Electrophoresis Fundamentals: Essential Theory and Practice

Description

The electrophoresis techniques are used in medicine, biochemistry, analytical chemistry, and biology to separate soluble and insoluble proteins, nucleic acids, chromosomes, viruses, as well as lysosomes, mitochondria, ribosomes and other cell organelles, red cells, tissue cells, and parasites. This book provides a view over the old electrophoresis techniques, as well as the recent developments in electrophoresis. Electrophoresis Fundamentals is based on the recent book Electrophoresis: Theory and Practice published in 2020 by De Gruyter. The previous book combines theory and technical applications with troubleshooting and problem solving. While Electrophoresis is intended for specialists, Electrophoresis Fundamentals is a book for laboratory technicians, students, biochemists, general practitioners, and more. Discusses modern electrophoresis methods such as the PCR technique, which is used to detect COVID-19. Contains troubleshooting and problem solving. Intended for students, chemists, biochemists, geneticists, physicians, and more. Cover Half Title Also of Interest Electrophoresis Fundamentals: Essential Theory and Practice Copyright Preface About the Author Contents Abbreviations 1. Fundamentals of electrophoresis Overview on electrophoresis Separation media Electrophoresis resolution and sharpness Detection of resolved bands References 1.1 Electric double layer of a charged particle 1.1.1 Electric charges of polyions 1.1.2 Model of Helmholtz 1.1.3 Model of Gouy–Chapman 1.1.4 Theory of Debye–Hückel 1.1.5 Model of Stern 1.1.6 Two radii and two electric potentials of a charged particle 1.2 Proteins and nucleic acids form polyions in solution 1.2.1 Structure and conformation of proteins 1.2.2 Structure and conformation of nucleic acids 1.3 Electrophoresis is running in buffers 1.3.1 Buffers 1.3.2 Buffers used in electrophoresis 1.3.3 Biological buffers References 1.4 Polyions are moving in electric field 1.4.1 Ionic and polyionic mobility 1.4.2 Equations of polyionic mobility 1.4.3 Mobilities of ions used in electrophoretic methods References 1.5 Electrophoresis is carried out in different solid media 1.5.1 Cellulose acetate 1.5.2 Agarose gel 1.5.3 Polyacrylamide gel References 1.6 General theory of electrophoresis 1.6.1 What is the polyionic mobility depending on? 1.6.2 Ionic boundaries 1.6.3 Regulating function 1.6.4 Diffusion 1.6.5 Joule heating 1.7 Electrophoresis instrumentation 1.7.1 Electrophoresis cells 1.7.2 Power supplies 1.7.3 Thermostats 1.7.4 Scanners and densitometers 1.7.5 Gel casting cassettes 1.7.6 Gradient makers 1.7.7 Buffer mixers 1.7.8 Blotters 1.7.9 Equipment for semi-automatic electrophoresis 1.8 Classification of electrophoretic methods 1.8.1 Zone electrophoresis 1.8.2 Isotachophoresis 1.8.3 Isoelectric focusing 1.8.4 Dielectrophoresis References 2. Electrophoresis of proteins References 2.1 Cellulose acetate electrophoresis of proteins 2.1.1 Theory of cellulose acetate electrophoresis of proteins 2.1.2 Practice of cellulose acetate electrophoresis of proteins References 2.2 Agarose gel electrophoresis of proteins 2.2.1 Theory of agarose gel electrophoresis of proteins 2.2.2 Agarose gel electrophoresis of serum proteins 2.2.3 Agarose gel electrophoresis of lipoproteins 2.2.4 Agarose gel electrophoresis of hemoglobins 2.2.5 Agarose gel