactic acid bacteria such as Lactococcus lactis and Streptococcus thermophilus were also found. Bacterial distribution was similar for all surface areas, while the blade guard exhibited the greatest diversity. This study provides a profile of the microbial ecology of slicers using DGGE to develop more specific sanitation practices and to reduce cross-contamination during slicing. PMID:23121623

Koo, O K; Mertz, A W; Akins, E L; Sirsat, S A; Neal, J A; Morawicki, R; Crandall, P G; Ricke, S C

2012-11-21

423

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

PubMed

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

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

2004-05-01

424

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

PubMed Central

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

Hernandez-Sanabria, Emma; Guan, Le Luo; Goonewardene, Laksiri A.; Li, Meiju; Mujibi, Denis F.; Stothard, Paul; Moore, Stephen S.; Leon-Quintero, Monica C.

2010-01-01

425

Molecular Typing of Penicillium marneffei Isolates from Thailand by NotI Macrorestriction and Pulsed-Field Gel Electrophoresis  

PubMed Central

Penicillium marneffei is recognized as one of the most frequently detected opportunistic pathogens of AIDS patients in northern Thailand. We undertook a genomic epidemiology study of 64 P. marneffei isolates collected from immunosuppressed patients by pulsed-field gel electrophoresis (PFGE) with restriction enzyme NotI. Among the 69 isolates fingerprinted by PFGE, 17 were compared by HaeIII restriction endonuclease typing. The PFGE method demonstrated a higher degree of discriminatory power than restriction endonuclease typing with HaeII. Moreover, an impressive diversity of P. marneffei isolates was observed, as there were 54 distinct macrorestriction profiles among the 69 isolates of P. marneffei. These profiles were grouped into two large clusters by computer-assisted similarity analysis: macrorestriction pattern I (MPI) and MPII, with nine subprofiles (MPIa to MPIf and MPIIa to MPIIc). We observed no significant correlation between the macrorestriction patterns of the P. marneffei isolates and geographical region or specimen source. It is interesting that all isolates obtained before 1995 were MPI, and we found an increase in the incidence of infections with MPII isolates after 1995. We conclude that PFGE is a highly discriminatory typing method and is well suited for computer-assisted analysis. Together, PFGE and NotI macrorestriction allow reliable identification and epidemiological characterization of isolates as well as generate a manageable database that is convenient for expansion with information on additional P. marneffei isolates.

Trewatcharegon, Sompong; Sirisinha, Stitaya; Romsai, Amparat; Eampokalap, Boonchaoy; Teanpaisan, Rawee; Chaiyaroj, Sansanee C.

2001-01-01

426

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

PubMed

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

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

2013-05-11

427

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

PubMed Central

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

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

1999-01-01

428

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

PubMed Central

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

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

2012-01-01

429

Application of temperature gradient gel electrophoresis to the study of yeast diversity in the estuary of the Tagus river, Portugal.  

PubMed

Temperature gradient gel electrophoresis (TGGE) was employed for the assessment of yeast diversity in the estuary of the Tagus river (Portugal). The molecular detection of yeasts was carried out directly from water samples and, in parallel, a cultivation approach by means of an enrichment step was employed. A nested PCR was employed to obtain a fungal amplicon containing the D2 domain of the 26S rRNA gene. For identification the TGGE bands were extracted, re-amplified, and sequenced. Fourteen fungal taxa were detected and all except one were yeasts. Most yeast sequences corresponded to members of the Ascomycota and only three belonged to the Basidiomycota. Five yeasts (four ascomycetes and one basidiomycete) could not be identified to the species level due to the uniqueness of their sequences. The number of species detected after enrichment was higher than the number of taxa found using the direct detection method. This suggests that some yeast populations are present in densities that are below the detection threshold of the method. With respect to the analysis of the yeast community structure, our results indicate that the dominant populations belong to Debaryomyces hansenii, Rhodotorula mucilaginosa, Cryptococcus longus, and to an uncultured basidiomycetous yeast phylogenetically close to Cr. longus. The combined analysis of direct detection and cultivation approaches indicates a similar community structure at the two sampled sites since nine species were present at both localities. PMID:15556087

Gadanho, Mário; Sampaio, José Paulo

2004-12-01

430

The protective role of curcumin on perfluorooctane sulfonate-induced genotoxicity: single cell gel electrophoresis and micronucleus test.  

PubMed

Perfluorooctane sulfonate (PFOS) is a man-made fluorosurfactant and global pollutant. PFOS a persistent and bioaccumulative compound, is widely distributed in humans and wildlife. Therefore, it was added to Annex B of the Stockholm Convention on Persistent Organic Pollutants in May 2009. Curcumin is a natural polyphenolic compound abundant in the rhizome of the perennial herb turmeric. It is commonly used as a dietary spice and coloring agent in cooking and anecdotally as an herb in traditional Asian medicine. In this study, male rats were treated with three different PFOS doses (0.6, 1.25 and 2.5 mg/kg) and one dose of curcumin, from Curcuma longa (80 mg/kg) and combined three doses of PFOS with 80 mg/kg dose of curcumin by gavage for 30 days at 48 h intervals. Here, we evaluated the DNA damage via single cell gel electrophoresis or comet assay and micronucleus test in bone marrow in vivo. PFOS induced micronucleus frequency and decreased the ratio of polychromatic erythrocyte to normochromatic erythrocyte in bone marrow. Using the alkaline comet assay, we showed that all doses of the PFOS strongly induced DNA damage in rat bone marrow and curcumin prevented the formation of DNA damage induced by PFOS. PMID:23246701

Çelik, Ayla; Eke, Dilek; Ekinci, Seda Yaprak; Y?ld?r?m, Seda

2012-12-12

431

Correlation of particular bacterial PCR-denaturing gradient gel electrophoresis patterns with bovine ruminal fermentation parameters and feed efficiency traits.  

