2D PAGE: Sample Preparation and Fractionation

1. Front Matter

   Pages i-xiii

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2. Sample Preparation Basics

   1. Front Matter

      Pages 1-1

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   2. Mechanical/Physical Methods of Cell Disruption and Tissue Homogenization

      • Stanley Goldberg

      Pages 3-22

   3. Bacteria and Yeast Cell Disruption Using Lytic Enzymes

      • Oriana Salazar

      Pages 23-34

   4. Sample Solublization Buffers for Two-Dimensional Electrophoresis

      • Walter Weiss, Angelika Görg

      Pages 35-42

   5. Quantitation of Protein in Samples Prepared for 2-D Electrophoresis

      • Tom Berkelman

      Pages 43-49

   6. Removal of Interfering Substances in Samples Prepared for Two-Dimensional (2-D) Electrophoresis

      • Tom Berkelman

      Pages 51-62

   7. Protein Concentration by Hydrophilic Interaction Chromatography Combined with Solid Phase Extraction

      • Ulrich Schneider

      Pages 63-69

3. Protein Labeling Techniques

   1. Front Matter

      Pages 71-71

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   2. Difference Gel Electrophoresis Based on Lys/Cys Tagging

      • Reiner Westermeier, Burghardt Scheibe

      Pages 73-85

   3. Isotope-Coded Two-Dimensional Maps: Tagging with Deuterated Acrylamide and 2-Vinylpyridine

      • Pier Giorgio Righetti, Roberto Sebastiano, Attilio Citterio

      Pages 87-99

   4. Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)

      • s Gruhler, Irina Kratchmarova

      Pages 101-111

   5. ICPL—Isotope-Coded Protein Label

      • Josef Kellermann

      Pages 113-123

   6. Radiolabeling for Two-Dimensional Gel Analysis

      • Hélian Boucherie, Aurélie Massoni, Christelle Monribot-Espagne

      Pages 125-135

4. Fractionation of Proteins by Chemical Reagents and Chromatography

   1. Front Matter

      Pages 137-137

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   2. Sequential Extraction of Proteins by Chemical Reagents

      • Stuart J. Cordwell

      Pages 139-146

   3. Reducing Sample Complexity by RP-HPLC: Beyond the Tip of the Protein Expression Iceberg

      • Gert Van den Bergh, Lutgarde Arckens

      Pages 147-156

   4. Enriching Basic and Acidic Rat Brain Proteins with Ion Exchange Mini Spin Columns Before Two-Dimensional Gel Electrophoresis

      • Ning Liu, Aran Paulus

      Pages 157-166

   5. Reducing the Complexity of the Escherichia coli Proteome by Chromatography on Reactive Dye Columns

      • Phillip Cash, Ian R. Booth

      Pages 167-185

   6. Reducing Sample Complexity in Proteomics by Chromatofocusing with Simple Buffer Mixtures

      • Hong Shen, Xiang Li, Charles J Bieberich, Douglas D Frey

      Pages 187-203

This book, split into two volumes, presents a broad coverage of the principles and recent developments of sample preparation and fractionation tools in Expression Proteomics in general and for two-dimensional electrophoresis (2-DE) in particular. 2-DE, with its unique capacity to resolve thousands of proteins in a single run, is still a fundamental research tool for nearly all protein-related scientific projects. The methods described here in detail are not limited to 2-DE and can also be applied to other protein separation techniques. Because each biological sample is unique, a suited sample preparation strategy has to consider the type of sample as well as the type of biological question being addressed. The complex nature of proteins often requires a multitude of sample preparation options. In addition, sample preparation is not only a prerequisite for a successful and reproducible Proteomics exp- iment, but also the key factor to meaningful data evaluation. Interestingly, not much attention was paid to this area during Proteomics methodology dev- opment and therefore this book is intended to explain in depth how proteins from various sources can be properly isolated and prepared for reproducible Proteome analysis.

• Amino acid
• Escherichia coli
• Lipid
• Organelle
• Polyacrylamid-Gelelektrophorese
• Proteomics
• biochemistry
• biomedicine
• enzymes
• glycoprotein
• membrane proteins
• polymer
• protein
• protein complexes
• proteins

• Bio-Rad Laboratories GmbH, Germany

  Anton Posch

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