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Two-Dimensional Electrophoresis and Immunological Techniques - Bonnie S. Dunbar

Two-Dimensional Electrophoresis and Immunological Techniques

Paperback Published: January 1987
ISBN: 9780306428395
Number Of Pages: 383

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This text is a summary of basic principles and techniques and is dedicated to all those students who have been told by their mentors, "Go forth and do two-dimensional gels and have the results on my desk tomorrow. " No attempt has been made in this text to provide exhaustive lists of references related to basic principles or techniques or to list every com- pany or supplier involved in this area of research. Nevertheless, it is hoped that sufficient information is given to help a new investigator or student appreciate the complexities but develop sufficient expertise to carry out these techniques successfully. The discussions are designed to instill in basic science and clinical investigators of all levels of expertise an appreciation of the power of combining a variety of techniques as well as to provide basic insight into the theories, complexities, and problems frequently encountered with electrophoretic and immunochemical meth- ods. Bonnie S. Dunbar Houston v Acknowledgments I wish to thank my students and staff for their patience and support throughout the preparation of this text. I would like to acknowledge my appreciation for my extensive discussions with Dr. David Sammons (Uni- versity of Arizona) and to Dr. N. L. and Dr. N. G. Anderson and their colleagues (Argonne National Laboratory) for their invaluable advice and suggestions in this area over the years. I thank my research assistant, Ms.

