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Protein Purification 2nd edition [Minkštas viršelis]

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(Nottingham Trent University, UK)
  • Formatas: Paperback / softback, 269 pages, aukštis x plotis: 254x178 mm, weight: 516 g, 33 Tables, black and white; 3 Illustrations, color; 89 Illustrations, black and white
  • Serija: THE BASICS (Garland Science)
  • Išleidimo metai: 16-Aug-2018
  • Leidėjas: CRC Press Inc
  • ISBN-10: 0815344880
  • ISBN-13: 9780815344889
Kitos knygos pagal šią temą:
Protein Purification 2nd editionDidesnis paveikslėlis
  • Formatas: Paperback / softback, 269 pages, aukštis x plotis: 254x178 mm, weight: 516 g, 33 Tables, black and white; 3 Illustrations, color; 89 Illustrations, black and white
  • Serija: THE BASICS (Garland Science)
  • Išleidimo metai: 16-Aug-2018
  • Leidėjas: CRC Press Inc
  • ISBN-10: 0815344880
  • ISBN-13: 9780815344889
Kitos knygos pagal šią temą:
Kitos knygos iš šio išskirtinio pasiūlymo: Taylor & Francis siūlo daug technologijos knygų ISE (International Student Edition) kainomis
Pastovi nuoroda: https://www.kriso.lt/db/9780815344889.html
Raktažodžiai:

This second edition of Protein Purification provides a guide to the major chromatographic techniques, including non-affinity absorption techniques, affinity procedures, non-absorption techniques and methods for monitoring protein purity. The new edition of the book has been organized to encourage incremental learning about the topic, starting with the properties of water, progressing through the characteristics of amino acids and proteins which relate to the purification process. There is an overview of protein strategy and equipment, followed by discussions and examples of each technique and their applications.

The basic theory and simple explanations given in Protein Purification make it an ideal handbook for final year undergraduates, and postgraduates, who are conducting research projects. It will also be a useful guide to more experienced researchers who need a good overview of the techniques and products used in protein purification.

Key Features

* Guide to the major techniques used in protein purification

* Includes flowcharts to help the reader slect the best purification strategy

* Contains step-by-step protocols that guide the reader through each technique and its use

