Dennison A Guide to Protein Isolation

2. Auflage 2007
ISBN: 978-0-306-46868-1
Verlag: Springer Netherlands
Format: PDF
Kopierschutz: 1 - PDF Watermark

E-Book, Englisch, 198 Seiten

This text takes the reader on a guided tour through the philosophical and physical foundations of protein isolation. Aimed at a student readership, it should also be very useful to life science researchers faced with the task of isolating a protein for the first time.

The logic of the overall approach to isolating a protein 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 described here. Examples are three-phase partitioning, non-linear electrophoresis, and a simple approach to buffer making.

Autoren/Hrsg.

Dennison, C.

Dennison, Clive

Inhaltsverzeichnis

1;Contents;5
2;Acknowledgements;9
3;Preface;11
4;Chapter 1 An overview of protein isolation;13
4.1;1.1 Why do it?;13
4.2;1.2 Properties of proteins that influence the methods used in their study;14
4.3;1.3 The conceptual basis of protein isolation;15
4.3.1;1.3.1 Where to start?;16
4.3.2;1.3.2 When to stop?;17
4.4;1.4 The purification table;18
4.5;1.5 Chapter 1 study questions;19
5;Chapter 2 Assay, extraction and subcellular fractionation;20
5.1;2.1 Buffers;20
5.1.1;2.1.1 Making a buffer;23
5.1.2;2.1.2 Buffers of constant ionic strength;25
5.2;2.2 Assays for activity;27
5.2.1;2.2.1 Enzyme assays;28
5.2.1.1;2.2.1.1 The progress curve;28
5.2.1.2;2.2.1.2 The enzyme dilution curve;29
5.2.1.3;2.2.1.3 The substrate dilution curve;30
5.2.1.4;2.2.1.4 The effect of pH on enzyme activity;31
5.2.1.5;2.2.1.5 The effect of temperature on enzyme activity;33
5.3;2.3 Assay for protein content;34
5.3.1;2.3.1 Absorption of ultraviolet light;34
5.3.2;2.3.2 The biuret assay;35
5.3.3;2.3.3 The Lowry assay;35
5.3.4;2.3.4 The bicinchoninic acid assay;36
5.3.5;2.3.5 The Bradford assay;36
5.4;2.4 Methods for extraction of proteins;36
5.4.1;2.4.1 Osmotic shock;37
5.4.2;2.4.2 Pestle homogenisers;38
5.4.3;2.4.3 The Waring blendor and Virtis homogeniser;39
5.4.4;2.4.4 The Polytron/UItra-Turrax-type homogeniser;40
5.4.5;2.4.5 Grinding;40
5.4.6;2.4.6 The Parr bomb;41
5.4.7;2.4.7 Extrusion under high pressure;41
5.4.8;2.4.8 Sonication;42
5.4.9;2.4.9 Enzymic digestion;42
5.5;2.5 Clarification of the extract;43
5.6;2.6 Centrifugal sub-cellular fractionation;43
5.6.1;2.6.1 Density gradient centrifugation;48
5.7;2.7 Chapter 2 study questions;52
6;Chapter 3 Concentration of the extract;53
6.1;3.1 Freeze drying;53
6.1.1;3.1.1 Theoretical and practical considerations in freeze-drying;54
6.1.2;3.1.2 Some tips on vacuum;58
6.2;3.2 Dialysis;60
6.2.1;3.2.1 The Donnan membrane effect;62
6.2.2;3.2.2 Counter-current dialysis;63
6.2.3;3.2.3 Concentration by dialysis (concentrative dialysis);64
6.2.4;3.2.4 Perevaporation;64
6.3;3.3 Ultrafiltration;65
6.3.1;3.3.1 Desalting or buffer exchange by ultrafiltration;68
6.3.2;3.3.2 Size fractionation by ultrafiltration;68
6.4;3.4 Concentration/fractionation by salting out;69
6.4.1;3.4.1 Why ammonium sulfate?;69
6.4.2;3.4.2 Empirical observations on protein salting out.;72
6.4.3;3.4.3 Three-phase partitioning (TPP);76
6.5;3.5 Fractional precipitation with polyethylene glycol;79
6.6;3.6 Precipitation with organic solvents;79
6.7;3.7 Dye precipitation;80
6.8;3.8 Chapter 3 study questions;82
7;Chapter 4 Chromatography;83
