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Gel electrophoresis

About: Gel electrophoresis is a research topic. Over the lifetime, 26026 publications have been published within this topic receiving 1113565 citations.


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Journal ArticleDOI
TL;DR: There are at least four distinct polypeptide species with apparent molecular masses of about 42,000 daltons in the outer membrane of E. coli O111, and these proteins may contain a small amount of carbohydrate.
Abstract: Previous studies have shown that the outer membrane of Escherichia coli O111 gives a single, major, 42,000-dalton protein peak when analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis at neutral pH. Further studies have shown that this peak consists of more than a single polypeptide species, and on alkaline SDS-gel electrophoresis this single peak is resolved into three subcomponents designated as proteins 1, 2, and 3. By chromatography of solubilized, outer membrane protein on diethylaminoethyl-cellulose followed by chromatography on Sephadex G-200 in the presence of SDS, it was possible to separate the 42,000-dalton major protein into four distinct protein fractions. Comparison of cyanogen bromide peptides derived from these fractions indicated that they represented at least four distinct polypeptide species. Two of these proteins migrated as proteins 1 and 2 on alkaline gels. The other two proteins migrated as protein 3 on alkaline gels and cannot be separated by SDS-polyacrylamide gel electrophoresis. In purified form, these major proteins do not contain bound lipopolysaccharide, phospholipid, or phosphate. These proteins may contain a small amount of carbohydrate, as evidenced by the labeling of these proteins by glucosamine, and to a lesser extent by glucose, under conditions where the metabolism of these sugars to amino acids and lipids is blocked. All of the proteins were labeled to the same extent by these sugars. Thus, it was concluded that there are at least four distinct polypeptide species with apparent molecular masses of about 42,000 daltons in the outer membrane of E. coli O111.

199 citations

BookDOI
24 Sep 1998
TL;DR: High-Resolution, IPG-Based, Mini Two-Dimensional Gel Electrophoresis, Jean-Charles Sanchez and Denis F. Hochstrasser, and Thierry Rabilloud - advantages of Immobilized pH Gradients.
Abstract: 2-D Protein Gel Electrophoresis: An Overview, Jenny Fichmann and Reiner Westermeier. Solubilization of Proteins in 2-D Electrophoresis: An Outline, Thierry Rabilloud. Preparation of Escherichia coli Samples for 2-D Gel Analysis, Ruth A. VanBogelen and Frederick C. Neidhardt. Preparing 2-D Protein Extracts from Yeast, David M. Schieltz. 2-D Protein Extracts from Drosophila melanogaster, Christer Ericsson. Preparing 2-D Protein Extracts from Caenorhabditis elegans, Robert Zwilling. Eukaryotic Cell Labeling and Preparation for 2-D, Nick Bizios. Differential Detergent Fractionation of Eukaryotic Cells: Analysis by Two-Dimensional Gel Electrophoresis, Melinda L. Ramsby and Gregory S. Makowski. Fractionated Extraction of Total Tissue Proteins from Mouse and Human for 2-D Electrophoresis, Joachim Klose. Preparation and Solubilization of Body Fluids for 2-D, Jean Charles Sanchez and Denis F. Hochstrasser. 2-D Electrophoresis of Plant Proteins, Akira Tsugita and Masaharu Kamo. Quantifying Protein in 2-D PAGE Solubilization Buffers, Louis S. Ramagli. Measuring the Radioactivity of 2-D Protein Extracts, Andrew J. Link and Nick Bizios. Advantages of Carrier Ampholyte IEF, Mary F. Lopez. 2-D Electrophoresis Using Carrier Ampholytes in the First Dimension (IEF), Mary F. Lopez. Nonequilibrium pH Gel Electrophoresis (NEPHGE), Mary F. Lopez. High Resolution, 2-D Protein Electrophoresis Using Nondedicated Equipment, Marion Sarmiento. Large-Gel 2-D Electrophoresis, Joachim Klose. Advantages of Immobilized pH Gradients, Jenny Fichmann. Casting Immobilized pH Gradients (IPGs), Elisabetta Gianazza. Analytical IPG-Dalt, Angelika Gorg and Walter Weiss. IPG-Dalt of Very Alkaline Proteins, Angelika Gorg. Running Preparative Carrier Ampholyte and Immobilized pH Gradient IEF Gels for 2-D, Neil M. Matsui, Diana M. Smith-Beckerman, Jenny Fichmann, and Lois B. Epstein. In-Gel Sample Rehydration of Immobilized pH Gradient, Jean-Charles Sanchez, Denis Hochstrasser, and Thierry Rabilloud.High-Resolution, IPG-Based, Mini Two-Dimensional Gel Electrophoresis, Jean-Charles Sanchez and Denis F. Hochstrasser. Horizontal SDS-PAGE for IPG-Dalt, Angelika Gorg and Walter Weiss. Casting and Running Vertical Slab-Gel Electrophoresis for 2D-PAGE, Bradley J. Walsh and Benjamin R. Herbert. Nonreducing 2-D Polyacrylamide Gel Electrophoresis, Hong Ji and Richard J. Simpson. 2-D Diagonal Gel Electrophoresis, Joan Goverman. 2-D Phosphopeptide Mapping, Hikaru Nagahara, Robert R. Latek, Sergei A. Ezhevsky, and Steven F. Dowdy. Internal Standards for 2-D, Andrew J. Link. Autoradiography of 2-D Gels, Andrew J. Link. Double-Label Analysis, Kelvin H. Lee and Michael G. Harrington. Silver Staining of 2-D Electrophoresis Gels, Thierry Rabilloud. Staining of Preparative 2-D Gels: Coomassie Blue and Imidazole-Zinc Negative Staining, Neil M. Matsui, Diana M. Smith-Beckerman, and Lois B. Epstein. Electroblotting of Proteins from 2-D Polyacrylamide Gels, Michael J. Dunn. Detection of Total Proteins on Western Blots of 2-D Polyacrylamide Gels, Michael J. Dunn. Protein Detection Using Reversible Metal Chelate Stains, Wayne F. Patton, Mark J. Lim, and David Shepro. Gylcoprotein Detection of 2-D Separated Proteins, Nicolle H. Packer, Malcolm S. Ball, and Peter L. Devine. Image Acquisition in 2-D Electrophoresis, Wayne F. Patton, Mark J. Lim, and David Shepro. Computer Analysis of 2-D Images, Ron D. Appel and Denis F. Hochstrasser. 2-D Databases on the World Wide Web, Ron D. Appel, Amos Bairoch, and Denis F. Hochstrasser. Comparing 2-D Electrophoresis Gels Across Internet Databases, Peter F. Lemkin. Constructing a 2-D Database for the World Wide Web, Ron D. Appel, Christine Hoogland, Amos Bairoch, and Denis F. Hochstrasser. Absolute Quantitation of 2-D Protein Spots, Steven P. Gygi and Ruedi Aebersold. Generating a Bacterial Genome Inventory: Identifying 2-D Spots by Comigrating Products of the Genome on 2-D Gels, Ruth A. VanBogelen. Immunoaffinity Identification of 2-DE Separated

