CiNii Books - The protein protocols handbook

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, 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 Contents

Protein Determination by UV Absorption.- The Lowry Method for Protein Quantitation.- The Bicinchoninic Acid (BCA) Assay for Protein Quantitation.- The Bradford Method for Protein Quantitation.- Ultrafast Protein Determinations Using Microwave Enhancement.- The Nitric Acid Methods for Protein Estimation in Biological Samples.- Quantitation of Tryptophan in Proteins.- Kinetic Silver Staining of Proteins.- Quantitation of cellular proteins by flow cytometry.- Quantitation of cellular proteins by laser scanning cytometry.- Protein Solubility in 2D Electrophoresis: Basic principles and issues.- Mouse and Human Tissues Sample Preparation for 2-D Electrophoresis.- Plant Protein sample preparation for 2DE.- Preparation of bacterial samples for 2-D PAGE.- Preparation of bodily fluids for 2-D PAGE.- Immunoaffinity Depletion of high abundance plasma and serum proteins.- Preparation of Yeast samples for 2D PAGE.- Membrane Protein Preparation Using Aqueous Polymer Two Phase Systems.- Subcellular fractionation of small sample amounts.- Nondenaturing Polyacrylamide Gel Electrophoresis of Proteins.- SDS Polyacrylamide Gel Electrophoresis of Proteins.- Gradient SDS Polyacrylamide Gel Electrophoresis of Proteins.- SDS-Polyacrylamide Gel Electrophoresis of Peptides.- Blue native gel electrophoresis (BN-PAGE).- Separation of proteins by gel electrophoresis in the Tris-Taurine-HCl system.- Cetyltrimethylammonium Bromide Discontinuous Gel Electrophoresis of Proteins: M -Based Separation of Proteins with Retained Native Activity.- Acetic Acid-Urea Polyacrylamide Gel Electrophoresis of Basic Proteins.- Acid-Urea-Triton Polyacrylamide Gel Electrophoresis of Histones.- Isoelectric Focusing of Proteins in Ultra-Thin Polyacrylamide Gels.- Serial immobilized pH gradient isoelectric focusing over pH 4-9.- Radiolabelling of Eukaryotic Cells and Subsequent Preparation for 2-D.- Two-Dimensional PAGE Using Carrier Ampholyte pH Gradients in the First Dimension.- Vertical agarose electrophoresis and electroblotting of high molecular weight proteins.- 2D PAGE of high molecular weight proteins.- Casting immobilised pH gradients.- Nonequilibrium pH Gel Electrophoresis.- Microchip capillary electrophoresis.- Protein Separations in Microfluidic Chips.- Difference gel electrophoresis (DIGE).- Comparing 2-D Electrophoresis Gels Across Internet Databases.- Quantification of Radiolabeled Proteins in Polyacrylamide Gels 25.4.07.- Differential ProteoTope radioactive quantification of protein abundance ratios.- Quantitation of Proteins on Polyacrylamide Gels.- Using SDS-PAGE and scanning laser densitometry to measure proteins.- Rapid and Sensitive Staining of Unfixed Proteins in Polyacrylamide Gels with Nile Red.- Zinc reverse staining technique.- Protein Staining with Calconcarboxylic Acid in Polyacrylamide Gels.- Detection of Proteins in Polyacrylamide Gels by Silver Staining.- Background-free Protein Detection on Polyacrylamide Gels and on Electroblots Using Transition Metal Chelate Stains.- Detection of Proteins in Polyacrylamide Gels by Fluorescent Staining.- Detection of Glycoproteins in Gels and Blots.- Staining of Glycoproteins/Proteoglycans on SDS-Gels.- Detection of Proteins and Sialoglycoproteins in Polyacrylamide Gels Using Eosin X stain.- Pro-Q Diamond phosphoprotein staining.- Electroelution of Proteins from Polyacrylamide Gels.- Autoradiography and Fluorography of Acrylamide Gels.- Proteolytic Activity Detection by Two-Dimensional Zymography.- Protein Blotting by Electroblotting.- Protein Blotting by the Semi-dry Method.- Protein Blotting by the Capillary Method.- Western Blotting of basic proteins electrophoretically resolved on acid-urea-Triton-polyacrylamide gels.- Immunoblotting of 2-DE Separated Proteins.- High efficiency blotting of high-molecular weight proteins.- Alkaline Phosphatase Labeling of IgG Antibody.- b-Galactosidase Labeling of IgG Antibody.- Horseradish Peroxidase Labeling of IgG Antibody.- Digoxigenin (DIG) Labelling of IgG Antibody.- Conjugation of fluorochromes to antibodies.- Coupling of Antibodies with Biotin.- Preparation of Avidin Conjugates.- MDPF Staining of Proteins on Western Blots.- Copper Iodided Staining of Proteins and its silver enhancement.- Detection of Proteins on Blots using Direct Blue 71.