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Western Blotting Guru provides researchers in molecular biology with a handy reference for approaching and solving challenging problems associated with immunoblotting setup and optimization. As a laboratory guide, it emphasizes the technical aspects of efficiently employing immunoblotting as a tool in molecular biology laboratories. The book covers the basic science underlying immunoblotting and detailed description of the method parameters, followed by good benchtop practices, tips and tricks for obtaining high-quality data and a detailed troubleshooting guide addressing a variety of problem types. Provides a benchtop reference that every molecular biologists will use to design, optimize, troubleshoot and analyze their immunoblotting experiments Contains unique good practices and tips that are indispensable for the beginner and expert alike Features special cases with applications of immunoblotting optimization Includes detailed appendices with tables, figures and key protocols Provides troubleshooting tips for various types of modifications of standard protocols Organized as a systematic, concentrated resource to save time when addressing an immunoblotting problem Front Cover Western Blotting Guru Copyright Page Contents Preface Note to the Reader 1 Introduction 1.1 What Is Western Blotting? 1.2 A Bit of History 2 Procedure 2.1 Sample Preparation 2.2 Gel Electrophoresis 2.2.1 Gel Selection and Preparation 2.2.2 Running the Gels 2.2.3 Molecular Weight Markers 2.3 Protein Transfer 2.3.1 Transfer Setup 2.3.2 Choosing the Right Membrane 2.3.3 Blotting Paper 2.3.4 Transfer Buffer 2.3.5 Transfer Power Settings 2.3.6 Visualization of Proteins After Transfer 2.4 Blocking the Membrane 2.5 Primary Antibodies 2.6 Secondary Antibodies 2.7 Blot Washes2.8 Developing Western Blots 3 Good Practices 4 Optimization and Troubleshooting 4.1 Optimization Rules 4.2 General Optimization Strategies 4.2.1 Antibody Concentration 4.2.2 Antibody Incubation Times 4.2.3 Wash Stringency 4.2.4 Blocking 4.2.5 Preclear the Antibodies 4.2.6 Supplement Antibody Solutions With Nonphosphorylated Peptides 4.3 Troubleshooting Specific Problems 4.3.1 Problem Type 1 4.3.1.1 Subtype 1 4.3.1.2 Subtype 2 4.3.2 Problem Type 2 4.3.3 Problem Type 3 4.3.4 Problem Type 4 4.3.5 Problem Type 5 4.3.6 Problem Type 6 4.3.7 Problem Type 7 4.3.8 Problem Type 84.3.9 Problem Type 9 4.3.10 Problem Type 10 5 Tips and Tricks 6 Special Cases 6.1 Quantitative Western Blotting 6.2 Overlay Assays 6.3 Phospho-Specific Antibodies 6.4 Phos-Tag 6.5 Nonreducing PAGE 6.6 Dot Blots 7 Data Analysis, Storage, Retrieval Appendix A: Buffers and Solutions Appendix B: SDS-PAGE Gel Tables Appendix C: SDS-PAGE Protocol Appendix D: Wet Transfer and Immunoblotting Protocol Appendix E: Home-Made Enhanced ChemiLuminescence (ECL) Detection Appendix F: Stripping Protocols Appendix G: Coomassie Staining Protocol Appendix H: Lysis of Cells Using Native ConditionsAppendix I: Quick Denaturing Lysis Protocol Appendix J: Protein Tags Appendix K: Covalent Crosslinking of Antibodies to Beads References Back Cover