2-D Proteome Analysis Protocols

A step-by-step tour through the complete process of doing proteomics. With easy-to-follow instructions, complete with many helpful hints and explanations, leading investigators and pioneers in the field show how to make protein extracts, reproducibly run them on 2-D gels, detect them, analyze the data, and precisely identify each protein. The book covers the latest methods of using carrier ampholytes in the 1st dimension, casting and running immobilized pH gradient 2-D gels, MALDI-TOF-based peptide mapping, automated tandem mass spectrometry, and nanoelectrospray ionization technology. For the 2nd dimension, there are methods for running flatbed or vertical gels and for protein detection using autoradiography, and Coomassie, silver, and reversible metal-chelate stains. 2-D Proteome Analysis Protocols is the most complete guide for using proteomics to answer biological questions.

In 2-D Gel Proteome Analysis Protocols, Andrew Link and his expert collaborators take today's researchers step-by-step through the complete process of doing proteomics. With easy-to-follow instructions, complete with many helpful hints and explanations, leading investigators and pioneers in the field show how to make protein extracts, reproducibly run them on a 2-D gels, detect them, analyze the data, and precisely identify each protein. The book covers the latest methods of using carrier ampholytes in the 1st dimension, casting and running immobilized pH gradient 2-D gels, MALDI-TOF-based peptide mapping, automated tandem mass spectrometry, and nanoelectrospray ionization technology. For the 2nd dimension, there are methods for running flatbed or vertical gels and for protein detection using autoradiography, and Coomassie, silver, and reversible metal-chelate stains. The book is a perfect complement to the genome sequencing project for answering biological questions.



2-D Gel Proteome Analysis Protocols is the most complete guide for using proteomics to answer biological questions. Whether it is a question of global protein analysis or evaluating a cell's response to internal or external stimuli, the advanced methods described here will enable today's researchers better to understand how cells work and open new possibilities for drug discovery.

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 Görg and Walter Weiss. IPG-Dalt of Very Alkaline Proteins, Angelika Görg. 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 Görg 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 Proteins, Barbara Magi, Luca Bini, Barbara Marzocchi, Sabrina Liberatori, Roberto Raggiaschi, and Vitaliano Pallini. 2-DE Spot Amino Acid Analysis with 9-Fluroenylmethyl Chloroformate, Marc R. Wilkins, Jun X. Yan, and Andrew A. Gooley. N-Terminal Amino Acid Sequencing of 2-DE Spots, Masaharu Kamo and Akira Tsugita. Characterizing Proteins from 2-DE Gels by Internal Sequence Analysis of Peptide Fragments: Strategies for Microsample Handling, Hediye Erdjument-Bromage, Mary Lui, Lynne Lacomis, and Paul Tempst. Obtaining Molecular Weights of Proteins and Their Cleavage Products by Directly Combining Gel Electrophoresis with Mass Spectrometry, Rachel R. Ogorzalek Loo, Joseph A. Loo, and Philip C. Andrews. Identification of Proteins by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Using Peptide and Fragment Ion Masses, Paul L. Courchesne and Scott D. Patterson. Sample Preparation Methods for Mass Spectrometric Peptide Mapping Directly from 2-DE Gels, Ole Nørregaard Jensen, Matthias Wilm, Andrej Shevchenko, and Matthias Mann. Protein Identification and Analysis Tools in the ExPASy Server, Marc R. Wilkins, Elisabeth Gasteiger, Amos Bairoch, Jean-Charles Sanchez, Keith L. Williams, Ron D. Appel, and Denis F. Hochstrasser. Automated Protein Identification Using Microcolumn Liquid Chromatography-Tandem Mass Spectrometry, John R. Yates III, Edwin Carmack, Lara Hays, Andrew J. Link, and Jimmy K. Eng. Peptide Sequencing of 2-DE Gel-Isolated Proteins by Nanoelectrospray Tandem Mass Spectrometry, Ole Nørregaard Jensen, Matthias Wilm, Andrej Shevchenko, and Matthias Mann. Index.

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