Description
Book SynopsisSince the publication of the bestselling second edition of John Walker’s widely acclaimed Protein Protocols Handbook, there have been continual methodological developments in the field of protein chemistry. This greatly enhanced third edition introduces 57 critically important new chapters, as well as significantly updating the previous edition's tried-and-true methods. Although the timely new chapters are spread throughout all of the book, the vital section on post-translational modifications has been expanded most to reflect the increasing importance of these modifications in the understanding of protein function.
Each readily reproducible method follows the highly praised format of the Methods in Molecular Biology™ series, offering a concise summary of its basic theory, a complete materials list, a step-by-step protocol for its successful execution, and extensive notes on avoiding pitfalls, or on modifying the method to function within your own experimental circumstances. The expert authors of each chapter have demonstrated a hands-on mastery of the methods described, fine-tuned here for optimal productivity.
Comprehensive, cutting-edge, and highly practical, The Protein Protocols Handbook, Third Edition is today's indispensable benchtop manual and guide, not only for all those new to the protein chemistry laboratory, but also for those established workers seeking to broaden their armamentarium of techniques in the urgent search for rapid and robust results
Table of ContentsPart I : QUANTITATION OF PROTEINS
1. Protein Determination by UV Absorption
A. Aitken and M.P. Learmonth
2. The Lowry Method for Protein Quantitation
J. H. Waterborg
3 The Bicinchoninic Acid (BCA) Assay for Protein Quantitation
J.M. Walker
4 The Bradford Method for Protein Quantitation
N.Kruger
5. Ultrafast Protein Determinations Using Microwave Enhancement
R. Akins and R. S. Tuan
6. The Nitric Acid Methods for Protein Estimation in Biological Samples
S.A. Boerner, Y.K. Lee, S.H. Kaufmann and K. Bible
7. Quantitation of Tryptophan in Proteins
A. Aitken and M.P. Learmonth
8. Kinetic Silver Staining of Proteins
D. D. Root and K. Wang
9. Quantitation of cellular proteins by flow cytometry
T. D. Friedrich , F.A. Ray, R.L. Smith and J. M. Lehman
10. Quantitation of cellular proteins by laser scanning cytometry
T.D. Friedrich, R.L.Smith and J.M. Lehman
Part II : ELECTROPHORESIS OF PROTEINS AND PEPTIDES AND DETECTION IN GELS
11 Protein Solubility in 2D Electrophoresis: Basic principles and issues
T.Rabilloud
12 Mouse and Human Tissues Sample Preparation for 2-D Electrophoresis
C. Zabel and J. Klose
13 Plant Protein sample preparation for 2DE
S. Carpentier , R.Swennen and B. Panis
14 Preparation of bacterial samples for 2-D PAGE.
B. Vandahl, G. Christiansen and S. Birkelund
15 Preparation of bodily fluids for 2-D PAGE
Sullivan, H.Brzeski, J. Ganesalingam, and M. Mayr
16 Immunoaffinity Depletion of high abundance plasma and serum proteins
L.A. Echan and D. Speicher
17 Preparation of Yeast samples for 2D PAGE
J. Norbeck
18 Membrane Protein Preparation Using Aqueous Polymer Two Phase Systems
J. Schindler and H-G Nothwang
19 Subcellular fractionation of small sample amounts
H.G Nothwang, I. Guillemin and J. Schindler
20. Nondenaturing Polyacrylamide Gel Electrophoresis of Proteins
J.M. Walker
21 SDS Polyacrylamide Gel Electrophoresis of Proteins
J.M. Walker
