The Protein Protocols Handbook

John M. Walker (Redaktør)

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. Les mer
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Om boka

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

Fakta

Innholdsfortegnelse

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