2-D Proteome Analysis Protocols

Front Cover
Andrew J. Link
Springer Science & Business Media, Feb 2, 2008 - Science - 601 pages
With the completion of sequencing projects and the advancement of a- lytical tools for protein identification, proteomics—the study of the expressed part of the genome—has become a major region of the burgeoning field of functional genomics. High-resolution 2-D gels can reveal virtually all p- teins present in a cell or tissue at any given time, including posttranslationally modified proteins. Changes in the expression and structure of most cellular proteins caused by differentiation or external stimuli can be displayed and eventually identified using 2-D protein gels. 2-D Proteome Analysis Protocols covers all aspects of the use of 2-D protein electrophoresis for the analysis of biological problems. The contri- tors include many of the leaders in the fields of biochemistry and analytical chemistry who were instrumental in the development of high-resolution 2-D gels, immobilized pH gradients, computer analysis, and mass spectromet- based protein identification methodologies. This book is intended as a benchtop manual and guide both for novices to 2-D gels and for those aficionados who wish to try the newer techniques. Any group using protein biochemistry—especially in the fields of molecular biology, biochemistry, microbiology, and cell biology—should find this book eminently useful. 2-D Proteome Analysis Protocols takes the researcher through the c- plete process of working with 2-D protein gels from making the protein - tract to finally identifying the proteins of interest. It includes protocols for generating 2-D protein extracts from most of the standard model organisms, including bacteria, yeast, nematode, Drosophila, plants, mouse, and human.
 

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
Coomassie Blue
307
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

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
Kelvin H Lee and Michael G Harrington
291
Appel Christine Hoogland Amos Bairoch
411
Absolute Quantitation of 2D Protein Spots
417
Identifying 2D Spots
423
Barbara Magi Luca Bini Barbara Marzocchi
431
Chloroformate
445
NTerminal Amino Acid Sequencing of 2DE Spots
461
Characterizing Proteins from 2DE Gels by Internal
467
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
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