* Includes exercises and solutions

Preface xiii
Abbreviations xv
Chapter 1 Water, pH and Buffers 1(12)
1.1 Introduction
1(1)
1.2 The Structure Of Water
2(1)
1.3 Water As A Solvent
3(2)
1.4 The Ionisation Of Water
5(1)
1.5 The Ph Scale
6(1)
1.6 Acids And Bases
7(1)
1.7 Buffers
8(2)
1.7.1 Points to remember about buffers
9(1)
1.8 Summary
10(1)
Exercise 1.1
10(1)
Recommended Reading
11(2)
Research papers
11(1)
Books
11(2)
Chapter 2 Protein Structure and Properties 13(18)
2.1 Introduction
13(1)
2.2 Amino Acids
13(6)
2.3 The Peptide Bond
19(2)
2.4 Overview Of Protein Structure
21(4)
2.4.1 Primary structure
21(1)
2.4.2 Secondary structure
21(1)
2.4.3 Tertiary structure
22(2)
2.4.4 Quaternary structure
24(1)
2.5 Motifs And Domains (Super Secondary Structure)
25(1)
2.6 Post-Translational Modifications
25(1)
2.7 The Characteristics Of A Protein Which Can Be Exploited To Purify A Target Protein
26(3)
2.7.1 Surface charge
26(1)
2.7.2 Hydrophobic nature
27(1)
2.7.3 Solubility
27(1)
2.7.4 Biospecificity
28(1)
2.7.5 Molecular mass (Mr)
28(1)
2.7.6 Post-translational modifications
28(1)
2.7.7 Engineering proteins to aid purification
28(1)
2.8 A Range Of Techniques That Can Be Used In Protein Purification
29(1)
2.8.1 Surface charge
29(1)
2.8.2 Hydrophobicity
29(1)
2.8.3 Biospecificity
29(1)
2.8.4 Molecular mass
29(1)
2.9 Summary
29(1)
Recommended Reading
30(1)
Research papers
30(1)
Books
30(1)
Chapter 3 Chromatography and the Strategy of Protein Purification 31(28)
3.1 Introduction
31(1)
3.2 Chromatography
31(1)
3.3 The Theories Of Chromatography
32(2)
3.3.1 The plate theory of chromatography
32(1)
3.3.2 The rate theory of chromatography
33(1)
3.4 Parameters In Chromatography
34(1)
3.5 The Strategy Of Protein Purification
35(9)
3.5.1 Introduction
35(3)
3.5.2 Strategy
38(1)
3.5.3 The process of protein purification
39(5)
3.6 The Equipment Required For Protein Purification
44(7)
3.6.1 Columns
45(1)
3.6.2 Tubing
46(1)
3.6.3 Pumps
46(1)
3.6.4 Fraction collectors
46(1)
3.6.5 Detectors
47(1)
3.6.6 Gradient makers
47(1)
3.6.7 Different elution procedures
47(3)
3.6.8 Regeneration and storage of chromatographic resins (media)
50(1)
3.7 Automated Protein Purification Chromatography Systems
51(1)
3.8 Simulated Moving Bed Chromatography (SMBC)
52(1)
3.9 Protein Purification Chromatographic Runs
52(3)
3.9.1 Pre-packed chromatographic columns
52(1)
3.9.2 Empty chromatography columns
53(1)
3.9.3 Flow rates and elution of the sample
54(1)
3.10 Scaling Up The Purification And Some Considerations About Industrial Protein Purification
55(1)
3.11 Summary
55(1)
Protocol For
Chapter 3
56(1)
Protocol 3.1 The preparation of a sample and buffers fora protein purification chromatographic run using an automated protein purification system
56(1)
Recommended Reading
57(2)
Research papers
57(1)
Books
57(2)
Chapter 4 The Groundwork 59(60)
4.1 Introduction
59(1)
4.2 Assay To Identify A Target Protein
59(4)
4.2.1 Temperature
60(1)
4.2.2 Substrate concentration
61(2)
4.2.3 pH
63(1)
4.3 Protein Assays
63(2)
4.4 The Extraction Of Protein From Cells Or Tissue
65(9)
4.4.1 Introduction
65(1)
4.4.2 Reducing agents
66(1)
4.4.3 Chelating agents
67(1)
4.4.4 Enzyme Substrates/Inhibitors/ Activators/Cofactors
68(1)
4.4.5 Inhibitors of peptidase enzymes
68(1)
4.4.6 Phosphatase inhibitors
69(1)
4.4.7 Removal of nucleic acids and nucleoproteins
70(1)
4.4.8 Removal of lipoproteins
