2-D Proteome Analysis Protocols / Edition 1

by Andrew J. Link

Overview

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 Prools 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 Prools 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 prools for generating 2-D protein extracts from most of the standard model organisms, including bacteria, yeast, nematode, Drosophila, plants, mouse, and human.

Product Details

ISBN-13:	9781617370601
Publisher:	Springer-Verlag New York, LLC
Publication date:	11/10/2010
Series:	Methods in Molecular Biology , #112
Edition description:	Softcover reprint of hardcover 1st ed. 1999
Pages:	601
Product dimensions:	5.98(w) x 9.02(h) x 0.35(d)

About the Author

Link, Andrew J. (Univ of Washington)

Table of Contents

2-D Protein Gel Electrophoresis.- Solubilization of Proteins in 2-D Electrophoresis.- Preparation of Escherichia coli Samples for 2-D Gel Analysis.- Preparing 2-D Protein Extracts from Yeast.- 2-D Protein Extracts from Drosophila melanogaster.- Preparing 2-D Protein Extracts from Caenorhabditis elegans.- Eukaryotic Cell Labeling and Preparation for 2-D.- Differential Detergent Fractionation of Eukaryotic Cells.- Fractionated Extraction of Total Tissue Proteins from Mouse and Human for 2-D Electrophoresis.- Preparation and Solubilization of Body Fluids for 2-D.- 2-D Electrophoresis of Plant Proteins.- Quantifying Protein in 2-D PAGE Solubilization Buffers.- Measuring the Radioactivity of 2-D Protein Extracts.- Advantages and Disadvantages of Carrier Ampholyte IEF.- 2-D Electrophoresis Using Carrier Ampholytes in the First Dimension (IEF).- Nonequilibrium pH Gel Electrophoresis (NEPHGE).- High-Resolution, 2-D Protein Electrophoresis Using Nondedicated Equipment.- Large-Gel 2-D Electrophoresis.- Advantages of Immobilized pH Gradients.- Casting Immobilized pH Gradients (IPGs).- Analytical IPG-Dalt.- IPG-Dalt of Very Alkaline Proteins.- Running Preparative Carrier Ampholyte and Immobilized pH Gradient IEF Gels for 2-D.- In-Gel Sample Rehydration of Immobilized pH Gradient.- High-Resolution, IPG-Based, Mini Two-Dimensional Gel Electrophoresis.- Horizontal SDS-PAGE for IPG-Dalt.- Casting and Running Vertical Slab-Gel Electrophoresis for 2D-PAGE.- Nonreducing 2-D Polyacrylamide Gel Electrophoresis.- 2-D Diagonal Gel Electrophoresis.- 2-D Phosphopeptide Mapping.- Internal Standards for 2-D.- Autoradiography of 2-D Gels.- Double-Label Analysis.- Silver Staining of 2-D Electrophoresis Gels.- Staining of Preparative 2-D Gels.- Electroblotting of Proteins from 2-D Polyacrylamide Gels.- Detection of Total Proteins on Western Blots of 2-D Polyacrylamide Gels.- Protein Detection Using Reversible Metal Chelate Stains.- Glycoprotein Detection of 2-D Separated Proteins.- Image Acquisition in 2-D Electrophoresis.- Computer Analysis of 2-D Images.- 2-D Databases on the World Wide Web.- Comparing 2-D Electrophoretic Gels Across Internet Databases.- Constructing a 2-D Database for the World Wide Web.- Absolute Quantitation of 2-D Protein Spots.- Generating a Bacterial Genome Inventory.- Immunoaffinity Identification of 2-DE Separated Proteins.- 2-DE Spot Amino Acid Analysis with 9-Fluorenylmethyl Chloroformate.- N-Terminal Amino Acid Sequencing of 2-DE Spots.- Characterizing Proteins from 2-DE Gels by Internal Sequence Analysis of Peptide Fragments.- Obtaining Molecular Weights of Proteins and Their Cleavage Products by Directly Combining Gel Electrophoresis with Mass Spectrometry.- Identification of Proteins by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Using Peptide and Fragment Ion Masses.- Sample Preparation Methods for Mass Spectrometric Peptide Mapping Directly from 2-DE Gels.- Protein Identification and Analysis Tools in the ExPASy Server.- Automated Protein Identification Using Microcolumn Liquid Chromatography-Tandem Mass Spectrometry.- Peptide Sequencing of 2-DE Gel-Isolated Proteins by Nanoelectrospray Tandem Mass Spectrometry.