22 Gradient SDS Polyacrylamide Gel Electrophoresis of Proteins
J.M. Walker
23. SDS-Polyacrylamide Gel Electrophoresis of Peptides
R. C. Judd
24 Blue native gel electrophoresis (BN-PAGE)
Elke A. Dian, J. Rassow and Christian Motz
25 Separation of proteins by gel electrophoresis in the Tris-Taurine-HCl system
S.Luche, M. Chevallet C. Lelong and T. Rabilloud
26 Cetyltrimethylammonium Bromide Discontinuous Gel Electrophoresis of Proteins: M -Based Separation of Proteins with Retained Native Activity
R.E. Akins and R. S. Tuan
27 Acetic Acid-Urea Polyacrylamide Gel Electrophoresis of Basic Proteins
J. H. Waterborg
28 Acid-Urea-Triton Polyacrylamide Gel Electrophoresis of Histones
J. H. Waterborg
29 Isoelectric Focusing of Proteins in Ultra-Thin Polyacrylamide Gels
J. M. Walker
30 Serial immobilized pH gradient isoelectric focusing over pH 4-9
S.Poznanovic, W. Wozny, H. Zengerling, G.P. Schwall and M. A. Cahill
31. Radiolabelling of Eukaryotic Cells and Subsequent Preparation for 2-D <
N. Bizios
32 Two-Dimensional PAGE Using Carrier Ampholyte pH Gradients in the First Dimension
P.Gravel
33 Vertical agarose electrophoresis and electroblotting of high molecular weight proteins
M. Greaser and C.M. Warren
34 2D PAGE of high molecular weight proteins
M. Oh-Ishi and T.Maeda
35 Casting immobilised pH gradients
E. Gianazza
36 Nonequilibrium pH Gel Electrophoresis
J. Carroll
37 Microchip capillary electrophoresis
B.A. Fogarty, N.A.Lacher and S.M. Lunte
38 Protein Separations in Microfluidic Chips
A. Chow and B.Fathollahi
39 Difference gel electrophoresis (DIGE)
D. Friedman and K.S. Lilley
40 Comparing 2-D Electrophoresis Gels Across Internet Databases
P. Lemkin, G.C.Thornwall, and J.A. Evans
41 Quantification of Radiolabeled Proteins in Polyacrylamide Gels 25.4.07
W.R. Springer
42 Differential ProteoTope radioactive quantification of protein abundance ratios
W.Wozny, G.P.Schwall, C.S. Sastri, S.Poznanovic, W. Stegmann, Hunzinger, K. Groebe and M.A. Cahill
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43 Quantitation of Proteins on Polyacrylamide Gels
J. Smith
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44 Using SDS-PAGE and scanning laser densitometry to measure proteins
Aaron P. Miles and A. Saul
45 Rapid and Sensitive Staining of Unfixed Proteins in Polyacrylamide Gels with Nile Red
J-R. Daban, S. B.A. Bermudez amd F.J. Alba
46 Zinc reverse staining technique
C. Fernandez-Patron
47 Protein Staining with Calconcarboxylic Acid in Polyacrylamide Gels
Wei-Tao Cong, Sun-Young Hwang, Li-Tai Jin and Jung-Kap Choi
48 Detection of Proteins in Polyacrylamide Gels by Silver Staining
M. J. Dunn
49 Background-free Protein Detection on Polyacrylamide Gels and on Electroblots Using Transition Metal Chelate Stains
W.F. Patton
50 Detection of Proteins in Polyacrylamide Gels by Fluorescent Staining
M. J. Dunn
51 Detection of Glycoproteins in Gels and Blots
Nicolle Packer, Malcolm S Ball, Peter L Devine, and Wayne F Patton
52 Staining of Glycoproteins/Proteoglycans on SDS-Gels
H.J. Møller and J.H. Poulsen
53. Detection of Proteins and Sialoglycoproteins in Polyacrylamide Gels Using Eosin X stain
F. Lin & G. Wise
54 Pro-Q Diamond phosphoprotein staining
G.K. Agrawal and J.J. Thelen
55. Electroelution of Proteins from Polyacrylamide Gels
P. Jenö and Martin Horst
56 Autoradiography and Fluorography of Acrylamide Gels
A. Circolo and S. Gulati
57 Proteolytic Activity Detection by Two-Dimensional Zymography