1 Basic Theories and Principles of Electrophoresis.- I. Introduction.- II. Basic Principles of Electrolysis.- III. Movement of Molecules in an Electrical Field.- IV. Choice of Buffers for Electrophoresis.- V. Basic Types of Electrophoresis.- A. Moving Boundary Electrophoresis.- B. Zone Electrophoresis.- C. Continuous Electrophoresis.- VI. Movement of Molecules in Support Media during Electrophoresis.- VII. Basic Principles of Isoelectric Focusing.- A. Definition of Isoelectric Point.- B. Definition of Ampholytes.- C. Experimental Parameters Affecting Measurement to Apparent Isoelectric Points of Proteins.- D. Determination of pH in Isoelectric Focusing Gels.- E. Equilibrium versus Nonequilibrium Isoelectric Focusing.- VIII. Basic Principles of Polyacrylamide Gel Electrophoresis.- A. Acrylamide Gel Matrix.- B. Role of Catalysts (Initiators) in Acrylamide Polymerization.- C. Acrylamide Pore Size.- D. Effect of Acrylamide Pore Size on Separation of Proteins.- IX. Principles of a Stacking Gel.- X. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis.- XI. High-Resolution Two-Dimensional Polyacrylamide Gel Electrophoresis.- References.- 2 Applications and Strategies for Use of Polyacrylamide Electrophoresis.- I. Introduction.- II. Nonreducing Polyacrylamide Gel Electrophoresis.- III. One-Dimensional Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis.- IV. When to Use Two-Dimensional Polyacrylamide Gel Electrophoresis.- V. Peptide Mapping.- A. One-Dimensional Peptide Mapping.- B. Two-Dimensional Peptide Mapping.- C. Limitations of Peptide-Mapping Techniques.- VI. Purification of Proteins from Polyacrylamide Gels.- VII. Obtaining Peptides for Amino Acid Sequence.- VIII. Preparation and Characterization of Antibodies Purified by Polyacrylamide Gel Electrophoresis.- IX. Analysis of Posttranslational Modification of Proteins.- X. Comparison of Complex Protein Samples.- XI. Use of Modified Electrophoresis Systems to Analyze Specific Proteins.- XII. Clinical Applications of Two-Dimensional Polyacrylamide Gel Electrophoresis.- References.- 3 Sample Preparation for Electrophoresis.- I. Quantitation of Protein in Samples.- II. Principles of Protein Solubilization.- III. Experimental Solubilization of Protein Samples.- A. Denaturation by Urea.- B. Denaturation by Guanidinium Chloride.- C. Denaturation by Detergents.- D. Solubilization of Membrane Proteins.- IV. Factors Critical for Sample Solubilization prior to Isoelectric Focusing.- V. Artifacts of Protein Solubilization.- A. Detergent Concentration and Interaction.- B. Chemical Modification of Proteins.- C. "Insoluble" Proteins.- VI. Subfractionation of Samples for Electrophoresis.- A. Organelle Fractionation Using Centrifugation and Electrophoresis.- B. Water-Soluble Fractionation.- C. Chemical Extraction Methods for Protein Analysis.- D. Differential Absorption of Proteins to Charged Matrices.- References.- 4 Protein Detection in Polyacrylamide Gel Electrophoresis.- I. Introduction.- II. Coomassie Blue Staining Methods.- III. Silver Staining of Proteins in Polyacrylamide Gels.- A. Introduction.- B. Factors Affecting Silver Stain Reactions.- C. Molecules and Artifacts Stained by Silver Stain Methods.- IV. Miscellaneous Protein-Staining Methods.- References.- 5 Basic Principles of Posttranslational Modification of Proteins and Their Analysis Using High-Resolution Two-Dimensional Polyacrylamide Gel Electrophoresis.- I. Introduction.- II. Protein Glycosylation.- A. Definitions and Basic Structures.- B. Protein-Carbohydrate Linkages.- C. Synthesis and Processing of Glycoproteins.- D. Effects of Glycosylation on the Physicochemical and Biochemical Properties of Proteins.- E. Carbohydrate Side-Chain Heterogeneity.- F. Analysis of Glycoproteins Using Two-Dimensional Polyacrylamide Gel Electrophoresis.- G. Deglycosylation of Glycoproteins.- III. Protein Phosphorylation.- A. Introduction.- B. cAMP-Dependent Protein Phosphorylation.- C. cAMP-Independent Protein Phosphorylation.- D. Experimental Identification of cAMP-Dependent Protein Kinases Using Polyacrylamide Gel Electrophoresis.- E. Experimental Identification of Endogenous Substrates for Protein Kinases.- F. Tyrosine-Specific Protein Kinases.- References.- 6 Isotopic Labeling of Proteins for Electrophoretic Analysis.- I. Introduction.- II. Amino Acid Labeling during Protein Synthesis.- A. Advantages of Radiolabeling Proteins.- B. Disadvantages of Radiolabeling Proteins.- C. Equilibrium, Pulse, and Pulse-Chase Labeling Studies.- D. Use of Protein Synthesis Inhibitors in Radiolabel Studies.- E. Interpretation of Radiolabeling Experiments.- III. Radiolabeling Using Protein Phosphorylation Methods.- IV. Carbohydrate Labeling of Glycoproteins.- V. Labeling of Proteins by Chemical Modification.- References.- 7 Use of Autoradiography in Polyacrylamide Gel Electrophoresis.- I. Introduction.- II. Isotopes Commonly Used in Autoradiography.- III. Emulsions and Films Used in Autoradiography.