PubMed

The influence of rumen microbial structure and functions on host physiology remains poorly understood. This study aimed to investigate the interaction between the ruminal microflora and the host by correlating bacterial diversity with fermentation measurements and feed efficiency traits, including dry matter intake, feed conversion ratio, average daily gain, and residual feed intake, using culture-independent methods. Universal bacterial partial 16S rRNA gene products were amplified from ruminal fluid collected from 58 steers raised under a low-energy diet and were subjected to PCR-denaturing gradient gel electrophoresis (DGGE) analysis. Multivariate statistical analysis was used to relate specific PCR-DGGE bands to various feed efficiency traits and metabolites. Analysis of volatile fatty acid profiles showed that butyrate was positively correlated with daily dry matter intake (P < 0.05) and tended to have higher concentration in inefficient animals (P = 0.10), while isovalerate was associated with residual feed intake (P < 0.05). Our results suggest that particular bacteria and their metabolism in the rumen may contribute to differences in host feed efficiency under a low-energy diet. This is the first study correlating PCR-DGGE bands representing specific bacteria to metabolites in the bovine rumen and to host feed efficiency traits. PMID:20709849

Hernandez-Sanabria, Emma; Guan, Le Luo; Goonewardene, Laksiri A; Li, Meiju; Mujibi, Denis F; Stothard, Paul; Moore, Stephen S; Leon-Quintero, Monica C

2010-08-13

432

Two-dimensional gel electrophoresis: discovering neuropathic pain-associated synaptic biomarkers in spinal cord dorsal horn.  

PubMed

Nerve injury-induced neuropathic pain is a major public health problem worldwide. Current treatment for neuropathic pain has had limited success because the mechanisms that underlie the induction and maintenance of neuropathic pain are incompletely understood. However, recent advances in proteomics may allow us to uncover complicated biological mechanisms that occur under neuropathic pain conditions. Here, we introduce a combined approach of two-dimensional gel electrophoresis (2-DE) with mass spectrometry (MS) to identify the expression changes in synaptosome-associated proteins in spinal cord dorsal horn after unilateral fifth spinal nerve injury. In 2-DE, a set of highly abundant synaptic proteins with a pI range of 4-7 are separated and compared by size fractionation (25-100 kDa). Then, the differentially expressed proteins are identified and validated by MS, and their potential involvement in physiological and pathological processes is searched. Thus, proteomic analysis can provide expression profiles of synaptic proteins and their posttranslational modifications in cells, tissues, and organs of the nervous system under neuropathic pain conditions. PMID:22351081

Singh, Om V; Tao, Yuan-Xiang

2012-01-01

433

Direct observation of the gene organization of the complement C4 and 21- hydroxylase loci by pulsed field gel electrophoresis  

PubMed Central

Pulsed field gel electrophoresis and enzymes that cut genomic DNA infrequently have been used to define large RFLPs at the human C4 loci. With the enzymes BssH II or Sac II, and C4 or 21-hydroxylase DNA probes, it has been possible to observe directly the number of C4 genes present on a haplotype, and also whether the C4 genes are long (6-7-kb intron present) or short (6-7-kb intron absent). Haplotypes that have either two long C4 genes or one long and one short C4 gene generate BssH II fragments of approximately 115 or approximately 105 kb, respectively. Haplotypes that have either a single long or a single short C4 gene generate BssH II fragments of approximately 80 or approximately 70 kb, respectively. This technique has been used to analyze the DNA isolated from PBMC and allows the complete definition of the C4 gene organization of an individual without the need for family studies.

1989-01-01

434

Molecular analysis of Salmonella enteritidis and Typhimurium clinical and food isolates by pulsed-field gel electrophoresis in Bogotá, Colombia.  

PubMed

In 1997, the Microbiology Group of the Colombian Instituto Nacional de Salud set up a surveillance program with the National Public Health Laboratories to monitor the principal etiological agents responsible for acute diarrheal disease. The main goal of this study was to determine the XbaI DNA digestion patterns of clinical and food Salmonella spp. isolates recovered in Bogotá from 1997 to 2002, and related them to the susceptibility patterns to antimicrobial agents. Two hundred and twenty-four Salmonella spp. isolates were studied, 153 (63%) S. Enteritidis and 71 (37%) S. Typhimurium. Pulsed-field gel electrophoresis (PFGE) was done using the XbaI restriction enzyme and Salmonella Braenderup H9812 as the molecular weight marker. For S. Enteritidis, pattern S. En0001 was found to be prevalent in 127 (83%) isolates, 78 (61%) susceptible to the antimicrobial agents tested. For S. Typhimurium, pattern S. Typ0001 was predominant in 18 (25%) isolates with differen