2-D Proteome Analysis Protocols

1. Front Matter

   Pages i-xvii

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2. 2-D Protein Gel Electrophoresis

   • Jenny Fichmann, Reiner Westermeier

   Pages 1-7

3. Solubilization of Proteins in 2-D Electrophoresis

   • Thierry Rabilloud

   Pages 9-19

4. Preparation of Escherichia coli Samples for 2-D Gel Analysis

   • Ruth A. VanBogelen, Frederick C. Neidhardt

   Pages 21-29

5. Preparing 2-D Protein Extracts from Yeast

   • David M. Schieltz

   Pages 31-34

6. 2-D Protein Extracts from Drosophila melanogaster

   • Christer Ericsson

   Pages 35-41

7. Preparing 2-D Protein Extracts from Caenorhabditis elegans

   • Robert Zwilling

   Pages 43-48

8. Eukaryotic Cell Labeling and Preparation for 2-D

   • Nick Bizios

   Pages 49-52

9. Differential Detergent Fractionation of Eukaryotic Cells

   • Melinda L. Ramsby, Gregory S. Makowski

   Pages 53-66

10. Fractionated Extraction of Total Tissue Proteins from Mouse and Human for 2-D Electrophoresis

    • Joachim Klose

    Pages 67-85

11. Preparation and Solubilization of Body Fluids for 2-D

    • Jean-Charles Sanchez, Denis F. Hochstrasser

    Pages 87-93

12. 2-D Electrophoresis of Plant Proteins

    • Akira Tsugita, Masaharu Kamo

    Pages 95-97

13. Quantifying Protein in 2-D PAGE Solubilization Buffers

    • Louis S. Ramagli

    Pages 99-103

14. Measuring the Radioactivity of 2-D Protein Extracts

    • Andrew J. Link, Nick Bizios

    Pages 105-107

15. Advantages and Disadvantages of Carrier Ampholyte IEF

    • Mary F. Lopez

    Pages 109-110

16. 2-D Electrophoresis Using Carrier Ampholytes in the First Dimension (IEF)

    • Mary F. Lopez

    Pages 111-127

17. Nonequilibrium pH Gel Electrophoresis (NEPHGE)

    • Mary F. Lopez

    Pages 129-131

18. High-Resolution, 2-D Protein Electrophoresis Using Nondedicated Equipment

    • Marion Sarmiento

    Pages 133-145

19. Large-Gel 2-D Electrophoresis

    • Joachim Klose

    Pages 147-172

20. Advantages of Immobilized pH Gradients

    • Jenny Fichmann

    Pages 173-174

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.

"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 protocols, 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 protocols 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

• Department of Molecular Biotechnology, University of Washington, Seattle

  Andrew J. Link

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