Electrophoresis in Practice: A Guide to Methods and Applications of DNA and Protein Separations

Foreword. Preface. Abbreviations. Part I Fundamentals. Introduction. 1 Electrophoresis. 1.0 General. 1.1 Electrophoresis in non-restrictive gels. 1.2 Electrophoresis in restrictive gels. 2 Isotachophoresis. 2.1 Migration with the same speed. 2.2 "Ion train" separation. 2.3 Zone sharpening effect. 2.4 Concentration regulation effect. 3 Isoelectric focusing. 3.1 Principles. 3.2 Gels for IEF. 3.3 Temperature. 3.4 Controlling the pH gradient. 3.5 The kinds of pH gradients. 3.6 Protein detection in IEFgels. 3.7 Preparative isoelectric focusing. 3.8 Titration curve analysis. 4 Blotting. 4.1 Principle. 4.2 Transfer methods. 4.3 Blotting membranes. 4.4 Buffers for electrophoretic transfers. 4.5 General staining. 4.6 Blocking. 4.7 Specific detection. 4.8 Protein sequencing. 4.9 Transfer problems. 5 Interpretation of electropherograms. 5.1 Introduction. 5.2 Image analysis. 6 Proteome Analysis. 6.1 General. 6.2 Sample preparation. 6.3 Two-dimensional electrophoresis. 6.4 Detection techniques. 6.5 Image analysis. 6.6 Protein spot identification. 6.7 Bioinformatics. 6.8 Functional proteomics. 7 Instrumentation. 7.1 Current and voltage conditions. 7.2 Power supply. 7.3 Separation chambers. 7.4 Staining apparatus for gels and blots. 7.5 Automated electrophoresis. 7.6 Instruments for 2-D electrophoresis. 7.7 Safety measures. 7.8 Environmental aspects. Part II Equipment and Methods. Equipment for Part II. Instrumentation. Special laboratory equipment. Consumables. Chemicals. Method 1: PAGE of dyes. 1 Sample preparation. 2 Stock solutions. 3 Preparing the casting cassette. 4 Casting ultrathin-layer gels. 5 Electrophoretic separation. Method 2: Agarose and immuno electrophoresis. 1 Sample preparation. 2 Stock solutions. 3 Preparing the gels. 4 Electrophoresis. 5 Protein detection. Method 3: Titration curve analysis. 1 Sample preparation. 2 Stock solutions. 3 Preparing the blank gels. 4 Titration curve analysis. 5 Coomassie and silver staining. 6 Interpreting the curves. Method 4: Native PAGE in amphoteric buffers. 1 Sample preparation. 2 Stock solutions. 3 Preparing the empty gels. 4 Electrophoresis. 5 Coomassie and silver staining. Method 5: Agarose IEF. 1 Sample preparation. 2 Preparing the agarose gel. 3 Isoelectric focusing. 4 Protein detection. Method 6: PAGIEF in rehydrated gels. 1 Sample preparation. 2 Stock solutions. 3 Preparing the blank gels. 4 Isoelectric focusing. 5 Coomassie and silver staining. 6 Perspectives. Method 7: Horizontal SDS-PAGE. 1 Sample preparation. 2 Stock solutions for gel preparation. 3 Preparing the casting cassette. 4 Gradient gel. 5 Electrophoresis. 6 Protein detection. 7 Blotting. 8 Perspectives. Method 8: Vertical PAGE. 1 Sample preparation. 2 Stock solutions. 3 Single gel casting. 4 Multiple gel casting. 5 Electrophoresis. 6 SDS electrophoresis of small peptides. 7 Two-dimensional electrophoresis. 8 DNA electrophoresis. 9 Long shelflife gels. 10 Detection of bands. Method 9: Semi-dry blotting of proteins. 1 Transfer buffers. 2 Technical procedure. 3 Staining of blotting membranes. Method 10: IEF in immobilized pH gradients. 1 Sample preparation. 2 Stock solutions. 3 Immobiline recipes. 4 Preparing the casting cassette. 5 Preparing the pH gradient gels. 6 Isoelectric focusing. 7 Staining. 8 Strategies for IPG focusing. Method 11: High-resolution 2-D electrophoresis. 1 Sample preparation. 2 Stock solutions. 3 Preparing the gels. 4 Separation conditions. 5 Staining procedures. Method 12: PAGE of double stranded DNA. 1 Stock solutions. 2 Preparing the gels. 3 Sample preparation. 4 Electrophoresis. 5 Silver staining. Method 13: Native PAGE of single stranded DNA. 1 Sample treatment. 2 Gel properties. 3 Buffers and additives. 4 Conditions for electrophoresis. 5 Strategy for SSCP analysis. Method 14: Denaturing gradient gel electrophoresis. 1 Sample preparation. 2 Rehydration solutions. 3 Preparing the rehydration cassette. 4 Rehydration. 5 Electrophoresis. 6 Silver staining. Method 15: Denaturing PAGE of DNA. 1 Sample preparation. 2 Solutions. 3 Rehydration. 4 Electrophoresis. 5 Silver staining. Appendix Trouble-shooting. A1 Isoelectric focusing. A1.1 PAGIEFwith carrier ampholytes. A1.2 Agarose IEFwith carrier ampholytes. A1.3 Immobilized pH gradients. A2 SDS electrophoresis. A3 Vertical PAGE. A4 Semidry blotting. A5 2-D electrophoresis. A6 DNA electrophoresis. References.