Editorial Reviews

5 Stars! from Doody

This book is a detailed compilation of the theory and practice of analyzing total cellular protein by two-dimensional gel electrophoresis. According to the editor, it is intended as a benchtop manual for anyone who uses these techniques. With the number of sophisticated techniques now in use in many areas of life sciences research, this will be a valuable volume and it fully meets the editor's stated objectives. It is written for beginners and for more experienced researchers who may wish to expand on or improve their use of the 2-D gel electrophoretic techniques. The 55 chapters are written by over 100 contributors, all of whom are quite expert in their specialty. This book contains a detailed description of the techniques (reagents, equipment, and trouble-shooting) that are widely used for the analysis of total cellular proteins. The illustrations are very helpful and easy to understand. Several of the chapters explain how the techniques can be standardized so valid comparisons of results can be made from laboratory to laboratory. This is a very worthwhile guide to the technique of two-dimensional gel electrophoresis. It would be a valuable addition to a lab carrying out these procedures and would make a very useful addition to any life science library.

Alvin Telser

Comprises 55 contributions that are intended as a guide both for novices to 2-D gels and for experienced practitioners who want to try the newer techniques. The contributions guide researchers through the complete process of working with 2-D protein gels from making the protein extract to finally identifying the proteins of interest. The volume includes protocols for generating 2-D protein extracts from most of the standard model organisms, including bacteria, yeasts, nematodes, plants, mice, and humans. Each chapter includes a description of the protocol, the basic theory behind it, a list of the equipment and reagents necessary for the protocol, a step-by-step guide to the experiment, and hints for dealing with problems. Annotation c. by Book News, Inc., Portland, Or.

Booknews

Reviewer: Alvin Telser, PhD (Northwestern University Feinberg School of Medicine)
Description: This book is a detailed compilation of the theory and practice of analyzing total cellular protein by two-dimensional gel electrophoresis.
Purpose: According to the editor, it is intended as a benchtop manual for anyone who uses these techniques. With the number of sophisticated techniques now in use in many areas of life sciences research, this will be a valuable volume and it fully meets the editor's stated objectives.
Audience: It is written for beginners and for more experienced researchers who may wish to expand on or improve their use of the 2-D gel electrophoretic techniques. The 55 chapters are written by over 100 contributors, all of whom are quite expert in their specialty.
Features: This book contains a detailed description of the techniques (reagents, equipment, and trouble-shooting) that are widely used for the analysis of total cellular proteins. The illustrations are very helpful and easy to understand. Several of the chapters explain how the techniques can be standardized so valid comparisons of results can be made from laboratory to laboratory.
Assessment: This is a very worthwhile guide to the technique of two-dimensional gel electrophoresis. It would be a valuable addition to a lab carrying out these procedures and would make a very useful addition to any life science library.

Doody's Review Service

"Our readers will find in this book with its 55 chapters, written by 78 highly well-trained specialists, all they need to come to a good result, going out from the theory of the subject to the precise description of the prools, the instrumentation, to additive notes, the problems and the eventual difficulties. In one word, an excellent and practical book."-Cellular and Molecular Biology

"...There are 55 chapters covering everything you really need to know to get you to speed in the analysis of the proteome. I really liked the layout of each chapter with a pithy introduction followed by materials and methods sections...an absolute must for the proteomic laboratory."-Microbiology Today

". . .the book covers practical aspects , is well documented, and clearly written . . . recommended for researchers in the field. This collection of prools is an invaluable starting point for people embarking on attempts to develop new proteomes."-FEBS Letters

"The wealth of techniques and helpful hints presented in this book should be useful to both neophytes and advanced researchers engaged in the area of high resolution 2-D protein electrophoresis."-Analytical Biochemistry

From the Publisher