70(1)
4.4.9 Additions for the extraction of plant tissue
70(1)
4.4.10 Additions for the extraction of membrane proteins
71(3)
4.5 Techniques Used To Disrupt Tissue Or Cells
74(2)
4.5.1 Animal cells or tissue
74(1)
4.5.2 Plant cells (typically 100 μm in diameter)
75(1)
4.5.3 Bacterial cells (0.7-4.0 μ in diameter)
75(1)
4.5.4 Fungal cells
76(1)
4.6 The Extraction Methods Used With Small Amounts Of Tissue Or Cells
76(1)
4.6.1 Liquid nitrogen
76(1)
4.6.2 Acid washed sand
76(1)
4.6.3 Osmotic shock
76(1)
4.6.4 Homogenisers/Tissue grinders
76(1)
4.6.5 Sonicators
77(1)
4.7 Extraction Methods For Large Amounts Of Animal/Plant Tissue Or Cells
77(2)
4.7.1 Blenders
77(1)
4.7.2 Blenders with beads
78(1)
4.7.3 Ultra-Turrax/Polytron Homogenisers
78(1)
4.8 The Extraction Methods Used With Bacterial Or Yeast Cells
79(1)
4.8.1 Abrasive materials
79(1)
4.8.2 Compression/Expansion
79(1)
4.8.3 Freezing and thawing
79(1)
4.8.4 Enzyme treatment
80(1)
4.9 Points To Remember About Extraction Procedures
80(1)
4.10 The Techniques Used To Clarify Homogenised Extracts
81(6)
4.10.1 Centrifugation
81(5)
4.10.2 Aqueous two-phase partitioning
86(1)
4.11 The Techniques That Can Be Used To Concentrate Proteins From Dilute Solutions (Laboratory Scale)
87(11)
4.11.1 Salt precipitation
88(4)
4.11.2 Organic solvent precipitation
92(1)
4.11.3 Polymer precipitation
92(1)
4.11.4 Aqueous two-phase partitioning
92(1)
4.11.5 Isoelectric precipitation
92(1)
4.11.6 Chromatography
93(1)
4.11.7 Hygroscopic material
93(1)
4.11.8 Dried acrylamide
93(1)
4.11.9 Lyophilisation (freeze drying)
93(2)
4.11.10 Ultrafiltration
95(3)
4.12 Clarification Of Process Scale Extracts
98(3)
4.12.1 Process scale centrifugation
98(1)
4.12.2 Stirred bed
99(1)
4.12.3 Fluidised bed
99(1)
4.12.4 Expanded bed
100(1)
4.13 Membrane Chromatography
101(1)
4.14 The Storage Of Protein Samples
101(1)
4.15 Summary
102(1)
Exercise 4.1
103(1)
Protocols for
Chapter 4
104(12)
Protocol 4.1 The measurement of protein concentration using the absorbance of light at 280 nm
104(1)
Protocol 4.2 Bicinchininic acid (BCA) protein assay (0-1.0 mg ml-1)
105(2)
Protocol 4.3 Bicinchoninic acid (BCA) protein assay (0.5-10 μg ml-1)
107(1)
Protocol 4.4 Coomassie blue dye binding protein assay
108(1)
Protocol 4.5 Azocasein assay to determine the peptidase profile in crude extracts
109(1)
Protocol 4.6 Extraction of plant tissue material using a blender and concentration using ammonium sulphate
110(1)
Protocol 4.7 Extraction of animal tissue material using a Potter-Elvehjem homogeniser
111(2)
Protocol 4.8 The total (a) and fractional (b) precipitation of protein by ammonium sulphate
113(1)
Protocol 4.9 Preparation of dialysis tubing
114(1)
Protocol 4.10 Precipitation of protein from a small volume (0.1-5.0 ml) using acetone
115(1)
Recommended Reading
116(3)
Research papers
116(1)
Books
117(2)
Chapter 5 Non-Affinity Absorption Techniques Used to Purify Proteins 119(26)
5.1 Introduction
119(1)
5.2 Ion Exchange (IEX) Chromatography
120(6)
5.2.1 Ion exchange resins
121(1)
5.2.2 Binding and elution of protein to IEX resins
122(4)
5.2.3 Regeneration and storage of IEX resins
126(1)
5.3 Chromatofocusing
126(1)
5.4 Hydroxyapatite (HA) Chromatography
127(1)
5.5 Hydrophobic Interaction Chromatography (HIC)
128(4)
5.5.1 The binding and elution conditions for HIC
130(2)
5.5.2 The regeneration and storage of HIC resins
132(1)
