2-D Proteome Analysis Protocols

About this book

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.

Contents

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 Grg and Walter Weiss. IPG-Dalt of Very Alkaline Proteins Angelika Grg. 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 Grg 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 N>rregaard 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 N>rregaard Jensen Matthias Wilm Andrej Shevchenko and Matthias Mann. Index.

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