9780199636747

Protein Purification Techniques A Practical Approach

by
  • ISBN13:

    9780199636747

  • ISBN10:

    0199636745

  • Edition: 2nd
  • Format: Hardcover
  • Copyright: 2001-04-05
  • Publisher: Oxford University Press

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Summary

Proteins are an integral part of molecular and cellular structure and function and are probably the most purified type of biological molecule. In order to elucidate the structure and function of any protein it is first necessary to purify it. Protein purification techniques have evolved over the past ten years with improvements in equipment control, automation, and separation materials, and the introduction of new techniques such as affinity membranes and expanded beds. These developments have reduced the workload involved in protein purification, but there is still a need to consider how unit operations linked together to form a purification strategy, which can be scaled up if necessary. The two Practical Approach books on protein purification have therefore been thoroughly updated and rewritten where necessary. The core of both books is the provision of detailed practical guidelines aimed particularly at laboratory scale purification. Information on scale-up considerations is given where appropriate. The books are not comprehensive but do cover the major laboratory techniques and common sources of protein. Protein Purification Techniques focuses on unit operations and analytical techniques. It starts with an overview of purification strategy and then covers initial extraction and clarification techniques. The rest of the book concentrates on different purification methods with the emphasis being on chromatography. The final chapter considers general scale-up considerations. Protein Purification Applications describes purification strategies from common sources: mammalian cell culture, microbial cell culture, milk, animal tissue, and plant tissue. It also includes chapters on purification of inclusion bodies, fusion proteins, and purification for crystallography. A purification strategy that can produce a highly pure single protein from a crude mixture of proteins, carbohydrates, lipids, and cell debris to is a work of art to be admired. These books (available individually or as a set) are designed to give the laboratory worker the information needed to undertake the challenge of designing such a strategy.