electrophoresis of cerebrospinal fluid proteins 2.2.6 Electrophoresis of creatine kinase isoenzymes 2.2.7 Electrophoresis of lactate dehydrogenase isoenzymes 2.2.8 Agarose gel electrophoresis of urinary proteins 2.3 Immunoelectrophoresis 2.3.1 Immunodiffusion electrophoresis according to Grabar and Williams 2.3.2 Rocket immunoelectrophoresis according to Laurell 2.3.3 Immunofixation and immunoprinting References 2.4 Affinity electrophoresis 2.4.1 Theory of affinity electrophoresis 2.4.2 Lectin affinity electrophoresis 2.4.3 Saccharide affinity electrophoresis 2.4.4 Affinity supported molecular matrix electrophoresis 2.4.5 Phosphate affinity electrophoresis 2.4.6 Capillary affinity electrophoresis 2.4.7 Affinity-trap electrophoresis 2.4.8 Charge shift electrophoresis 2.4.9 Mobility shift electrophoresis 2.4.10 Protocols References 2.5 Polyacrylamide gel zone electrophoresis of proteins 2.5.1 Homogeneous gel zone electrophoresis of proteins 2.5.2 Gradient gel zone electrophoresis of proteins 2.5.3 Blue native polyacrylamide gel electrophoresis 2.6 Isotachophoresis of proteins 2.6.1 Theory of isotachophoresis of proteins References 2.7 Disc-electrophoresis of proteins 2.7.1 Theory of disc-electrophoresis 2.7.2 Native disc-electrophoresis 2.7.3 Denatured SDS disc-electrophoresis 2.7.4 Protocols 2.8 Isoelectric focusing of proteins 2.8.1 Theory of isoelectric focusing 2.8.2 Isoelectric focusing with carrier ampholytes 2.8.3 Isoelectric focusing in immobilized pH gradients 2.8.4 Isoelectric focusing in the clinical laboratory 2.8.5 Protocols References 2.9 Free-flow electrophoresis of proteins 2.9.1 Theory of free-flow electrophoresis 2.9.2 Types of free-flow electrophoresis 2.9.3 Device technology of free-flow electrophoresis 2.9.4 Detection system of free-flow electrophoresis 2.9.5 Applications of free-flow electrophoresis References 2.10 Capillary electrophoresis of proteins 2.10.1 Theory of capillary electrophoresis of proteins 2.10.2 Instrumentation 2.10.3 Practice of capillary electrophoresis 2.10.4 Types of capillary electrophoresis 2.10.5 Applications of capillary electrophoresis 2.10.6 Protocols 2.11 Two-dimensional electrophoresis 2.11.1 Theory of 2D electrophoresis 2.11.2 Isoelectric focusing in the first dimension 2.11.3 SDS disc-electrophoresis in the second dimension 2.11.4 Detection and evaluation of proteins in 2D pherograms 2.11.5 Protocols References 2.12 Preparative electrophoresis of proteins 2.12.1 Preparative disc-electrophoresis 2.12.2 Preparative isoelectric focusing 2.12.3 QPNC-PAGE 2.12.4 Protocols 2.13 Microchip electrophoresis of proteins 2.13.1 Microchip materials 2.13.2 Microchip fabrication 2.13.3 Zone electrophoresis on microchip 2.13.4 Isotachophoresis on microchip 2.13.5 Isoelectric focusing on microchip 2.13.6 Two-dimensional electrophoresis on microchip 2.13.7 Protein separation technique on microchip 2.13.8 Microchips in clinical diagnostics References 2.14 Blotting of proteins 2.14.1 Theory of protein blotting 2.14.2 Blot membranes 2.14.3 Transfer of proteins 2.14.4 Blocking 2.14.5 Detection 2.14.6 Making the blot membranes transparent 2.14.7 Blotting techniques 2.15 Evaluation of protein pherograms 2.15.1 Qualitative evaluation of protein pherograms 2.15.2 Quantitative evaluation of a protein pherogram 2.15.3 Protocols References 2.16 Precast gels for protein electrophoresis. Rehydratable gels 2.16.1 