5.6 Hydrophobic Charge Induction Chromatography (HCIC)
132(1)
5.7 Introduction To Mixed Mode (Multimodal) Chromatography (MMC)
133(3)
5.7.1 Capto™ MMC (GE Healthcare)
134(1)
5.7.2 Mercapto-benzimidazole sulfonic acid (MBI)
135(1)
5.7.3 Nuvia cPrime (Bio-Rad)
135(1)
5.8 Summary
136(1)
Protocols for
Chapter 5
137(7)
Protocol 5.1 Small-scale screening experiment to establish the appropriate binding and elution conditions for chromatographic resins
137(2)
Protocol 5.2 The purification of calcium-dependent proteins using hydrophobic interaction chromatography (HIC)
139(2)
Protocol 5.3 The affinity elution (displacement) of enzymes from an ion exchange column
141(3)
Recommended Reading
144(1)
Research papers
144(1)
Books
144(1)
Chapter 6 Affinity-Based Procedures Used to Purify Proteins 145(32)
6.1 Introduction To Affinity Chromatography
145(1)
6.2 The Resins Used In Affinity Chromatography
146(1)
6.3 The Affinity Ligand
147(2)
6.4 The Binding And Elution Conditions For Affinity Chromatography
149(3)
6.5 The Regeneration And Storage Of Affinity Resins
152(1)
6.6 Group-Specific Ligands
153(1)
6.7 Covalent Chromatography
153(2)
6.7.1 Introduction to covalent chromatography
153(1)
6.7.2 The binding and elution conditions for covalent chromatography
154(1)
6.7.3 The regeneration and storage of covalent chromatography resins
155(1)
6.8 Dye Ligand Affinity Chromatography
155(1)
6.9 Immunoaffinity Chromatography
156(4)
6.9.1 Introduction to immunoaffinity chromatography
156(2)
6.9.2 The binding and elution conditions for immunoaffinity resins
158(1)
6.9.3 Regeneration and storage of immunoaffinity resins
159(1)
6.10 Lectin Affinity Chromatography
160(2)
6.10.1 Introduction to lectin affinity chromatography
160(1)
6.10.2 The binding and elution conditions for lectin affinity chromatography
161(1)
6.10.3 Regeneration and storage of lectin resins
161(1)
6.11 Immobilised Metal (ION) Affinity Chromatography (IMAC)
162(3)
6.11.1 Introduction to immobilised metal affinity chromatography
162(1)
6.11.2 The binding and elution conditions for IMAC
163(2)
6.11.3 The regeneration and storage of IMAC resins
165(1)
6.12 The Purification Of Recombinant Proteins
165(4)
6.12.1 Introduction to the purification of recombinant proteins
165(1)
6.12.2 The use of IMAC to purify recombinant proteins
166(2)
6.12.3 The use of fusion proteins to purify recombinant proteins
168(1)
6.13 Affinity Partitioning (Precipitation)
169(1)
6.14 Summary
170(1)
Protocols for
Chapter 6
171(4)
Protocol 6.1 Purification of IgG using protein A agarose
171(2)
Protocol 6.2 Purification of anti-transglutaminase 2 (TG2) monoclonal antibody ID10 (NTU) from a monoclonal cell culture supernatant using cation exchange chromatography
173(2)
Recommended Reading
175(2)
Research papers
175(1)
Books
175(2)
Chapter 7 Non-Absorption Techniques for Purifying Proteins 177(16)
7.1 Introduction To Size Exclusion Chromatography (SEC)
177(1)
7.2 The Theory Of Size Exclusion Chromatography
178(4)
7.3 Factors To Consider In Size Exclusion Chromatography
182(1)
7.3.1 The resin
182(1)
7.3.2 The bead size of the resin
182(1)
7.3.3 The flow rate
182(1)
7.3.4 The size of the sample
182(1)
7.3.5 The sample viscosity
182(1)
7.3.6 The composition of the buffer
183(1)
7.4 Size Exclusion Media Preparation And Storage
183(1)
7.5 Applications Of Size Exclusion Chromatography
183(3)
7.5.1 Analytical SEC