- IV. Formation of an Autoradiographic Image.- V. Exposure Methods for Autoradiography.- A. Direct Exposure Method.- B. Fluorography Method.- C. Intensifying Screens.- D. Use of Hypersensitized or Preexposed Film.- E. Exposure of X-Ray Film at Low Temperature.- References.- 8 Strategies for Use of Polyacrylamide Gel Electrophoresis in the Preparation and Characterization of Antibodies.- I. Introduction.- II. Strategies for Developing Antibodies.- A. Polyclonal Antibodies.- B. Monoclonal Antibodies.- III. Immunization Protocols.- A. Choice of Antibody.- B. Guidelines for Choosing Animal Species to Be Immunized.- C. Choice of Adjuvant.- D. Preparation of Polyacrylamide Gel for Immunization.- E. Routes of Administration.- F. Dosages for Immunization.- G. Enhancement of Immunogenicity of Proteins.- H. Immunization Procedures for Obtaining Ascites Fluid.- IV. Collection of Blood and Ascites Fluid for Antibody Preparation.- A. Bleeding Procedures.- B. Collection of Ascites Fluid.- V. Immunoglobulin Purification.- A. Immunoglobulin Fractionation.- B. Immunoglobulin Characterization by One- and Two-Dimensional Polyacrylamide Gel Electrophoresis.- VI. Methods for Antibody Detection.- A. Introduction.- B. Immunoprecipitation Methods.- C. Solid-Phase Assays.- References.- 9 Practical Methods for Laboratory Photography.- I. Introduction.- A. Cost Effectiveness and Efficiency.- B. Immediate and Complete Data Storage.- C. Accurate Data Recording.- II. Choice of Photographic Methods.- A. Choice of Camera.- B. Choice of Lens.- C. Choice of Film.- D. Choice of Filters.- III. Bulk Loading Film.- IV. Photographing Polyacrylamide Gels.- A. Photographic Setup.- B. Photographic Composition.- C. Film Exposure Conditions.- V. Developing Film.- A. Photographic Paper.- B. Print Exposure.- C. Print Development.- VI. Printing Photographs from Negatives.- A. Equipment.- VII. Direct Contact Prints of Color-Based Silver-Stained Polyacrylamide Gels.- References.- 10 Troubleshooting and Artifacts in Two-Dimensional Polyacrylamide Gel Electrophoresis.- I. Introduction.- II. Reagent Quality and Equipment Maintenance.- A. Water.- B. Chemicals.- C. Equipment Maintenance.- III. Isoelectric Focusing.- A. Sample Solubilization.- B. Problems with Gel Polymerization.- C. Problems with Removal of Gels from Tubes.- D. Gel Breakage during Isoelectric Focusing.- E. Irreproducible Isoelectric Focusing Patterns.- IV. Sodium Dodecyl Sulfate Slab Gel Electrophoresis.- A. Gel Polymerization.- V. Problems with Staining Gels.- A. Coomassie Blue Staining.- B. Problems Associated with Silver-Staining Procedures.- C. Staining Artifacts.- VI. Troubleshooting Immunoblotting Methods.- A. High Background.- B. Uneven Background or Splotches on Transfer.- C. Little or No Antibody Binding to Proteins.- D. Irregular Transfer of Protein to Nitrocellulose.- VII. Conclusions.- References.- 11 Advances in Technology of High-Resolution Two-Dimensional Polyacrylamide Gel Electrophoresis.- I. Introduction.- II. Equipment and Reagents for Isoelectric Focusing.- A. Isoelectric Focusing Using Horizontal Slab Apparatus.- B. Isoelectric Focusing Using a Gel-Tube Apparatus.- C. Reagents Used for Isoelectric Focusing.- III. Equipment for Second-Dimension Polyacrylamide Slab Gel Electrophoresis.- A. Uses of Standardized Equipment.- B. Standardization of High-Resolution Two-Dimensional Polyacrylamide Gel Electrophoresis.- C. Specialized Equipment for Slab Gel Electrophoresis.- IV. Computerized Analysis of One- and Two-Dimensional Polyacrylamide Gel Electrophoresis.- V. Commercial Availability of Precast Gradient Gels and Gel Analysis Services.- References.- Appendixes.- 1. Protein Assays.- 2. Sample Preparation and Solubilization for One-Dimensional Polyacrylamide Gel Electrophoresis and Isoelectric Focusing.- 3. Method for Isoelectric Focusing.- 4. Preparation of Slab Gels for One- or Two-Dimensional Polyacrylamide Sodium Dodecyl Sulfate Gel Electrophoresis.- 5. Loading the Isoelectric Focusing Gel and Carrying Out Second-Dimension Electrophoresis.- 6. Methods for Studying Protein Phosphorylation.- 7. Fluorography.- 8. Drying Gels for Autoradiography.- 9. Methods for Staining Proteins in Polyacrylamide Gels.- 10. Methods for Deglycosylation of Glycoproteins.- 11. Antibody Preparation, Detection, and Characterization Techniques.- 12. Electrophoretic Transfer of Proteins to Nitrocellulose: Immunoblotting and Lectin Blotting.- 13. Electroelution of Proteins from Polyacrylamide Gels.- 14. Photography of Polyacrylamide Gels and Development of 35-mm Film.- 15. Direct Color Contact Printing from Polyacrylamide Gels.- 16. Addresses of Major Equipment Suppliers.

ISBN: 9780306428395
ISBN-10: 0306428393
Audience: General
Format: Paperback
Language: English
Number Of Pages: 383
Published: January 1987
Country of Publication: US
Dimensions (cm): 22.86 x 15.24  x 2.06
Weight (kg): 0.52
Edition Type: New edition

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