J.Wilkesman
Part III : BLOTTING AND DETECTION METHODS
58. Protein Blotting by Electroblotting
M. Page and R.Thorpe
59. Protein Blotting by the Semi-dry Method
P. Gravel
60. Protein Blotting by the Capillary Method
J.M. Walker
61. Western Blotting of basic proteins electrophoretically resolved on acid-urea-Triton-polyacrylamide gels
G.P. Delcuve and J. R. Davie
62 Immunoblotting of 2-DE Separated Proteins
Magi and L Bianchi
63 High efficiency blotting of high-molecular weight proteins
M. Greaser and D.R. Swartz
64. Alkaline Phosphatase Labeling of IgG Antibody
G. B.Wisdom
65. b-Galactosidase Labeling of IgG Antibody
G. B. Wisdom
66. Horseradish Peroxidase Labeling of IgG Antibody
G. B. Wisdom
67. Digoxigenin (DIG) Labelling of IgG Antibody
G. B. Wisdom
68. Conjugation of fluorochromes to antibodies
S-Y. Mao
69. Coupling of Antibodies with Biotin
R.P. Haugland and Wendy W. You
70. Preparation of Avidin Conjugates
R.P. Haugland and M.K. Bhalgat
71. MDPF Staining of Proteins on Western Blots
F. Alba and J.R. Daban
72. Copper Iodided Staining of Proteins and its silver enhancement
D. D. Root and K. Wang
73. Detection of Proteins on Blots using Direct Blue 71
Wei-Tao Cong, Sun-Young Hwang, Li-Tai Jin and Jung-Kap Choi
74 Detection of proteins on Western blots using colorimetric and radiometric vusialization of Secondary Ligands
N. J. Kruger
75. Identification of Glycoproteins on Nitrocellulose Membranes Using Lectin Blotting
P. Gravel
76. A Sensitive Method to Quantitatively Detect Total Protein on Membranes after Electrophoretic Transfer Using Avidin- or Streptavidin-Biotin
W.J. LaRochelle
77. Detection and Quantification of Proteins on Immunoblots using Enhanced Chemiluminescence
J. Young
78 Reutilization of Western Blots After Chemiluminescent Detection or Autoradiography
S. Kaufmann
79 The use of quantum dot luminescent probes for Western blot analysis
S. Makrides, C. Gasbarro and J.M.Bello
80 The use of infrared fluorescent dyes in quantitative immunoblotting
C-H Yang, C. Kasbek, and H. A. Fisk
81 The use of infrared fluorescent dyes in immunofluorescence microscopy
C. Kasbek, C-H Yang and H. A. Fisk
Part IV : CHEMICAL MODIFICATION OF PROTEINS AND PEPTIDE
PRODUCTION, PURIFICATION AND CHARACTERISATION
82 Carboxymethylation of Cysteine Using Iodoacetamide/Iodoacetic Acid
A. Aitken and Michele Learmonth
83 Performic Acid Oxidation
A. Aitken and Michele Learmonth
84 Succinylation of Protein
A. Aitken and Michele Learmonth
85 Pyridylethylation of Cysteine Residues
M. Ward
86. Side-Chain Selective Chemical Modifications of Proteins
D. S. Tawfik
87 Nitration of Tyrosines
D. S. Tawfik
88 Ethoxyformylation of Histidine
D. S. Tawfik
89 Modification of Arginine Side Chains with p-Hydroxyphenylglyoxal
D. S. Tawfik
90 Amidination of Carboxyl Groups
D. S. Tawfik
91 Amidination of Lysine Side Chains
D.S. Tawfik
92 Modification of tryptophan with 2-Hydroxy-5-Nitrogenzylbromide
D. S. Tawfik
93. Modification of Sulhydryl Groups with DTNB
D. S. Tawfik
94 Chemical Cleavage of Proteins at Methionyl-X Peptide Bonds
B.J. Smith
95 Chemical Cleavage of Proteins at Tryptophanyl-X Peptide Bonds
B. J. Smith
96 Chemical Cleavage of Proteins at Aspartyl-X Peptide Bonds
B. J.Smith
97 Chemical Cleavage of Proteins at Cysteinyl-X Peptide Bonds
B. J. Smith
98 Chemical Cleavage of Proteins at Asparaginyl-Glycyl Peptide Bonds
B.J. Smith
99 Enzymatic Digestion of Proteins in Solution and in SDS Polyacrylamide Gels
