The Protein Protocols Handbook

Front Cover
John M. Walker
Springer, Oct 2, 2007 - Science - 1146 pages
The Protein Protocols Handbook, Second Edition aims to provide a cross-section of analytical techniques commonly used for proteins and peptides, thus providing a benchtop manual and guide for those who are new to the protein chemistry laboratory and for those more established workers who wish to use a technique for the first time. All chapters are written in the same format as that used in the Methods in Molecular BiologyTM series. Each chapter opens with a description of the basic theory behind the method being described. The Materials section lists all the chemicals, reagents, buffers, and other materials necessary for carrying out the protocol. Since the principal goal of the book is to provide experimentalists with a full account of the practical steps necessary for carrying out each protocol successfully, the Methods section contains detailed st- by-step descriptions of every protocol that should result in the successful execution of each method. The Notes section complements the Methods material by indicating how best to deal with any problem or difficulty that may arise when using a given technique, and how to go about making the widest variety of modifications or alterations to the protocol. Since the first edition of this book was published in 1996 there have, of course, been significant developments in the field of protein chemistry.
 

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Contents

Amino Acid Analysis by Precolumn Derivatization with 1Fluoro24Dinitrophenyl5LAlanine Amide Marfeys Reagent
567
Molecular Weight Estimation for Native Proteins Using HighPerformance SizeExclusion Chromatography
573
Detection of DisulfideLinked Peptides by HPLC
581
Detection of DisulfideLinked Peptides by Mass Spectrometry
585
Diagonal Electrophoresis for Detecting Disulfide Bridges
589
Estimation of Disulfide Bonds Using Ellmans Reagent
595
Quantitation of Cysteine Residues and Disulfide Bonds by Electrophoresis
597
Analyzing Protein Phosphorylation
603

Kinetic Silver Staining of Proteins
51
ELECTROPHORESIS OF PROTEINS AND PEPTIDES AND DETECTION IN GELS
55
Nondenaturing Polyacrylamide Gel Electrophoresis of Proteins
57
SDS Polyacrylamide Gel Electrophoresis of Proteins
61
Gradient SDS Polyacrylamide Gel Electrophoresis of Proteins
69
SDSPolyacrylamide Gel Electrophoresis of Peptides
73
Identification of Nucleic Acid Binding Proteins Using Nondenaturing Sodium Decyl Sulfate Polyacrylamide Gel Electrophoresis SDecSPAGE
81
Cetyltrimethylammonium Bromide Discontinuous Gel Electrophoresis of Proteins MrBased Separation of Proteins with Retained Native Activity
87
Acetic AcidUrea Polyacrylamide Gel Electrophoresis of Basic Proteins
103
AcidUreaTriton Polyacrylamide Gel Electrophoresis of Histones
113
Isoelectric Focusing of Proteins in UltraThin Polyacrylamide Gels
125
Protein Solubility in TwoDimensional Electrophoresis Basic Principles and Issues
131
Preparation of Protein Samples from Mouse and Human Tissues for 2D Electrophoresis
141
Radiolabeling of Eukaryotic Cells and Subsequent Preparation for 2Dimensional Electrophoresis
159
TwoDimensional Polyacrylamide Gel Electrophoresis of Proteins Using Carrier Ampholyte pH Gradients in the First Dimension
163
Casting Immobilized pH Gradients IPGs
169
Nonequilibrium pH Gel Electrophoresis NEPHGE
181
Difference Gel Electrophoresis
185
Comparing 2D Electrophoresis Gels Across Internet Databases
197
Immunoblotting of 2D Electrophoresis Separated Proteins
215
Quantification of Radiolabeled Proteins in Polyacrylamide Gels
231
Quantification of Proteins on Polyacrylamide Gels
237
Rapid and Sensitive Staining of Unfixed Proteins in Polyacrylamide Gels with Nile Red
243
ZincReverse Staining Technique
251
Protein Staining with Calconcarboxylic Acid in Polyacrylamide Gels
259
Detection of Proteins in Polyacrylamide Gels by Silver Staining