7.1;4.1 Principles of chromatography;83
7.1.1;4.1.1The effect of particle size;88
7.1.2;4.1.2 The effect of the mobile phase flow rate;90
7.1.2.1;4.1.2.1 The relationship between linear and volumetric flow rates.;91
7.2;4.2 Equipment required for low pressure liquid chromatography;92
7.2.1;4.2.1 The column;92
7.2.2;4.2.2 Moving the mobile phase;94
7.2.3;4.2.3 Monitoring the effluent and collecting fractions.;97
7.2.4;4.2.4 Refrigeration;98
7.3;4.3 Ion-exchange chromatography (IEC);99
7.3.1;4.3.1 Ion-exchange “resins”;101
7.3.2;4.3.2 Gradient generators;104
7.3.3;4.3.3 Choosing the pH;106
7.3.4;4.3.4 An ion-exchange chromatography run;107
7.4;4.4 Chromatofocusing;109
7.5;4.5 Molecular exclusion chromatography (MEC);109
7.5.1;4.5.1 The effect of gel sphere size on Vo;112
7.5.2;4.5.2 The manufacture of small, uniform, gel spheres;114
7.5.3;4.5.3 Determination of MW by MEC;114
7.5.4;4.5.4 Gels used in MEC;116
7.5.5;4.5.5 An MEC run;120
7.6;4.6 Hydroxyapatite chromatography;120
7.6.1;4.6.1 The mechanism of hydroxyapatite chromatography;121
7.7;4.7 Affinity chromatography;122
7.8;4.8 Hydrophobic interaction (HI) chromatography;123
7.9;4.9 Chapter 4 study questions;124
8;Chapter 5 Principles of Electrophoresis;127
8.1;5.1 Principles of electrophoresis;127
8.1.1;5.1.1 The effect of the buffer;131
8.2;5.2 Boundary (Tiselius) electrophoresis;134
8.3;5.3 Paper electrophoresis;135
8.3.1;5.3.1 Electroendosmosis;136
8.4;5.4 Cellulose acetate membrane electrophoresis (CAM-E);137
8.5;5.5 Agarose gel electrophoresis;138
8.6;5.6 Starch gel electrophoresis;139
8.7;5.7 Polyacrylamide gel electrophoresis (PAGE);141
8.7.1;5.7.1 Disc electrophoresis;141
8.7.1.1;5.7.1.1 Isotachophoresis;144
8.8;5.8 SDS-PAGE;145
8.8.1;5.8.1 An SDS-PAGE zymogram for proteinases;147
8.9;5.9 Pore gradient gel electrophoresis;147
8.10;5.10 Isoelectric focusing;148
8.10.1;5.10.1 Establishing a pH gradient;149
8.10.2;5.10.2 Control of convection;152
8.10.3;5.10.3 Applying the sample and measuring the pH gradient;152
8.10.3.1;5.10.3.1 An analytical IEF system;152
8.10.3.2;5.10.3.2 Preparative IEF;154
8.11;5.11 2-D Electrophoresis;155
8.12;5.12 Non-linear electrophoresis;155
8.13;5.13 Chapter 5 study questions;160
9;Chapter 6 Immunological methods;162
9.1;6.1 The structure of antibodies;162
9.2;6.2 Antibody production;163
9.2.1;6.2.1 Making an antiserum;166
9.3;6.3 Immunoprecipitation;168
9.3.1;6.3.1 Immuno single diffusion;170
9.3.1.1;6.3.1.1 Mancini radial diffusion;171
9.3.2;6.3.2 Immuno double diffusion;172
9.3.2.1;6.3.2.1 Ouchterlony double diffusion analysis;173
9.3.2.2;6.3.2.2 Determination of diffusion coefficients;174
9.4;6.4 Immunoelectrophoresis;176
9.4.1;6.4.1 Cross-over electrophotesis;176
9.4.2;6.4.2 Rocket electrophoresis;177
9.4.3;6.4.3 Grabar-Williams immunoelectrophoresis;177
9.4.4;6.4.4 Clarke-Freeman 2-D immunoelectrophoresis;178
9.5;6.5 Amplification methods;180
9.5.1;6.5.1 Complement fixation;180
9.5.2;6.5.2 Radioimmunoassay (RIA);182
9.5.3;6.5.3 Enzyme amplification;183
9.5.3.1;6.5.3.1 Enzyme linked immunosorbent assay (ELISA);183
9.5.3.2;6.5.3.2 Immunoblotting;185
9.5.4;6.5.4 Immunogold labeling with silver amplification;187
9.5.5;6.5.5 Colloid agglutination;188
9.6;6.6 Chapter 6 study questions;191
10;Index;194
11;More eBooks at www.ciando.com;0

