Immobilized pH Gradients: Theory and Methodology

(Chapter headings) List of abbreviations. Preface. Acknowledgements. Chapter 1. The Chemicals 1.1 Isoelectric focusing: an evolving concept 1.2 Comparison between carrier ampholyte and Immobiline chemicals 1.3 Structure and synthesis of acidic Immobiline 1.4 Structure and synthesis of basic Immobiline 1.5 Physico-chemical properties of Immobiline 1.6 Problems with the Immobiline chemicals 1.7 Storage of Immobiline: a new generation 1.8 Conclusions 2. Theory 2.1 Per aspera ad astra 2.2 Narow and ultra-narrow gradients 2.3 Extended pH gradients 2.4 How to deal with experimental errors 2.5 Ionic strength (I) 2.6 Buffering capacity (Beta) 2.7 Conductivity 2.8 Electroendomosis 2.9 The limits of IPGs: how to break barriers 2.10 Resolving power: Rilbe's equation 2.11 Alternatives for buffer focusing 2.12 Conclusions 3. Analytical IPGs 3.1 General considerations 3.2 Equipment 3.3 The polyacrylamide gel matrix 3.4 Gel preparation and electrophoresis 3.5 General experimental considerations 3.6 Blotting from Immobiline gels 3.7 Mixed-bed, CA-IPG gels 3.8 Effect of salts on protein patterns 3.9 General staining procedures 3.10 Trouble-shooting 3.11 Conclusions 4. Two-dimensional (2-D) maps 4.1 Introduction 4.2 Sample preparation 4.3 Sample solubilization 4.4 Choice of detergent 4.5 Radiolabelling techniques 4.6 The first (IPG) dimension 4.7 The second dimension 4.8 Reproducibility of spot positions in 2-D gels 4.9 Computer data acquisition 4.10 Silver staining 4.11 Blotting of spots from a 2-D map 4.12 General experimental aspects 4.13 Trouble shooting 4.14 Examples of some applications 4.15 To be or not to be? 5. Preparative aspects of Immobilized pH gradients 5.1 Introduction 5.2 Theory 5.3 Methodology 5.4 Immobiline canal technique 5.5 Reversible polyacrylamide gels 5.6 Segmented immobilized pH gradients 5.7 Alternative preparative methods 5.8 Conclusions 6. Some Applications of IPGs 6.1 Analysis of hemoglobins 6.2 Polymorphism of human proteins 6.3 Zymograms 6.4 Focusing at pH extremes 6.5 Conclusions. On protein heterogeneity.