198 citations

Journal ArticleDOI
TL;DR: In this review DGGE and the several modifications of the original protocol are presented and its applications in human molecular genetics are summarized together with a preliminary comparison with other mutation detection technologies such as chemical cleavage, RNase protection, and single‐strand conformation polymorphism.
Abstract: The molecular analysis of genetic diseases relies on several technical approaches which allow genetic and physical mapping, characterization of the gene structure, expression studies, and identification of disease-causing mutations. Denaturing gradient gel electrophoresis (DGGE) allows the rapid screening for single base changes in enzymatically amplified DNA. The technique is based on the migration of double-stranded DNA molecules through polyacrylamide gels containing linearly increasing concentrations of a denaturing agent. In this review DGGE and the several modifications of the original protocol are presented. Moreover, its applications in human molecular genetics are summarized together with a preliminary comparison with other mutation detection technologies such as chemical cleavage, RNase protection, and single-strand conformation polymorphism.

198 citations

Journal ArticleDOI
TL;DR: A new method is described for the purification of a cystic fibrosis plasmid vector of clinical grade, which includes an ammonium sulfate precipitation followed by hydrophobic interaction chromatography (HIC) using a Sepharose gel derivatized with 1,4‐butanediol‐diglycidylether.
Abstract: The success and validity of gene therapy and DNA vaccination in in vivo experiments and human clinical trials depend on the ability to produce large amounts of plasmid DNA according to defined specifications. A new method is described for the purification of a cystic fibrosis plasmid vector (pCF1-CFTR) of clinical grade, which includes an ammonium sulfate precipitation followed by hydrophobic interaction chromatography (HIC) using a Sepharose gel derivatized with 1,4-butanediol-diglycidylether. The use of HIC took advantage of the more hydrophobic character of single-stranded nucleic acid impurities as compared with double-stranded plasmid DNA. RNA, denatured genomic and plasmid DNAs, with large stretches of single strands, and lipopolysaccharides (LPS) that are more hydrophobic than supercoiled plasmid, were retained and separated from nonbinding plasmid DNA in a 14-cm HIC column. Anion-exchange HPLC analysis proved that >70% of the loaded plasmid was recovered after HIC. RNA and denatured plasmid in the final plasmid preparation were undetectable by agarose electrophoresis. Other impurities, such as host genomic DNA and LPS, were reduced to residual values with the HIC column (<6 ng/microg pDNA and 0.048 EU/microg pDNA, respectively). The total reduction in LPS load in the combined ammonium acetate precipitation and HIC was 400,000-fold. Host proteins were not detected in the final preparation by bicinchoninic acid (BCA) assay and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Plasmid identity was confirmed by restriction analysis and biological activity by transformation experiments. The process presented constitutes an advance over existing methodologies, is scaleable, and meets quality standards because it does not require the use of additives that usually pose a challenge to validation and raise regulatory concerns.

198 citations

Journal ArticleDOI
TL;DR: Northern blotting analyses of rat adult tissues showed that α1-6FucT was highly expressed in brain, and no sequence homology was found with other previously cloned fucosyl transferases, but the enzyme appears to be a type II transmembrane protein like the other glycosyltransferases.

198 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
202364
2022116
2021108
2020104
2019120
2018147