Leseproben

Chapter 1 An overview of protein isolation (p. 1-2)

Isolating a protein may be compared to playing a game of golf. In golf, the player is faced with a series of problems, each unique and yet similar to problems previously encountered. In facing each problem the player must analyse the situation and decide, from experience, which club is likely to give the best result in the given circumstances. Similarly, in attempting to isolate proteins, researchers face a series of similar-yetunique problems. To solve these they must dip into their bags and select an appropriate technique. The purpose of this book is thus to fill the beginnerís "golf bag" with techniques relevant to protein isolation, hopefully to improve their game.

Developing a protein isolation is also somewhat like finding a route up a mountainside. Different routes have to be explored and base-camps established at each stage. Occasionally it will be necessary to return to the base of the mountain for further supplies, and haul these up to the established camps, before the next stage can be attacked. A successful climb is always rewarding and if an efficient route is established, it may become a pass, opening the way to further discoveries
1.1 Why do it?

This book is about the methods that biochemists use to isolate proteins, and so it may be asked, "why isolate proteins?" Looked at in one way, living organisms may be regarded as machines with features in common with the entities that we commonly think of as "machines". A typical machine is made of a number of parts which interact, transduce energy, and bring about some desired effect. Mechanical machines have moving parts, while electronic machines move electrons. "Engines" convert energy to mechanical motion. Internal combustion engines, for example, convert chemical energy to mechanical motion. Similarly, living organisms such as the human body are complex machines made up of many interacting systems. Proteins constitute the majority of the working parts of these systems and there are thus diverse reasons for isolating proteins, viz.;

• To gain insight. As with any mechanism, to study the way in which a living system works it is necessary to dismantle the machine and to isolate the component parts so that they may be studied, separately and in their interaction with other parts. The knowledge that is gained in this way may be put to practical use, for example, in the design of medicines, diagnostics, pesticides, or industrial processes. Many proteins may themselves be used as "medicines" to make up for losses or inadequate synthesis. Examples are hormones, such as insulin, which is used in the therapy of diabetes, and blood fractions, such as the so-called Factor VIII, which is used in the therapy of haemophilia. Other proteins may be used in medical diagnostics, an example being the enzymes glucose oxidase and peroxidase, which are used to measure glucose levels in biological fluids, such as blood and urine.

• For use in Industry. Many enzymes are used in industrial processes, especially where the materials being processed are of biological origin. In every case a pure protein is desirable as impurities may either be misleading, dangerous or unproductive, respectively. Protein isolation is, therefore, a very common, almost central, procedure in biochemistry.

• For use in Medicine. Many enzymes are used in industrial processes, especially where the materials being processed are of biological origin.

In every case a pure protein is desirable as impurities may either be misleading, dangerous or unproductive, respectively. Protein isolation is, therefore, a very common, almost central, procedure in biochemistry.