265
BackgroundFree Protein Detection in Polyacrylamide Gels and on Electroblots Using Transition Metal Chelate Stains
273
Detection of Proteins in Polyacrylamide Gels by Fluorescent Staining
287
Detection of Proteins and Sialoglycoproteins in Polyacrylamide Gels Using Eosin Y Stain
295
Electroelution of Proteins from Polyacrylamide Gels
299
Autoradiography and Fluorography of Acrylamide Gels
307
BLOTTING AND DETECTION METHODS
315
Protein Blotting by Electroblotting
317
Protein Blotting by the Semidry Method
321
Protein Blotting by the Capillary Method
335
Protein Blotting of Basic Proteins Resolved on AcidUreaTritonPolyacrylamide Gels
337
Alkaline Phosphatase Labeling of IgG Antibody
343
βGalactosidase Labeling of IgG Antibody
345
Horseradish Peroxidase Labeling of IgG Antibody
347
Digoxigenin DIG Labeling of IgG Antibody
349
Conjugation of Fluorochromes to Antibodies
351
Coupling of Antibodies with Biotin
355
Preparation of Avidin Conjugates
365
MDPF Staining of Proteins on Western Blots
375
Copper Iodide Staining of Proteins and Its Silver Enhancement
381
Detection of Proteins on Blots Using Direct Blue 71
387
Protein Staining and Immunodetection Using Immunogold
393
Detection of Polypeptides on Immunoblots Using Enzyme Conjugated or Radiolabeled Secondary Ligands
405
Utilization of Avidin or StreptavidinBiotin as a Highly Sensitive Method to Stain Total Protein on Membranes
415
Detection of Proteins on Western Blots Using Chemifluorescence
421
Quantification of Proteins on Western Blots Using ECL
429
Reutilization of Western Blots After Chemiluminescent Detection or Autoradiography
439
CHEMICAL MODIFICATION OF PROTEINS AND PEPTIDE PRODUCTION AND PURIFICATION
453
Carboxymethylation of Cysteine Using Iodoacetamide Iodoacetic Acid
455
Performic Acid Oxidation
457
Succinylation of Proteins
459
Pyridylethylation of Cysteine Residues
461
Side Chain Selective Chemical Modifications of Proteins
465
Nitration of Tyrosines
469
Ethoxyformylation of Histidine
473
Modification of Arginine Side Chains with pHydroxyphenylglyoxal
475
Amidation of Carboxyl Groups
477
Amidination of Lysine Side Chains
479
Modification of Tryptophan with 2Hydroxy5 Nitrobenzylbromide
481
Modification of Sulfhydryl Groups with DTNB
483
Chemical Cleavage of Proteins at MethionylX Peptide Bonds
485
Chemical Cleavage of Proteins at TryptophanylX Peptide Bonds
493
Chemical Cleavage of Proteins at AspartylX Peptide Bonds
499
Chemical Cleavage of Proteins at CysteinylX Peptide Bonds
503
Chemical Cleavage of Proteins at AsparaginylGlycyl Peptide Bonds
507
Enzymatic Digestion of Proteins in Solution and in SDS Polyacrylamide Gels
511
Enzymatic Digestion of MembraneBound Proteins for Peptide Mapping and Internal Sequence Analysis
523
ReversePhase HPLC Separation of Enzymatic Digests of Proteins
533
PROTEINPEPTIDE CHARACTERIZATION
541
Peptide Mapping by TwoDimensional ThinLayer ElectrophoresisThinLayer Chromatography
543
Peptide Mapping by Sodium Dodecyl SulfatePolyacrylamide Gel Electrophoresis
553
Peptide Mapping by HighPerformance Liquid Chromatography
559
Production of Protein Hydrolysates Using Enzymes
563
Mass Spectrometric Analysis of Protein Phosphorylation
609
Identification of Proteins Modified by Protein DAspartylLIsoaspartyl Carboxyl Methyltransferase
623
Analysis of Protein Palmitoylation
633
Incorporation of Radiolabeled Prenyl Alcohols and Their Analogs into Mammalian Cell Proteins
641
The Metabolic Labeling and Analysis of Isoprenylated Proteins
657
2D Phosphopeptide Mapping
673
Detection and Characterization of Protein Mutations by Mass Spectrometry
681
Peptide Sequencing by Nanoelectrospray Tandem Mass Spectrometry
693
MatrixAssisted Laser DesorptionIonization Mass Spectrometry for Protein Identification Using Peptide and Fragment Ion Masses
711
Protein Ladder Sequencing
733
Sequence Analysis with WinGeneWinPep
741
Isolation of Proteins Crosslinked to DNA by Cisplatin
