Two-Dimensional Gel Electrophoresis of Proteins

Two-Dimensional Gel Electrophoresis of Proteins: Methods and Applications reviews current methods and clinical applications of two-dimensional gel electrophoresis of proteins, including the QUEST system, silver staining, and peptide mapping. Two-dimensional gel electrophoresis are applied to the study of diseases ranging from inborn errors of metabolism to human germ-line mutation rates, cancer, and mistranslation in animal and bacterial cells.

This volume is organized into three sections encompassing 14 chapters and begins with an overview of the methodology of two-dimensional gel electrophoresis, followed by a discussion of computerized two-dimensional gel electrophoresis, silver staining, immunoblotting, and one- and two-dimensional peptide mapping. In most cases, a step-by-step guide to the techniques is given so that procedures may be easily repeated. A catalog of mouse fibroblast proteins is also given. The chapters that follow focus mainly on applications of two-dimensional gel electrophoresis in areas such as clinical and cancer research, human genetics, protein biosynthesis, and gene expression in plants. The final section presents current protein catalogs of Escherichia coli and human HeLa cells.
This book is suitable for young researchers as well as for senior scientists working with a wide variety of problems in molecular and cell biology, basic biochemistry, genetics, and clinical research.

Contenu

Contributors
Preface
Part I. Methods and Satellite Techniques

1. Two-Dimensional Gel Electrophoresis: A Guide for the Beginner
   I. Introduction
   II. Materials and Solutions
   III. Experimental Procedures
   IV. Hints and Comments
   V. Concluding Remarks
   References
2. The QUEST System for Computer-Analyzed Two-Dimensional Electrophoresis of Proteins
   I. Introduction
   II. Problems to Be Faced in Development of Computerized Protein Data Bases
   III. Strategy for the QUEST System
   IV. The QUEST System As Seen by the User
   V. Program Descriptions
   VI. Perspectives
   References
3. Detection of Polypeptides in Two-Dimensional Gels Using Silver Staining
   I. Introduction
   II. Staining Procedures
   III. Mechanisms of Silver Stains
   IV. Potential and Real Problems in Silver Staining
   References
4. Applicability of Color Silver Stain (GELCODE® System) to Protein Mapping with Two-Dimensional Gel Electrophoresis
   I. Introduction
   II. Variables that Affect Sensitivity and Reproducibility of the Color Silver Stain (GELCODE System)
   III. Application of Color Silver Staining (GELCODE System) to Two-Dimensional Mapping of Proteins
   IV. Computerized Data Acquisition of Color-Stained Images: Test for Stoichiometric Staining
   V. Concluding Remarks
   References
5. Electrophoretic Transfer of Proteins from Two-Dimensional Gels to Sheets and Their Detection
   I. Introduction
   II. Electrophoretic Transfer of Proteins
   III. Probing the Transfer
   IV. Removal of Probes
   V. Examples and Applications of Protein Transfer Analysis
   VI. Concluding Remarks
   References
6. Correlation between Mouse and Human Two-Dimensional Gel Patterns: Peptide Mapping of Proteins Extracted from Two-Dimensional Gels
   I. Introduction
   II. Methods
   III. Correlation between the Mouse and Human Two-Dimensional Gel Patterns
   IV. Concluding Remarks
   References
   Part II. Applications of Two-Dimensional Gel Electrophoresis
7. Clinical Applications of Two-Dimensional Gel Electrophoresis
   I. Introduction
   II. The Electrophoresis System
   III. Serum and Plasma
   IV. Urine
   V. Blood Cells
   VI. Solid Tissue
   VII. Other Specimens
   VIII. Conclusions
   References
8. Two-Dimensional Gel Electrophoresis for Studies of Inborn Errors of Metabolism
   I. Introduction
   II. Detection of Protein Polymorphism
   III. Protein Mutation by Two-Dimensional Gel Electrophoresis
   IV. Characteristic Patterns of Polypeptide Modulation
   V. Conclusion
   References
9. Adapting Two-Dimensional Gel Electrophoresis to the Study of Human Germ-Line Mutation Rates
   I. Introduction
   II. Some Basic Facts Concerning Mutation
   III. Why Attempt to Use 2-D Gels to Study Mutation
   IV. Some General Considerations in the Design of a Program to Detect Mutations
   V. Selection of Most Appropriate Material for Study
   VI. Technical Considerations
   VII. A Statistical Analysis of Factors Influencing Spot Location
   VIII. Ability of the System to Detect Genetic Variants
   IX. Nomenclature
   X. Gel Analysis
   XI. Steps Necessary to Identify a Putative Mutant
   XII. Data Management
   XIII. The Selection of Populations for Study
   XIV. Concluding Comments
   References
10. Expression of Cellular Proteins in Normal and Transformed Human Cultured Cells and Tumors: Two-Dimensional Gel Electrophoresis as a Tool to Study Neoplastic Transformation and Cancer
    I. Introduction
    II. Transformation-Sensitive Polypeptides in Cultured Epithelial and Fibroblast Human Cells: Work in This Laboratory
    III. Transformation-Sensitive Polypeptides of Known Identity
    IV. Variation in the Expression of Mutant Actins in Chemically Transformed Human Diploid Fibroblasts: Actin in General
    V. Phosphoproteins
    VI. Polypeptide Synthesis in Human Tumors and Normal Tissue
    VII. Conclusions
    References
11. Application of Two-Dimensional Polyacrylamide Gel Electrophoresis to Studies of Mistranslation in Animal and Bacterial Cells
    I. Introduction
    II. Detection of Error-Containing Proteins by Two-Dimensional Polyacrylamide Gel Electrophoresis
    III. Quantification of the Error Frequency of Protein Synthesis
    IV. Assessment of the Error Frequencies of Protein Synthesis under Varying Physiological Conditions
    V. Other Uses of the Two-Dimensional Gel Assay
    VI. Two Out of Three Reading
    References
12. Application of Two-Dimensional Gel Electrophoresis in Studies of Gene Expression during Early Plant Development
    I. Introduction
    II. Gene Expression during Early Embryogenesis
    III. Somatic Embryogenesis
    IV. Gene Expression during Somatic Embryogenesis
    V. Coordinate Regulation of Sets of Biochemical Phenotypes
    VI. Gene Expression in Temperature-Sensitive Cell Lines Impaired Embryogeny
    VII. Gene Expression during Late Embryogeny
    VIII. Gene Expression in Cotton Cotyledons
    IX. Gene Expression in Germinating Seeds
    X. Conclusion
    References
    Part III. Protein Catalogs
13. The Protein Catalog of Escherichia coli
    I. Introduction
    II. Identification of Proteins
    III. Current E. coli Catalog
    IV. Applications in E. coli Research
    References
14. Catalog of HeLa Cell Proteins
    I. Introduction
    II. Two-Dimensional Gel Electrophoretic Separation of HeLa Cell Proteins
    III. Some Characteristics of HeLa Cell Proteins
    IV. Transformation-Sensitive Polypeptides
    V. Conclusions
    References
    Index