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| Acknowledgements | |
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| Preface | |
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| Preface to the 2nd edition | |
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| Basic physical concepts applicable to the isolation of proteins | |
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| Chapter 1 study questions | |
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| An overview of protein isolation | |
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| Why do it? | |
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| Properties of proteins that influence the methods used in their study | |
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| The conceptual basis of protein isolation | |
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| Where to start? | |
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| When to stop? | |
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| The purification table | |
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| Chapter 2 study questions | |
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| Assay, extraction and subcellular fractionation | |
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| Buffers | |
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| Making a buffer | |
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| Buffers of constant ionic strength | |
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| Calculating the ionic strength of a buffer | |
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| Assays for activity | |
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| Enzyme assays | |
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| The progress curve | |
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| The enzyme dilution curve | |
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| The substrate dilution curve | |
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| The effect of pH on enzyme activity | |
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| The effect of temperature on enzyme activity | |
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| Assay for protein content | |
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| Absorption of ultraviolet light | |
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| The biuret assay | |
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| The Lowry assay | |
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| The bicinchoninic acid assay | |
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| The Bradford assay | |
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| Methods for extraction of proteins | |
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| Osmotic shock | |
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| Pestle homogenisers | |
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| The Waring blendor and Virtis homogeniser | |
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| The Polytron/Ultra-Turrax-type homogeniser | |
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| Grinding | |
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| The Parr bomb | |
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| Extrusion under high pressure | |
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| Sonication | |
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| Enzymic digestion | |
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| Clarification of the extract | |
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| Centrifugal subcellular fractionation | |
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| The centrifuge | |
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| Principles of centrifugation | |
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| Sub-cellular fractionation | |
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| Density gradient centrifugation | |
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| Chapter 3 study questions | |
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| Concentration of the extract | |
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| Freeze drying | |
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| Theoretical and practical considerations in freeze-drying | |
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| Some tips on vacuum | |
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| Dialysis | |
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| The Donnan membrane effect | |
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| Counter-current dialysis | |
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| Concentration by dialysis (concentrative dialysis) | |
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| Perevaporation | |
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| Ultrafiltration | |
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| Desalting or buffer exchange by ultrafiltration | |
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| Size fractionation by ultrafiltration | |
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| Concentration/fractionation by salting out | |
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| Why ammonium sulfate? | |
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| Empirical observations on protein salting out | |
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| Three-phase partitioning (TPP) | |
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| Homogenisation in 30% t-butanol | |
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| Fractional precipitation with polyethylene glycol | |
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| Precipitation with organic solvents | |
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| Dye precipitation | |
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| Chapter 4 study questions | |
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| Chromatography | |
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| Principles of chromatography | |
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| The effect of particle size | |
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| The effect of the mobile phase flow rate | |
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| Linear and volumetric flow rates | |
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| Equipment required for low pressure liquid chromatography | |
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| The column | |
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| Moving the mobile phase | |
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| Monitoring the effluent and collecting fractions | |
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| Refrigeration | |
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| Ion-exchange chromatography (IEC) | |
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| Ion-exhcange "resins" | |
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| Gradient generators | |
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| Choosing the pH | |
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| An ion-exchange chromatography run | |
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| Chromatofocusing | |
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| Molecular exclusion chromatography (MEC) | |
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| The effect of gel sphere size on V[subscript o] | |
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| The manufacture of small, uniform, gel spheres | |
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| Determination of MW by MEC | |
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| Gels used in MEC | |
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| An MEC run | |
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| Hydroxyapatite chromatography | |
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| The mechanism of hydroxyapatite chromatography | |
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| Affinity chromatography | |
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| Hydrophobic interaction (HI) chromatography | |
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| HPLC | |
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| Concepts and terms relevant to HPLC | |
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| Stationary phase materials | |
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| Solvent systems | |
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| Preparative HPLC | |
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| Chapter 5 study questions | |
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| Electrophoresis | |
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| Principles of electrophoresis | |
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| The effect of the buffer | |
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| Boundary (Tiselius) electrophoresis | |
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| Paper electrophoresis | |
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| Electroendosmosis | |
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| Cellulose acetate membrane electrophoresis (CAM-E) | |
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| Agarose gel electrophoresis | |
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| Starch gel electrophoresis | |
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| Polyacrylamide gel electrophoresis (PAGE) | |
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| Disc electrophoresis | |
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| Isotachophoresis | |
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| SDS-PAGE | |
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| An SDS-PAGE zymogram for proteinases | |
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| Pore gradient gel electrophoresis | |
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| Isoelectric focusing | |
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| Establishing a pH gradient | |
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| Control of buoyancy-driven fluid flow | |
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| Applying the sample and measuring the pH gradient | |
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| An analytical IEF system | |
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| Preparative IEF | |
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| 2-D Electrophoresis | |
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| Non-linear electrophoresis | |
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| Chapter 6 study questions | |
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| Immunological methods | |
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| The structure of antibodies | |
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| Antibody production | |
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| Making an antiserum | |
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| Monoclonal antibodies | |
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| Immunoprecipitation | |
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| Immuno single diffusion | |
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| Immuno double diffusion | |
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| Determination of diffusion coefficients | |
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| Immunoprecipitation methods of historical interest | |
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| Mancini radial diffusion | |
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| Ouchterlony double diffusion analysis | |
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| Immunoelectrophoresis | |
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| Cross-over electrophoresis | |
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| Rocket electrophoresis | |
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| Grabar-Williams immunoelectrophoresis | |
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| Clarke-Freeman 2-D immunoelectrophoresis | |
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| Amplification methods | |
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| Complement fixation | |
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| Radioimmunoassay (RIA) | |
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| Enzyme amplification | |
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| Enzyme linked immunosorbent assay (ELISA) | |
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| Immunoblotting | |
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| Immunogold labeling with silver amplification | |
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| Colloid agglutination | |
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| Chapter 7 study questions | |
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| Some common practical methods | |
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| The Bradford dye-binding assay | |
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| Reagents | |
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| Procedure | |
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| Methods for concentrating protein solutions | |
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| Dialysis against sucrose or PEG | |
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| SDS/KCl precipitation | |
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| Reagents | |
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| Procedure | |
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| SDS-PAGE | |
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| Tris-glycine SDS-PAGE | |
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| Reagents | |
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| Procedure | |
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| Tris-tricine SDS-PAGE | |
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| Reagents | |
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| Procedure | |
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| Serva blue G rapid stain | |
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| Reagents | |
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| Procedure | |
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| Silver staining of electrophoretic gels | |
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| Reagents | |
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| Procedure | |
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| Protease zymography | |
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| Reagents | |
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| Procedure | |
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| Western blotting | |
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| Reagents | |
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| Procedure | |
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| Fractionation of IgG and IgY | |
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| Reagents | |
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| Isolation of IgG from rabbit serum | |
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| Isolation of IgY from chicken egg yolk | |
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| Enzyme-linked immunosorbent assay (ELISA) | |
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| Reagents | |
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| Procedure | |
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| Answers to study questions | |
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| Chapter 1 | |
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| Chapter 2 | |
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| Chapter 3 | |
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| Chapter 4 | |
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| Chapter 5 | |
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| Chapter 6 | |
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| Chapter 7 | |
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| Further sources of information | |
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| Index | |