Table of Contents

List of protocols page
xiii
Abbreviations xv
Purification strategy
1(10)
Simon D. Roe
Key considerations
1(1)
Aims of purification---why?
2(1)
The target protein and contaminants
3(1)
Protein structure
4(1)
Source of the product
5(1)
Key steps in purification
6(1)
Process integration
6(1)
Scale-up
7(1)
Monitoring the purification---assays
8(1)
Time and temperature
8(3)
References
10(1)
Getting started
11(16)
Simon D. Roe
Overview of lab equipment
11(3)
Ancillary equipment
12(1)
Detection and analysis equipment
12(1)
Seperations equipment
13(1)
Control of pH---buffers---principles, selection, and use
14(3)
Purification strategy
17(6)
Ordering of steps
17(1)
Nature of starting material
17(1)
Storage of material
18(1)
Fermentation products
19(1)
Other sources of proteins
19(1)
Making an extract
19(2)
Separation of particulates
21(1)
Protein concentration
22(1)
Adjusting sample composition between steps
22(1)
Protein lability and structure---implications for purification
23(4)
Introduction
23(1)
Denaturation
24(1)
Catalytic site inactivation
24(1)
Proteolysis
24(1)
Other causes of yield loss
25(1)
References
26(1)
Analysis of purity
27(24)
Dev Baines
Introduction
27(1)
Considering yield and purity
27(1)
Scope of the methods included
28(1)
Total protein quantitation
28(6)
Ultraviolet absorption protein assay
28(2)
Lowry (Folin-Ciocalteau) protein assay
30(1)
Bradford (Coomassie Brilliant Blue) dye-binding protein assay
31(1)
Bicinchoninic acid protein assay
32(1)
Colloidal gold protein assay
32(1)
General comments
33(1)
Specific quantitation
34(4)
Activity assays
34(2)
Binding assays
36(2)
Detection of impurities
38(11)
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
38(2)
Detection of protein bands in electrophoresis gels
40(2)
Analysis by isoelectric focusing (IEF)
42(2)
Glycoprotein analysis
44(1)
Western blotting
45(2)
Chromatographic methods
47(2)
Other biochemical and biophysical methods
49(2)
Protein mass spectrometry
49(1)
References
49(2)
Clarification techniques
51(32)
Ian Reed
Duncan Mackay
Introduction
51(1)
Sedimentation
52(2)
Principles of sedimentation
52(2)
Gravity sedimentation
54(1)
Centrifugal sedimentation
54(6)
Centrifuge equipment
55(1)
Laboratory centrifuge test work
56(3)
Suspension conditioning
59(1)
Filtration
60(5)
Background
60(2)
Principles of dead-end filtration
62(2)
Laboratory scale filtration equipment
64(1)
Application of filtration
65(1)
Membrane processes
65(15)
Background
65(2)
Principles of operation
67(4)
Membrane equipment
71(2)
Membrane selection
73(3)
Membrane operation
76(4)
Choice of clarification process
80(3)
Cell disintegration and extraction techniques
83(28)
R. H. Cumming
G. Iceton
Overview
83(8)
Stability of the released protein
84(1)
Location of target protein within the cell
84(1)
The yield and kinetics of the process
85(1)
Continuous or batch disruption
86(1)
The need to consider subsequent steps
87(1)
Assessing the extent of disruption
88(1)
Marker substances for cell disruption
89(1)
Containment of the process
90(1)
Scale-up
91(1)
Methods of disruption
91(17)
Pre-treatment of material
91(1)
General procedure notes
92(1)
Mixers and blenders
92(2)
Coarse grinding: pestle and mortar
94(1)
Fine grinding: the bead mill
94(2)
Homogenization
96(3)
Ultrasonication
99(3)
Heat shock
102(1)
Freezing and thawing
102(1)
Osmotic shock
103(1)
Lytic enzymes
103(2)
Chemical treatments
105(1)
Detergents
105(2)
Solvents
107(1)
Conclusions: choice of methods
108(3)
References
108(3)
Concentration of the extract
111(44)
E. L. V. Harris
Introduction
111(1)
Addition of a dry matrix polymer
112(1)
Ultrafiltration
112(17)
Equipment
116(8)
Operation
124(3)
Other applications of ultrafiltration
127(2)
Freeze-drying or lyophilization
129(1)
Removal of salts and exchange of buffer
130(2)
Dialysis
130(2)
Diafiltration
132(1)
Gel filtration
132(1)
Purification and concentration by precipitation
132(11)
Precipitation by alteration of the pH
134(1)
Precipitation by decreasing the ionic strength
135(1)
Precipitation by increasing the ionic strength (salting-out)
135(4)
Precipitation by organic solvents
139(2)
Precipitation by organic polymers
141(1)
Precipitation by denaturation
141(2)
Aqueous two-phase partitioning
143(12)
B. A. Andrews
J. A. Asenjo
Equipment and materials
146(1)
Optimization
146(2)
Using phase partitioning in a purification procedure
148(2)
Affinity partitioning
150(2)
Acknowledgements
152(1)
References
152(3)
Separation on the basis of chemistry
155(32)
Lars Hagel
Introduction
155(1)
Chemical interaction between surfaces
155(3)
Basic properties of proteins
158(2)
Properties of the solvent
160(1)
Properties of chromatography media
161(26)
Ion exchange chromatography
166(6)
Hydrophobic interaction chromatography
172(3)
Reversed-phase chromatography
175(2)
Charge-transfer chromatography
177(2)
Covalent chromatography
179(3)
Design, optimization, and scale-up of a chromatographic purification procedure
182(1)
Acknowledgements
183(1)
References
183(4)
Chromatography on the basis of size
187(26)
P. Cutler
Introduction
187(10)
Principle
187(5)
Applications of size exclusion chromatography
192(5)
Materials
197(5)
Equipment
197(2)
Mobile phase
199(2)
Non-ideal size exclusion
201(1)
Methods
202(11)
Flow rates
202(1)
Preparation and packing
202(2)
Equilibrium
204(1)
Sample preparation and application
205(1)
Evaluation and calibration
205(2)
Separation of proteins
207(1)
Column cleaning and storage
208(1)
Trouble shooting
209(1)
References
209(4)
Purification by exploitation of activity
213(28)
S. Angal
P. D. G. Dean
Simon D. Roe
Introduction
213(1)
Design and preparation of affinity adsorbents
214(9)
Choice of ligand
214(1)
Selection of the matrix
215(2)
Choice of spacer
217(1)
Activation and coupling chemistry
218(4)
Estimation of ligand concentration
222(1)
Use of affinity adsorbents
223(7)
Initial questions
223(1)
Selection of conditions for operation
223(3)
Estimation of capacity
226(1)
Ligand efficiency
227(1)
Application to protein purification
227(3)
Immunopurification
230(11)
C. R. Hill
L. G. Thompson
A. C. Kenney
Antibody selection
231(1)
Immobilization of antibodies on CNBr-activated Sepharose
232(3)
Procedures for immunopurification
235(4)
References
239(2)
Scale-up considerations
241(14)
John B. Noble
Scale-up of purification processes and unit operations
241(14)
Choice of scale
241(1)
Overview of critical issues
242(3)
Practical aspects of scale-up
245(4)
Case study 1
249(1)
Case study 2
250(3)
References
253(2)
A1 List of suppliers 255(4)
Index 259

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