- Detection of proteins on Western blots using colorimetric and radiometric vusialization of Secondary Ligands.- Identification of Glycoproteins on Nitrocellulose Membranes Using Lectin Blotting.- A Sensitive Method to Quantitatively Detect Total Protein on Membranes after Electrophoretic Transfer Using Avidin- or Streptavidin-Biotin.- Detection and Quantification of Proteins on Immunoblots using Enhanced Chemiluminescence.- Reutilization of Western Blots After Chemiluminescent Detection or Autoradiography.- The use of quantum dot luminescent probes for Western blot analysis.- The use of infrared fluorescent dyes in quantitative immunoblotting.- The use of infrared fluorescent dyes in immunofluorescence microscopy.- Carboxymethylation of Cysteine Using Iodoacetamide/Iodoacetic Acid.- Performic Acid Oxidation.- Succinylation of Protein.- Pyridylethylation of Cysteine Residues.- Side-Chain Selective Chemical Modifications of Proteins.- Nitration of Tyrosines.- Ethoxyformylation of Histidine.- Modification of Arginine Side Chains with p-Hydroxyphenylglyoxal.- Amidination of Carboxyl Groups.- Amidination of Lysine Side Chains.- Modification of tryptophan with 2-Hydroxy-5-Nitrogenzylbromide.- Modification of Sulhydryl Groups with DTNB.- Chemical Cleavage of Proteins at Methionyl-X Peptide Bonds.- Chemical Cleavage of Proteins at Tryptophanyl-X Peptide Bonds.- Chemical Cleavage of Proteins at Aspartyl-X Peptide Bonds.- Chemical Cleavage of Proteins at Cysteinyl-X Peptide Bonds.- Chemical Cleavage of Proteins at Asparaginyl-Glycyl Peptide Bonds.- Enzymatic Digestion of Proteins in Solution and in SDS Polyacrylamide Gels.- On-PVDF protein digestions for N-terminal sequencing and peptide mass fingerprinting.- Enzymatic Digestion of Proteins on PVDF membranes.- Reverse Phase HPLC Separation of Enzymatic Digests of Proteins.- Peptide Mapping by Two-Dimensional Thin-Layer Electrophoresis-Thin-Layer Chromatography.- Peptide Mapping by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis.- Peptide Mapping by High-Performance Liquid Chromatography.- Production of Protein Hydrolysates Using Enzymes.- Amino Acid Analysis by Precolumn Derivatization with 1- Fluoro-2,4-Dinitrophenyl-5-L-Alanine Amide (Marfey’s Reagent).- Amino acid analysis in protein hydrolysates using anion exchange chromatography and IPAD detection.- Validation of amino acid analysis.- Molecular Weight Estimation for Native Proteins Using High-Performance Size Exclusion Chromatography.- Detection of Disulfide-Linked Peptides by HPLC.- Detection of Disulfide-Linked Peptides by Mass Spectrometry.- Diagonal Electrophoresis for Detecting Disulfide Bridges.- Estimation of Disulfide Bonds Using Ellman’s Reagent.- Quantitation of Cysteine Residues and Disulfide Bonds by Electrophoresis.- N-Terminal sequencing of the N-terminally Modified Proteins.- De-blocking of Proteins containing N-Terminal Pyroglutamic Acid.- Detection and characterization of protein mutations by mass spectrometry.- Peptide Sequencing by Nanoelectrospray Tandem Mass Spectrometry.- Protein identification by Peptide mass fingerprinting using MALDI-TOFF Mass spectrometry.- Protein ladder sequencing.- Sequence Analysis with WinGene/WinPep.- HPLC and MS of integral membrane proteins.- Enrichment of Serum Peptides and Analysis by MALDI-TOF Mass Spectrometry.- Computational Methods for the Analysis of MALDI-TOF Spectra to Discover Peptide Serum Biomarkers.- Simple tools for complex N-glycan analysis.- A Lectin-Binding Assay for the Rapid Characterization of the Glycosylation of Purified Gycoproteins.- Chemical Methods of Analysis of Glycoproteins.- Monosaccharide Analysis by HPAEC.- Monosaccharide Analysis by GC.- Determination ofmonosaccharide linkage and Substitution Patterns by GC.MS Methylation Analysis.- Sialic Acid Analysis by HPAEC-PAD.- Chemical Release of O-Linked Oligosaccharide Chains.- O-Linked Oligosaccharide Profiling by HPLC.- O-Linked Oligosaccharide Profiling by HPAEC-PAD.- Release of N-Linked Oligosaccharide Chains by Hydrazinolysis.- Enzymatic Release of O- and N-Linked Oligosaccharide Chains.- N-Linked Oligosaccharide Profiling by HPLC on Porous Graphitized Carbon (PGC).- N-linked Oligosaccharide Profiling by HPAEC-PAD.- HPAE-PAD analysis of monosaccharides released by exoglycosidase digestion.- Microassay Analyses of Protein Glycosylation.- Polyacrylamide gel electrophoresis of fluorophore-labeled carbohydrates from glycoproteins.- HPLC Analysis of fluorescently labelled glycans.