183(2)
7.5.2 Separation of aggregates or removal of low amounts of contaminating material by size exclusion chromatography
185(1)
7.5.3 Desalting (group separation)
186(1)
7.5.4 Refolding of denatured proteins
186(1)
7.6 Dynamic Light Scattering
186(1)
7.7 Summary
187(1)
Exercise 7.1
188(1)
Exercise 7.2
188(1)
Protocol For
Chapter 7
189(2)
Protocol 7.1 Desalting: The exchange of buffer ions in a protein sample using size exclusion chromatography
189(2)
Recommended Reading
191(2)
Research papers
191(1)
Books
191(1)
Web page
191(2)
Chapter 8 Methods for Monitoring the Purity of Protein Solutions 193(38)
8.1 Introduction
193(1)
8.2 Quality Control: Information For A Protein Purification Balance Sheet
193(2)
8.3 The Theory Of Electrophoresis
195(8)
8.3.1 Polyacrylamide gel electrophoresis (PAGE) of proteins
197(1)
8.3.2 The stacking gel
198(1)
8.3.3 Denaturing polyacrylamide gel electrophoresis of proteins
199(2)
8.3.4 Non-denaturing (native) polyacrylamide gel electrophoresis
201(1)
8.3.5 Visualising the polypeptides/Proteins in polyacrylamide gels
202(1)
8.4 Preparative Gel Electrophoresis And Methods To Isolate Proteins From Polyacrylamide Gels
203(2)
8.4.1 Electroelution of proteins from polyacrylamide gels
205(1)
8.4.2 Gel homogenisation
205(1)
8.4.3 Gel solubilisation
205(1)
8.5 Introduction To Western Blotting
205(2)
8.5.1 Recovery Of Proteins From Nitrocellulose
207(1)
8.6 Introduction To Isoelectric Focusing
207(2)
8.6.1 Preparative Isoelectric Focusing (IEF)
209(1)
8.7 Introduction To Capillary Electrophoresis (CE)
209(2)
8.8 Introduction To Reversed-Phase High-Pressure Liquid Chromatography Of Proteins
211(1)
8.9 Summary
212(1)
Exercise 8.1
213(2)
Exercise 8.2
215(1)
Protocols for
Chapter 8
216(14)
Protocol 8.1 The procedure and solutions for denaturing PAGE
216(3)
Protocol 8.2 The procedures and solutions to run non-denaturing PAGE
219(3)
Protocol 8.3 Western blotting using a semi-dry blotter
222(2)
Protocol 8.4 The preparation of enhanced chemiluminescent (ECL) reagent for developing Western blots probed with horseradish peroxidase conjugated secondary antibodies
224(1)
Protocol 8.5 Silver stain (adapted from GE Healthcare protocol) compatible with mass spectrometry
225(1)
Protocol 8.6 Fluorescent staining for polypeptides in polyacrylamides gels
226(1)
Protocol 8.7 SDS-Imidazole zinc stain for polypeptides in polyacrylamide gels
227(1)
Protocol 8.8 The detection of polypeptides in SDS-PAGE gels without the use of additional staining reagents
228(1)
Protocol 8.9 Homogenisation of polyacrylamide gels containing a target protein
229(1)
Recommended Reading
230(1)
Research papers
230(1)
Books
230(1)
Appendix 1: The Laws of Thermodynamics and the Gibbs Free Energy Equation 231(2)
Appendix 2: Properties of Amino Acids 233(2)
Appendix 3:The Common Units Used in Biology 235(2)
Appendix 4: Chromatographic Runs 237(2)
Appendix 5: Determining the Concentration of a Compound in a Solution in PPM 239(2)
Appendix 6:The Answers to the Exercises in the Various
Chapters		 241(4)
Appendix 7: An Alphabetical List of Chromatography (C), Electrophoresis (E), Filtration Equipment (F) and Laboratory Suppliers (LS) 245(4)
Appendix 8: Buffer Tables to Prepare Buffers at a Required pH Value 249(4)
Glossary Of Terms 253(8)
Index 261
Dr Philip Bonner is Programme Leader for the MRes in Applied Biosciences at Nottingham Trent University.