K.L. Stone, E.E.Gulcicek and K.R. Williams
100 On-PVDF protein digestions for N-terminal sequencing and peptide mass fingerprinting
Victoria Pham, William Henzel & Jennie R Lill
101 Enzymatic Digestion of Proteins on PVDF membranes
J. Fernandez and S. Mische
102 Reverse Phase HPLC Separation of Enzymatic Digests of Proteins
K.L. Stone and K.R. Williams
103 Peptide Mapping by Two-Dimensional Thin-Layer Electrophoresis-Thin-Layer Chromatography
R. C. Judd
104 Peptide Mapping by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
R. C. Judd
105 Peptide Mapping by High-Performance Liquid Chromatography
P. Højrup
106 Production of Protein Hydrolysates Using Enzymes
J. M. Walker and P. J. Sweeney
107 Amino Acid Analysis by Precolumn Derivatization with 1- Fluoro-2,4-Dinitrophenyl-5-L-Alanine Amide (Marfey’s Reagent)
S. Kochhar and P. Christen
108 Amino acid analysis in protein hydrolysates using anion exchange chromatography and IPAD detection
P. Jandik, Jun Cheng and N. Avdalovic
109 Validation of amino acid analysis
A. Reason
110 Molecular Weight Estimation for Native Proteins Using High-Performance Size Exclusion Chromatography
G. B. Irvine
111 Detection of Disulfide-Linked Peptides by HPLC
A. Aitken and M. Learmonth
112 Detection of Disulfide-Linked Peptides by Mass Spectrometry
A.Aitken and M. Learmonth
113 Diagonal Electrophoresis for Detecting Disulfide Bridges
A. Aitken and M. Learmonth
114. Estimation of Disulfide Bonds Using Ellman’s Reagent
A. Aitken and M. Learmonth
115 Quantitation of Cysteine Residues and Disulfide Bonds by Electrophoresis
A. Aitken and M. Learmonth
116 N-Terminal sequencing of the N-terminally Modified Proteins
R. Kamp and Hisashi Hirano
117 De-blocking of Proteins containing N-Terminal Pyroglutamic Acidc
J. Mozdzanowski
118 Detection and characterization of protein mutations by mass spectrometry
Y. Wada
119 Peptide Sequencing by Nanoelectrospray Tandem Mass Spectrometry
O. Nørregaard Jensen and M. Wilm
120 Protein identification by Peptide mass fingerprinting using MALDI-TOFF Mass spectrometry
J. Webster and D. Oxley
121 Protein ladder sequencing
Rong Wang and Brian T. Chait
122. Sequence Analysis with WinGene/WinPep
L. Hennig
123 HPLC and MS of integral membrane proteins
J. Whitelegge
124 Enrichment of Serum Peptides and Analysis by MALDI-TOF Mass Spectrometry
H.W. Ressom, R.S.Varghese and R. Goldman
125 Computational Methods for the Analysis of MALDI-TOF Spectra to Discover Peptide Serum Biomarkers
Y. An, H.W. Ressom and R. Goldman
Part V : POST-TRANSLATIONAL MODIFICATIONS
126 Simple tools for complex N-glycan analysis
A-C. Fitchette, M. Benchabane, T. Paccalet, L. Faye and V. Gomord
127 A Lectin-Binding Assay for the Rapid Characterization of the Glycosylation of Purified Gycoproteins
M.T. Goodarzi, A. Fotinopoulou and G. A. Turner
128 Chemical Methods of Analysis of Glycoproteins
E.F. Hounsell, M.J. Davies and K.D. Smith
129 Monosaccharide Analysis by HPAEC
E.F. Hounsell, M.J. Davies and K.D. Smith
130 Monosaccharide Analysis by GC
E.F. Hounsell, M.J. Davies and K.D. Smith
131 Determination of monosaccharide linkage and Substitution Patterns by GC.MS Methylation Analysis
E.F. Hounsell, M.J. Davies and K.D. Smith
132 Sialic Acid Analysis by HPAEC-PAD
E.F. Hounsell, M.J. Davies and K.D. Smith
133 Chemical Release of O-Linked Oligosaccharide Chains
E.F. Hounsell, M.J. Davies and K.D. Smith