- Glycoprofiling purified glycoproteins using Surface Plasmon Resonance.- Sequencing Heparan Sulphate Saccharides.- Analysis of glycoprotein heterogeneity by Capillary Electrophoresis and Mass Spectrometry.- Affinity chromatography of oligosaccharides and glycopeptides with immobilized lectins.- In-Gel Enzymatic Release of N-glycans.- Analysis of N-Linked glycans by mass spectrometry.- MS Analysis of Protein glycosylation.- Mapping protein N-glycosylation by COFRADIC.- Mass spectrometric analysis of O-linked glycans released directly from Glycoproteins in gels using ß-elimination.- Glycopeptide analysis using LC/MS and LC/MSn.- Identification of Vitamin K-Dependent Proteins Using a Gla-specific Monoclonal Antibody.- The identification of protein S-Nitrocysteine.- Detection of nitrotyrosine-containing proteins.- Mass spectrometric determination of protein Ubiquitination.- Detection of Sumoylated Proteins.- Efficient enrichment of intact phosphorylated proteins by modified immobilized metal-affinity chromatography.- Analyzing Protein Phosphorylation.- Mass spectrometric analysis of protein phosphorylation.- Protein microarrays for phosphorylation studies.- TwoDimensional Phosphopeptide Mapping.- Identification of Proteins Modified by Protein (D-Aspartyl/L-Isoaspartyl) Carboxyl Methyltransferase.- Analysis of Tyrosine-o- sulfation.- Analysis of Protein Palmitoylation by metabolic radiolabeling methods.- Incorporation of radiolabeled prenyl alcohols and their analogs into mammalian cell proteins: a useful tool for studying protein prenylation.- Labelling and analysis of isoprenylated proteins in cells.- Antibody Production.- Production of Antibodies Using Proteins in Gel Bands.- Raising Highly Specific Polyclonal Antibodies Using Biocompatible Support Bound Antigens.- Production of antisera using peptide conjugates.- Small-molecule protein conjugation procedures.- The Chloramine T Method for Radiolabeling Protein.- The Lactoperoxidase Method for Radiolabeling Protein.- The Bolton and Hunter Method for Radiolabeling Protein.- Preparation of 125I-labelled peptides and proteins with high specific activity using iodogen.- Purification and Assessment of Quality of Radioiodinated Protein.- Purification of IgG by Precipitation with Sodium Sulfate or Ammoniun Sulfate.- Purification of IgG Using Caprylic Acid.- Purification of IgG Using DEAE-Sepharose Chromatography.- Purification of IgG Using Ion-Exchange HPLC.- Purification of IgG by Precipitation with Polyethylene Glycol (PEG).- Purification of IgG Using Protein A or Protein G.- Purification of IgG Using Size Exclusion High Performance Liquid Chromatography (SE-HPLC).- Purification of IgG Using Affinity Chromatography on Antigen-Ligand Columns.- Purification of IgG Using Thiophilic Chromatography.- Analysis of IgG Fractions by Electrophoresis.- Purification of Immunoglobulin Y (IgY) from Chicken Eggs.- Affinity Purification of Immunoglobulins Using PAM (Protein A Mimetic).- Detection of Serological Cross Reactions by Western Cross Blotting.- Enzymatic Digestion of Monoclonal Antibodies.- How to Make Bispecific Antibodies.- Antigen Measurements Using ELISA.- Enhancedchemiluminescence immunoassay.- Immunoprecipitation and Blotting.- Determination of Epitope by Mass Spectrometry.- Immunogen Preparation and Immunization Procedures for Rats and Mice.- Making Hybridomas.- Growing hybridomas.- Mouse Hybridomas as an Entryway to Monoclonal Antibody Design and Production.- Pitfalls.- Recombinant antibody expression and purification.- Screening Hybridoma Culture Supernatants Using Solid-Phase Radiobinding Assay.- Screening Hybridoma Culture Supernatants Using ELISA.- Growth and Purification of Murine Monoclonal Antibodies.- Affinity purification techniques for monoclonal antibodies.- A rapid method for generating large numbers of high-affinity monoclonal antibodies from a single mouse.

Volume: [: pbk] ISBN 9781603274746

Description

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 (TM) 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 Contents

Part 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 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. Jenoe 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. 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 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. Jenoe 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

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