Focus on Structural Biology

By: C. Dennison

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Hardcover | 30 April 2003 | Edition Number 2

At a Glance

Hardcover
272 Pages

Edition Type
Revised

Dimensions(cm)
24.13 x 16.51 x 1.27

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This text is aimed at a student readership but will also be useful to life science researchers faced with the task of isolating a protein. The logic of the overall approach to protein isolation is explained and the physical principles of each separation method are made clear by the use of simple models and analogies drawn from everyday experiences. The author's aim has been to deepen the readers' insight into protein isolation methods, so that they may tackle new problems and perhaps devise new approaches to old problems. many of the methods described are drawn from the author's own research and are thus uniquely described here; examples are three-phase partitioning, non-linear electrophoresis and a simple approach to buffer making. In this second edition, the treatment of the basic physical principles has been expanded and clarified, the importance of ionic strength in measuring enzyme pH optima is emphasised and a computer program for the calculation of buffers of defined ionic strength is provided. The section on three-phase partitioning has been expanded to include the latest research findings on the use of t-butanol to inhibit enzymes and minimise homogenisation artefacts, the treatment of HPLC has been expanded and the most common practical methods are explained in detail in a new chapter. Additional study questions are provided, as are the answers to all study questions.

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Acknowledgements	p. XI
Preface	p. XIII
Preface to the 2nd edition	p. XV
Basic physical concepts applicable to the isolation of proteins	p. 1
Chapter 1 study questions	p. 11
An overview of protein isolation	p. 13
Why do it?	p. 13
Properties of proteins that influence the methods used in their study	p. 14
The conceptual basis of protein isolation	p. 15
Where to start?	p. 16
When to stop?	p. 17
The purification table	p. 18
Chapter 2 study questions	p. 19
Assay, extraction and subcellular fractionation	p. 20
Buffers	p. 20
Making a buffer	p. 23
Buffers of constant ionic strength	p. 26
Calculating the ionic strength of a buffer	p. 28
Assays for activity	p. 30
Enzyme assays	p. 30
The progress curve	p. 30
The enzyme dilution curve	p. 32
The substrate dilution curve	p. 32
The effect of pH on enzyme activity	p. 33
The effect of temperature on enzyme activity	p. 36
Assay for protein content	p. 36
Absorption of ultraviolet light	p. 37
The biuret assay	p. 38
The Lowry assay	p. 38
The bicinchoninic acid assay	p. 39
The Bradford assay	p. 39
Methods for extraction of proteins	p. 39
Osmotic shock	p. 40
Pestle homogenisers	p. 42
The Waring blendor and Virtis homogeniser	p. 43
The Polytron/Ultra-Turrax-type homogeniser	p. 43
Grinding	p. 44
The Parr bomb	p. 44
Extrusion under high pressure	p. 44
Sonication	p. 45
Enzymic digestion	p. 46
Clarification of the extract	p. 46
Centrifugal subcellular fractionation	p. 46
The centrifuge	p. 46
Principles of centrifugation	p. 47
Sub-cellular fractionation	p. 51
Density gradient centrifugation	p. 53
Chapter 3 study questions	p. 57
Concentration of the extract	p. 58
Freeze drying	p. 58
Theoretical and practical considerations in freeze-drying	p. 59
Some tips on vacuum	p. 63
Dialysis	p. 65
The Donnan membrane effect	p. 67
Counter-current dialysis	p. 68
Concentration by dialysis (concentrative dialysis)	p. 69
Perevaporation	p. 69
Ultrafiltration	p. 70
Desalting or buffer exchange by ultrafiltration	p. 73
Size fractionation by ultrafiltration	p. 73
Concentration/fractionation by salting out	p. 74
Why ammonium sulfate?	p. 74
Empirical observations on protein salting out	p. 77
Three-phase partitioning (TPP)	p. 81
Homogenisation in 30% t-butanol	p. 84
Fractional precipitation with polyethylene glycol	p. 84
Precipitation with organic solvents	p. 85
Dye precipitation	p. 86
Chapter 4 study questions	p. 88
