2-d Proteome Analysis Protocols

Front Cover
Andrew J. Link
Springer Science & Business Media, 1999 - Science - 601 pages
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.
 

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Contents

An Outline
9
Preparing 2D Protein Extracts from Yeast
32
Eukaryotic Cell Labeling and Preparation for
49
Fractionated Extraction of Total Tissue Proteins from Mouse
67
Preparation and Solubilization of Body Fluids for
87
Measuring the Radioactivity of 2D Protein Extracts
105
Dimension IEF
111
Nonequilibrium pH Gel Electrophoresis NEPHGE
129
Detection of Total Proteins on Western Blots
319
Protein Detection Using Reversible Metal Chelate Stains
331
Gylcoprotein Detection of 2D Separated Proteins
341
Image Acquisition in 2D Electrophoresis
353
Computer Analysis of 2D Images
363
Appel Amos Bairoch and Denis F Hochstrasser
383
Comparing 2D Electrophoresis Gels Across Internet Databases
393
Constructing a 2D Database for the World Wide
411

LargeGel 2D Electrophoresis
147
Advantages of Immobilized pH Gradients
173
Analytical IPGDalt
189
IPGDalt of Very Alkaline Proteins
197
Running Preparative Carrier Ampholyte and Immobilized
211
InGel Sample Rehydration of Immobilized pH Gradient
221
Horizontal SDSPAGE for IPGDalt
235
Casting and Running Vertical SlabGel Electrophoresis
245
Nonreducing 2D Polyacrylamide Gel Electrophoresis
255
Joan Goverman
265
Hikaru Nagahara Robert R Latek Sergei A Ezhevsky
271
Internal Standards for
281
DoubleLabel Analysis
291
Coomassie Blue
307
Absolute Quantitation of 2D Protein Spots
417
Identifying 2D Spots
423
Immunoaffinity Identification of 2DE Separated Proteins
431
Chloroformate
445
NTerminal Amino Acid Sequencing of 2DE Spots
461
Characterizing Proteins from 2DE Gels by Internal
467
Obtaining Molecular Weights of Proteins and Their Cleavage
473
Identification of Proteins by MatrixAssisted Laser
487
Sample Preparation Methods for Mass Spectrometric Peptide
513
Protein Identification and Analysis Tools in the ExPASy Server
531
Automated Protein Identification Using Microcolumn Liquid
553
Peptide Sequencing of 2DE GelIsolated Proteins
571
Index
589
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About the author (1999)

Link, University of Washington School of Medicine.

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