134 O-Linked Oligosaccharide Profiling by HPLC
E.F. Hounsell, M.J. Davies and K.D. Smith
135 O-Linked Oligosaccharide Profiling by HPAEC-PAD
E.F. Hounsell, M.J. Davies and K.D. Smith
136 Release of N-Linked Oligosaccharide Chains by Hydrazinolysis
T.Mizuochi and E.F. Hounsell
137 Enzymatic Release of O- and N-Linked Oligosaccharide Chains
E.F. Hounsell, M.J. Davies and K.D. Smith
138 N-Linked Oligosaccharide Profiling by HPLC on Porous Graphitized Carbon (PGC)
E.F. Hounsell, M.J. Davies and K.D. Smith
139 N-linked Oligosaccharide Profiling by HPAEC-PAD
E.F. Hounsell, M.J. Davies and K.D. Smith
140 HPAE-PAD analysis of monosaccharides released by exoglycosidase digestion
Weitzhandler, M., J. Rohrer, J,R Thayer and N. Avdalovic
141 Microassay Analyses of Protein Glycosylation
N.K.C. Wong, N. Kanu, N. Thandrayen, G,J.Rademaker, C.I. Baldwin, D.V. Renouf and E.F. Hounsell
142 Polyacrylamide gel electrophoresis of fluorophore-labeled carbohydrates from glycoproteins
B. Brandley, J. Klock and C. Starr
143 HPLC Analysis of fluorescently labelled glycans
A. Merry and S. Astrautsova
144 Glycoprofiling purified glycoproteins using Surface Plasmon Resonance
A. Fotinopoulou and G.A. Turner
145 Sequencing Heparan Sulphate Saccharides
J. E. Turnbull
146 Analysis of glycoprotein heterogeneity by Capillary Electrophoresis and Mass Spectrometry
A. Hooker and D.C. James
147 Affinity chromatography of oligosaccharides and glycopeptides with immobilized lectins
K. Yamamoto
148 In-Gel Enzymatic Release of N-glycans
D. Harvey
149 Analysis of N-Linked glycans by mass spectrometry
D. Harvey
150 MS Analysis of Protein glycosylation
N.Takemori, N. Komori and H. Matsumoto
151 Mapping protein N-glycosylation by COFRADIC
B. Ghesquiere, J. Vandekerckhove, and K. Gevaert
152 Mass spectrometric analysis of O-linked glycans released directly from Glycoproteins in gels using ß-elimination
A.M. Taylor and J. Thomas-Oates
153 Glycopeptide analysis using LC/MS and LC/MSn
S.Itoh, D. Takakura, N. Kawasaki and T. Yamaguchi
154 Identification of Vitamin K-Dependent Proteins Using a Gla-specific Monoclonal Antibody
K. Hansson
155 The identification of protein S-Nitrocysteine
T.M Greco, S.L.Stamer, D.C. Liebler and H. Ischiropoulos
156 Detection of nitrotyrosine-containing proteins
Xianquan Zhan and D. Desiderio
157 Mass spectrometric determination of protein Ubiquitination
C. E. Parker, M.R.E. Warren, V. Mocanu, S.F. Greer and C.H. Borchers
158 Detection of Sumoylated Proteins
Ok-Kyong Park-Sarge and K. Sarge
159 Efficient enrichment of intact phosphorylated proteins by modified immobilized metal-affinity chromatography
A. Dubrovska
160 Analyzing Protein Phosphorylation
J. Colyer
161 Mass spectrometric analysis of protein phosphorylation
S. Gander, A. Cremonesi, J. Chicher, S. Moes and P. Jenö
162 Protein microarrays for phosphorylation studies
B. Kersten and T. Feilner
163. Two Dimensional Phosphopeptide Mapping
H. Nagahara, R.R. Latek, S.A. Ezhevsky and S.F. Dowdy
164 Identification of Proteins Modified by Protein (D-Aspartyl/L-Isoaspartyl) Carboxyl Methyltransferase
D. Weber and P.N. McFadden
165 Analysis of Tyrosine-o- sulfation
Jens R. Bundgaard, J.W. Sen, A.H. Johnsen and J.F. Rehfeld
166 Analysis of Protein Palmitoylation by metabolic radiolabeling methods
K.H. Pedone, L.S. Bernstein, Maurine E. Linder and J.R. Hepler
167 Incorporation of radiolabeled prenyl alcohols and their analogs into mammalian cell proteins: a useful tool for studying protein prenylation
