Contents	5
	Acknowledgements	9
	Preface	11
	Chapter 1 An overview of protein isolation	13
	1.1 Why do it?	13
	1.2 Properties of proteins that influence the methods used in their study	14
	1.3 The conceptual basis of protein isolation	15
	1.3.1 Where to start?	16
	1.3.2 When to stop?	17
	1.4 The purification table	18
	1.5 Chapter 1 study questions	19
	Chapter 2 Assay, extraction and subcellular fractionation	20
	2.1 Buffers	20
	2.1.1 Making a buffer	23
	2.1.2 Buffers of constant ionic strength	25
	2.2 Assays for activity	27
	2.2.1 Enzyme assays	28
	2.2.1.1 The progress curve	28
	2.2.1.2 The enzyme dilution curve	29
	2.2.1.3 The substrate dilution curve	30
	2.2.1.4 The effect of pH on enzyme activity	31
	2.2.1.5 The effect of temperature on enzyme activity	33
	2.3 Assay for protein content	34
	2.3.1 Absorption of ultraviolet light	34
	2.3.2 The biuret assay	35
	2.3.3 The Lowry assay	35
	2.3.4 The bicinchoninic acid assay	36
	2.3.5 The Bradford assay	36
	2.4 Methods for extraction of proteins	36
	2.4.1 Osmotic shock	37
	2.4.2 Pestle homogenisers	38
	2.4.3 The Waring blendor and Virtis homogeniser	39
	2.4.4 The Polytron/UItra-Turrax-type homogeniser	40
	2.4.5 Grinding	40
	2.4.6 The Parr bomb	41
	2.4.7 Extrusion under high pressure	41
	2.4.8 Sonication	42
	2.4.9 Enzymic digestion	42
	2.5 Clarification of the extract	43
	2.6 Centrifugal sub-cellular fractionation	43
	2.6.1 Density gradient centrifugation	48
	2.7 Chapter 2 study questions	52
	Chapter 3 Concentration of the extract	53
	3.1 Freeze drying	53
	3.1.1 Theoretical and practical considerations in freeze-drying	54
	3.1.2 Some tips on vacuum	58
	3.2 Dialysis	60
	3.2.1 The Donnan membrane effect	62
	3.2.2 Counter-current dialysis	63
	3.2.3 Concentration by dialysis (concentrative dialysis)	64
	3.2.4 Perevaporation	64
	3.3 Ultrafiltration	65
	3.3.1 Desalting or buffer exchange by ultrafiltration	68
	3.3.2 Size fractionation by ultrafiltration	68
	3.4 Concentration/fractionation by salting out	69
	3.4.1 Why ammonium sulfate?	69
	3.4.2 Empirical observations on protein salting out.	72
	3.4.3 Three-phase partitioning (TPP)	76
	3.5 Fractional precipitation with polyethylene glycol	79
	3.6 Precipitation with organic solvents	79
	3.7 Dye precipitation	80
	3.8 Chapter 3 study questions	82
	Chapter 4 Chromatography	83
	4.1 Principles of chromatography	83
	4.1.1The effect of particle size	88
	4.1.2 The effect of the mobile phase flow rate	90
	4.1.2.1 The relationship between linear and volumetric flow rates.	91
	4.2 Equipment required for low pressure liquid chromatography	92
	4.2.1 The column	92
	4.2.2 Moving the mobile phase	94
	4.2.3 Monitoring the effluent and collecting fractions.	97
	4.2.4 Refrigeration	98
	4.3 Ion-exchange chromatography (IEC)	99
	4.3.1 Ion-exchange “resins”	101
	4.3.2 Gradient generators	104
	4.3.3 Choosing the pH	106
	4.3.4 An ion-exchange chromatography run	107
	4.4 Chromatofocusing	109
	4.5 Molecular exclusion chromatography (MEC)	109
	4.5.1 The effect of gel sphere size on Vo	112
	4.5.2 The manufacture of small, uniform, gel spheres	114
	4.5.3 Determination of MW by MEC	114
	4.5.4 Gels used in MEC	116
	4.5.5 An MEC run	120
	4.6 Hydroxyapatite chromatography	120
	4.6.1 The mechanism of hydroxyapatite chromatography	121
	4.7 Affinity chromatography	122
	4.8 Hydrophobic interaction (HI) chromatography	123
	4.9 Chapter 4 study questions	124
	Chapter 5 Principles of Electrophoresis	127
	5.1 Principles of electrophoresis	127
	5.1.1 The effect of the buffer	131
	5.2 Boundary (Tiselius) electrophoresis	134
	5.3 Paper electrophoresis	135
	5.3.1 Electroendosmosis	136
	5.4 Cellulose acetate membrane electrophoresis (CAM-E)	137
	5.5 Agarose gel electrophoresis	138
	5.6 Starch gel electrophoresis	139
	5.7 Polyacrylamide gel electrophoresis (PAGE)	141
	5.7.1 Disc electrophoresis	141
	5.7.1.1 Isotachophoresis	144
	5.8 SDS-PAGE	145
	5.8.1 An SDS-PAGE zymogram for proteinases	147
	5.9 Pore gradient gel electrophoresis	147
	5.10 Isoelectric focusing	148
	5.10.1 Establishing a pH gradient	149
	5.10.2 Control of convection	152
	5.10.3 Applying the sample and measuring the pH gradient	152
	5.10.3.1 An analytical IEF system	152
	5.10.3.2 Preparative IEF	154
	5.11 2-D Electrophoresis	155
	5.12 Non-linear electrophoresis	155
	5.13 Chapter 5 study questions	160
	Chapter 6 Immunological methods	162
	6.1 The structure of antibodies	162
	6.2 Antibody production	163
	6.2.1 Making an antiserum	166
	6.3 Immunoprecipitation	168
	6.3.1 Immuno single diffusion	170
	6.3.1.1 Mancini radial diffusion	171
	6.3.2 Immuno double diffusion	172
	6.3.2.1 Ouchterlony double diffusion analysis	173
	6.3.2.2 Determination of diffusion coefficients	174
	6.4 Immunoelectrophoresis	176
	6.4.1 Cross-over electrophotesis	176
	6.4.2 Rocket electrophoresis	177
	6.4.3 Grabar-Williams immunoelectrophoresis	177
	6.4.4 Clarke-Freeman 2-D immunoelectrophoresis	178
	6.5 Amplification methods	180
	6.5.1 Complement fixation	180
	6.5.2 Radioimmunoassay (RIA)	182
	6.5.3 Enzyme amplification	183
	6.5.3.1 Enzyme linked immunosorbent assay (ELISA)	183
	6.5.3.2 Immunoblotting	185
	6.5.4 Immunogold labeling with silver amplification	187
	6.5.5 Colloid agglutination	188
	6.6 Chapter 6 study questions	191
	Index	194
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