The Protein Protocols Handbook - 3rd edition

Edited by J M Walker 
Springer  October 2009  

Hardcover  1464 pp  ISBN 9781588298805      £109.00

Softcover  1440 pp  ISBN 9781603274746      £79.00
Since 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 this book is an 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 armametarium of techniques in the urgent search for rapid and robust results.

Written for: Protein scientists, biochemists, molecular and cellular biologists.



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


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

43 Quantitation of Proteins on Polyacrylamide Gels , J. Smith

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. Moller 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


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


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. Hojrup

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. Norregaard 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


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


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

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