Precast agarose gels 2.16.2 Precast polyacrylamide gels 2.16.3 Rehydratable polyacrylamide gels 2.16.4 Protocols 3. Electrophoresis of nucleic acids Buffers for electrophoresis of nucleic acids Polymerase chain reaction Surface electrophoresis References 3.1 Agarose gel electrophoresis of nucleic acids 3.1.1 Zone polyacrylamide gel electrophoresis of native nucleic acids 3.1.2 Zone agarose gel electrophoresis of denatured nucleic acids 3.1.3 DNA sequencing 3.1.4 RNA separation 3.1.5 Protocols 3.2 Pulsed-field gel electrophoresis of nucleic acids 3.2.1 Theory of pulsed-field gel electrophoresis of nucleic acids 3.2.2 Types of pulsed-field gel electrophoresis 3.2.3 Mapping the human genome 3.2.4 Protocols 3.3 Capillary electrophoresis of nucleic acids 3.3.1 Theory of capillary electrophoresis of nucleic acids 3.3.2 Instrumentation for capillary electrophoresis 3.3.3 Running capillary electrophoresis 3.3.4 Pulsed-field capillary electrophoresis 3.3.5 Applications of capillary electrophoresis 3.3.6 Protocols References 3.4 Polyacrylamide gel electrophoresis of nucleic acids 3.4.1 Zone polyacrylamide gel electrophoresis of native nucleic acids 3.4.2 Disc-electrophoresis of native nucleic acids 3.4.3 Electrophoretic mobility shift assay 3.4.4 Clinical applications 3.4.5 Protocols References 3.5 Microchip electrophoresis of nucleic acids 3.5.1 Theory of microchip electrophoresis of nucleic acids 3.5.2 Construction of a microchip for electrophoresis of nucleic acids 3.5.3 Running microchip electrophoresis of nucleic acids 3.5.4 Applications of DNA microchip electrophoresis References 3.6 Blotting of nucleic acids 3.6.1 Blotting principles 3.6.2 Southern blotting 3.6.3 Northern blotting 3.6.4 Middle-Eastern blotting References 3.7 Evaluation of nucleic acid pherograms 3.7.1 Counter-ion dye staining of nucleic acids 3.7.2 Silver staining of nucleic acids 3.7.3 Fluorescence methods for detecting nucleic acids 3.7.4 Autoradiography of nucleic acids 3.7.5 Labeling of nucleic acids with proteins 3.7.6 Absorption spectroscopy of nucleic acids 3.7.7 Protocols 3.8 Precast gels for nucleic acid electrophoresis 3.8.1 Precast agarose gels 3.8.2 Precast polyacrylamide gels 4. Iontophoresis 4.1 Theory of iontophoresis 4.2 Factors affecting iontophoresis 4.3 Calculating the iontophoretic current 4.4 Iontophoresis device 4.5 Diagnostic iontophoresis 4.6 Therapeutic iontophoresis 5. History of electrophoresis and iontophoresis 5.1 History of electrophoresis 5.2 History of iontophoresis 6. Troubleshooting 6.1 Protein electrophoresis troubleshooting 6.2 IEF troubleshooting 6.3 Nucleic acid electrophoresis troubleshooting 6.4 Blotting troubleshooting 6.5 Iontophoresis troubleshooting Problems 1. Fundamentals of electrophoresis 2. Electrophoresis of proteins 3. Electrophoresis of nucleic acids Solution of problems 1. Fundamentals of electrophoresis 2. Electrophoresis of proteins 3. Electrophoresis of nucleic acids Reagents for electrophoresis Recipes for electrophoresis solutions Buffers Solutions for agarose gel electrophoresis Solutions for affinity electrophoresis Solutions for native disc-electrophoresis Solutions for SDS disc-electrophoresis Solutions for IEF Blotting solutions Fixative solutions Staining solutions Destaining solutions Silver staining solutions Other solutions SI units and physical constants used in electrophoresis Electrophoresis terms Index