Chromatography	p. 89
Principles of chromatography	p. 89
The effect of particle size	p. 95
The effect of the mobile phase flow rate	p. 97
Linear and volumetric flow rates	p. 98
Equipment required for low pressure liquid chromatography	p. 99
The column	p. 99
Moving the mobile phase	p. 101
Monitoring the effluent and collecting fractions	p. 104
Refrigeration	p. 105
Ion-exchange chromatography (IEC)	p. 106
Ion-exhcange "resins"	p. 108
Gradient generators	p. 111
Choosing the pH	p. 113
An ion-exchange chromatography run	p. 114
Chromatofocusing	p. 116
Molecular exclusion chromatography (MEC)	p. 116
The effect of gel sphere size on V[subscript o]	p. 119
The manufacture of small, uniform, gel spheres	p. 121
Determination of MW by MEC	p. 121
Gels used in MEC	p. 123
An MEC run	p. 127
Hydroxyapatite chromatography	p. 128
The mechanism of hydroxyapatite chromatography	p. 128
Affinity chromatography	p. 129
Hydrophobic interaction (HI) chromatography	p. 130
HPLC	p. 131
Concepts and terms relevant to HPLC	p. 132
Stationary phase materials	p. 133
Solvent systems	p. 134
Preparative HPLC	p. 135
Chapter 5 study questions	p. 137
Electrophoresis	p. 139
Principles of electrophoresis	p. 139
The effect of the buffer	p. 144
Boundary (Tiselius) electrophoresis	p. 147
Paper electrophoresis	p. 148
Electroendosmosis	p. 150
Cellulose acetate membrane electrophoresis (CAM-E)	p. 151
Agarose gel electrophoresis	p. 152
Starch gel electrophoresis	p. 153
Polyacrylamide gel electrophoresis (PAGE)	p. 155
Disc electrophoresis	p. 155
Isotachophoresis	p. 158
SDS-PAGE	p. 160
An SDS-PAGE zymogram for proteinases	p. 161
Pore gradient gel electrophoresis	p. 162
Isoelectric focusing	p. 162
Establishing a pH gradient	p. 164
Control of buoyancy-driven fluid flow	p. 167
Applying the sample and measuring the pH gradient	p. 167
An analytical IEF system	p. 167
Preparative IEF	p. 169
2-D Electrophoresis	p. 170
Non-linear electrophoresis	p. 170
Chapter 6 study questions	p. 175
Immunological methods	p. 178
The structure of antibodies	p. 178
Antibody production	p. 179
Making an antiserum	p. 182
Monoclonal antibodies	p. 184
Immunoprecipitation	p. 184
Immuno single diffusion	p. 187
Immuno double diffusion	p. 188
Determination of diffusion coefficients	p. 189
Immunoprecipitation methods of historical interest	p. 191
Mancini radial diffusion	p. 191
Ouchterlony double diffusion analysis	p. 192
Immunoelectrophoresis	p. 194
Cross-over electrophoresis	p. 194
Rocket electrophoresis	p. 194
Grabar-Williams immunoelectrophoresis	p. 195
Clarke-Freeman 2-D immunoelectrophoresis	p. 196
Amplification methods	p. 197
Complement fixation	p. 197
Radioimmunoassay (RIA)	p. 199
Enzyme amplification	p. 201
Enzyme linked immunosorbent assay (ELISA)	p. 201
Immunoblotting	p. 202
Immunogold labeling with silver amplification	p. 205
Colloid agglutination	p. 205
Chapter 7 study questions	p. 209
Some common practical methods	p. 210
The Bradford dye-binding assay	p. 210
Reagents	p. 210
Procedure	p. 210
Methods for concentrating protein solutions	p. 211
Dialysis against sucrose or PEG	p. 211
SDS/KCl precipitation	p. 211
Reagents	p. 211
Procedure	p. 211
SDS-PAGE	p. 212
Tris-glycine SDS-PAGE	p. 212
Reagents	p. 212
Procedure	p. 214
Tris-tricine SDS-PAGE	p. 215
Reagents	p. 215
Procedure	p. 216
Serva blue G rapid stain	p. 216
Reagents	p. 217
Procedure	p. 217
Silver staining of electrophoretic gels	p. 217
Reagents	p. 217
Procedure	p. 218
Protease zymography	p. 218
Reagents	p. 219
Procedure	p. 219
Western blotting	p. 219
Reagents	p. 220
Procedure	p. 221
Fractionation of IgG and IgY	p. 223
Reagents	p. 223
Isolation of IgG from rabbit serum	p. 223
Isolation of IgY from chicken egg yolk	p. 224
Enzyme-linked immunosorbent assay (ELISA)	p. 224
Reagents	p. 225
Procedure	p. 226
Answers to study questions	p. 228
Chapter 1	p. 228
Chapter 2	p. 229
Chapter 3	p. 230
Chapter 4	p. 231
Chapter 5	p. 233
Chapter 6	p. 238
Chapter 7	p. 241
Further sources of information	p. 243
Index	p. 245
Table of Contents provided by Rittenhouse. All Rights Reserved.

Focus on Structural Biology

At a Glance

Hardcover

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