747
Isolation of Proteins Crosslinked to DNA by Formaldehyde
753
GLYCOPROTEINS
759
Detection of Glycoproteins in Gels and Blots
761
Staining of GlycoproteinsProteoglycans in SDSGels
773
Identification of Glycoproteins on Nitrocellulose Membranes Using Lectin Blotting
779
A LectinBinding Assay for the Rapid Characterization of the Glycosylation of Purified Glycoproteins
795
Chemical Methods of Analysis of Glycoproteins
803
Monosaccharide Analysis by HPAEC
805
Monosaccharide Analysis by Gas Chromatography GC
809
Determination of Monosaccharide Linkage and Substitution Patterns by GCMS Methylation Analysis
811
Sialic Acid Analysis by HPAECPAD
815
Chemical Release of OLinked Oligosaccharide Chains
817
OLinked Oligosaccharide Profiling by HPLC
819
OLinked Oligosaccharide Profiling by HPAECPAD
821
Release of NLinked Oligosaccharide Chains by Hydrazinolysis
823
Enzymatic Release of O and NLinked Oligosaccharide Chains
827
NLinked Oligosaccharide Profiling by HPLC on Porous Graphitized Carbon PGC
829
NLinked Oligosaccharide Profiling by HPAECPAD
831
HPAECPAD Analysis of Monosaccharides Released by Exoglycosidase Digestion Using the CarboPac MA1 Column
833
Microassay Analyses of Protein Glycosylation
841
Polyacrylamide Gel Electrophoresis of FluorophoreLabeled Carbohydrates from Glycoproteins
851
HPLC Analysis of Fluorescently Labeled Glycans
865
Glycoprofiling Purified Glycoproteins Using Surface Plasmon Resonance
885
Sequencing Heparan Sulfate Saccharides
893
Analysis of Glycoprotein Heterogeneity by Capillary Electrophoresis and Mass Spectrometry
905
Affinity Chromatography of Oligosaccharides and Glycopeptides with Immobilized Lectins
917
ANTIBODY TECHNIQUES
933
Antibody Production
935
Production of Antibodies Using Proteins in Gel Bands
941
Raising Highly Specific Polyclonal Antibodies Using Biocompatible SupportBound Antigens
945
Production of Antisera Using Peptide Conjugates
953
The Chloramine T Method for Radiolabeling Protein
963
The Lactoperoxidase Method for Radiolabeling Protein
967
The Bolton and Hunter Method for Radiolabeling Protein
969
Preparation of ⁵ILabeled Peptides and Proteins with High Specific Activity Using IODOGEN
971
Purification and Assessment of Quality of Radioiodinated Protein
979
Purification of IgG Using Caprylic Acid
985
Purification of IgG Using DEAESepharose Chromatography
987
Purification of IgG Using IonExchange HPLC
989
Purification of IgG by Precipitation with Polyethylene Glycol PEG
991
Purification of IgG Using Protein A or Protein G
993
Analysis and Purification of IgG Using SizeExclusion High Performance Liquid Chromatography SEHPLC
995
Purification of IgG Using Affinity Chromatography on AntigenLigand Columns
999
Purification of IgG Using Thiophilic Chromatography
1003
Analysis of IgG Fractions by Electrophoresis
1005
Purification of Immunoglobulin Y IgY from Chicken Eggs
1009
Affinity Purification of Immunoglobulins Using Protein A Mimetic PAM
1013
Detection of Serological CrossReactions by Western CrossBlotting
1025
Bacterial Expression Purification and Characterization of SingleChain Antibodies
1035
Enzymatic Digestion of Monoclonal Antibodies
1047
How to Make Bispecific Antibodies
1053
Phage Display
1059
Screening of Phage Displayed Antibody Libraries
1073
Antigen Measurement Using ELISA
1083
Enhanced Chemiluminescence Immunoassay
1089
Immunoprecipitation
1097
MONOCLONAL ANTIBODIES
1107
Immunogen Preparation and Immunization Procedures for Rats and Mice
1109
Hybridoma Production
1111
Screening Hybridoma Culture Supernatants Using SolidPhase Radiobinding Assay
1115
Screening Hybridoma Culture Supernatants Using ELISA
1117
Growth and Purification of Murine Monoclonal Antibodies
1119
Affinity Purification Techniques for Monoclonal Antibodies
1121
A Rapid Method for Generating Large Numbers of HighAffinity Monoclonal Antibodies from a Single Mouse
1129
Index
1139
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