A. Corsini, C.C. Farnsworth, P. McGeady, M.H. Gelb and J.A. Glomset
168 Labelling and analysis of isoprenylated proteins in cells
D.A. Andres, D.C. Crick, H.P.Spielmann and C. J. Waechter
Part VII : ANTIBODY TECHNIQUES
169 Antibody Production
R. Burns
170 Production of Antibodies Using Proteins in Gel Bands
S.A. Amero, T.C. James and S.C.R. Elgin
171 Raising Highly Specific Polyclonal Antibodies Using Biocompatible Support
Bound Antigens
M. Diano and A. Le Bivic
172 Production of antisera using peptide conjugates
T. E. Adrian
173 Small-molecule protein conjugation procedures
S. Thompson
174 The Chloramine T Method for Radiolabeling Protein
G.S. Bailey
175 The Lactoperoxidase Method for Radiolabeling Protein
G.S. Bailey
176 The Bolton and Hunter Method for Radiolabeling Protein
G.S. Bailey
177 Preparation of 125I-labelled peptides and proteins with high specific activity
using iodogen
M. Conlon
178 Purification and Assessment of Quality of Radioiodinated Protein
G.S. Bailey
179 Purification of IgG by Precipitation with Sodium Sulfate or Ammoniun Sulfate
M. Page and R. Thorpe
180 Purification of IgG Using Caprylic Acid
M. Page and R. Thorpe
181 Purification of IgG Using DEAE-Sepharose Chromatography
M. Page and R. Thorpe
182 Purification of IgG Using Ion-Exchange HPLC
M. Page and R. Thorpe
183 Purification of IgG by Precipitation with Polyethylene Glycol (PEG)
M. Page and R. Thorpe
184 Purification of IgG Using Protein A or Protein G
M. Page and R. Thorpe
185 Purification of IgG Using Size Exclusion High Performance Liquid
Chromatography (SE-HPLC)
C. Dolman and R. Thorpe
186 Purification of IgG Using Affinity Chromatography on Antigen-Ligand
Columns
M. Page and R. Thorpe
187 Purification of IgG Using Thiophilic Chromatography
M. Page and R. Thorpe
188 Analysis of IgG Fractions by Electrophoresis
M. Page and R. Thorpe
189 Purification of Immunoglobulin Y (IgY) from Chicken Eggs
C.R. Bird and R. Thorpe
190 Affinity Purification of Immunoglobulins Using PAM (Protein A Mimetic)
G. Fassina G. Palombo, A. Verdoliva and M. Ruvo
191 Detection of Serological Cross Reactions by Western Cross Blotting
P. Hemmerl, A. Hartl, J. Freund and J.Thalhamer
192 Enzymatic Digestion of Monoclonal Antibodies
S. Andrew
193 How to Make Bispecific Antibodies
R. French
194 Antigen Measurements Using ELISA
W. Jordan
195 Enhanced chemiluminescence immunoassay
R. Stott
196 Immunoprecipitation and Blotting
S. Thompson
197 Determination of Epitope by Mass Spectrometry
C.Hager-Braun and K. B. Tomer
198 Immunogen Preparation and Immunization Procedures for Rats and Mice
M. Page and R. Thorpe
199 Making Hybridomas
R. Burns
200 Growing hybridomas
G. Entrican, C. Jepson and D. Deane
201 Mouse Hybridomas as an Entryway to Monoclonal Antibody Design
and Production
E. Mechetner
202 Pitfalls
E. Mechetner
203 Recombinant antibody expression and purification
A. Knappik and R. Brundiers
204 Screening Hybridoma Culture Supernatants Using Solid-Phase Radiobinding
Assay
M. Page and R. Thorpe
205 Screening Hybridoma Culture Supernatants Using ELISA
M. Page and R. Thorpe
206 Growth and Purification of Murine Monoclonal Antibodies
M. Page and R. Thorpe
207 Affinity purification techniques for monoclonal antibodies
A. Schwarz
208 A rapid method for generating large numbers of high-affinity monoclonal antibodies from a single mouse
N.T. Man and G.E. Morris
