The Protein Protocols Handbook

The Protein Protocols Handbook\r......Page 1
Front Matter\r......Page 3
Preface\r......Page 5
Table of Contents\r......Page 6
Part 1:  Quantitation of Proteins\r......Page 23
Protein Determination by UV Absorption\r......Page 24
The Lowry Method for Protein Quantitation\r......Page 28
The Bicinchoninic Acid (BCA) Assay for Protein Quantitation\r......Page 31
Bradford Method\r......Page 35
Ultrafast Protein Determinations Using Microwave Enhancement\r......Page 42
Nitric Acid Method for Protein Estimation in Biological Samples\r......Page 50
Quantitation of Tryptophan in Proteins\r......Page 60
Flow Cytometric Quantitation of Cellular Proteins\r......Page 64
Kinetic Silver Staining of Proteins\r......Page 70
Part 2:  Electrophoresis of Proteins and Peptides and Detection in Gels\r......Page 74
Nondenaturing Polyacrylamide Gel Electrophoresis of Proteins\r......Page 75
SDS Polyacrylamide Gel Electrophoresis of Proteins\r......Page 79
Gradient SDS-PAGE of Proteins\r......Page 86
SDS-PAGE of Peptides\r......Page 90
Identification of Nucleic Acid Binding Proteins Using Nondenaturing SDS-PAGE (SDecS-PAGE)\r......Page 97
Cetyltrimethylammonium Bromide (CTAB)\r Discontinuous Gel Electrophoresis of Proteins......Page 103
Acetic Acid-Urea Polyacrylamide Gel Electrophoresis of Basic Proteins\r......Page 118
Acid-Urea-Triton Polyacrylamide Gel Electrophoresis of Histones\r......Page 127
Isoelectric Focusing of Proteins in Ultra-Thin Polyacrylamide Gels\r......Page 138
Protein Solubility in Two-Dimensional Electrophoresis\r......Page 143
Preparation of Protein Samples from Mouse and Human Tissues for 2-D Electrophoresis\r......Page 153
Radiolabeling of Eukaryotic Cells and Subsequent Preparation for 2-Dimensional Electrophoresis\r......Page 170
Two-Dimensional PAGE of Proteins Using Carrier Ampholyte pH Gradients in the First Dimension\r......Page 174
Casting Immobilized pH Gradients (IPGs)\r......Page 180
Nonequilibrium pH Gel Electrophoresis (NEPHGE)\r......Page 192
Part 3:  Blotting and Detection Methods\r......Page 317
Part 4:  Chemical Modification of Proteins and Peptide Production and Purification\r......Page 449
Carboxymethylation of Cys Using Iodoacetamide/Iodoacetic Acid\r......Page 450
Performic Acid Oxidation\r......Page 452
Succinylation of Proteins\r......Page 454
Pyridylethylation of Cys Residues\r......Page 456
Side Chain Selective Chemical Modifications of Proteins\r......Page 459
Nitration of Tyr\r......Page 462
Ethoxyformylation of His\r......Page 466
Modification of Arg Side Chains with p-Hydroxyphenylglyoxal\r......Page 468
Amidation of Carboxyl Groups\r......Page 470
Amidination of Lys Side Chains\r......Page 472
Modification of Trp with 2-Hydroxy-5-Nitrobenzylbromide\r......Page 474
Modification of Sulfhydryl Groups with DTNB\r......Page 476
Chemical Cleavage of Proteins at Methionyl-X-Peptide Bonds\r......Page 478
Chemical Cleavage of Proteins at Tryptophanyl-X Peptide Bonds\r......Page 485
Chemical Cleavage of Proteins at Aspartyl-X Peptide Bonds\r......Page 491
Chemical Cleavage of Proteins at Cysteinyl-X Peptide Bonds\r......Page 495
Chemical Cleavage of Proteins at Asparaginyl-X Peptide Bonds \r......Page 499
Enzymatic Digestion of Proteins in Solution and in SDS-PAGE gels\r......Page 503
Enzymatic Digestion of Membrane-Bound Proteins for Peptide Mapping and Internal Sequence Analysis\r......Page 514
Part 5:  Protein/Peptide Characterization\r......Page 532
Peptide Mapping by Two-Dimensional Thin-Layer Electrophoresis--Thin-Layer Chromatography\r......Page 533
Peptide Mapping by SDS-PAGE\r......Page 543
Peptide Mapping by HPLC\r......Page 548
Production of Protein Hydrolysates Using Enzymes\r......Page 551
Amino Acid Analysis of Precolumn Derivatization with 1-Fluor-2,4-Dinitrophenyl-5-L-Alanine Amide (Marfrey\'s Reagent)\r......Page 555
MW Estimation for Native Proteins Using High-Performance Size-Exclusion Chromatography\r......Page 561
Detection of Disulfide-Linked Peptides by HPLC\r......Page 568
Detection of Disulfide-Linked Peptides by Mass Spectrometry\r......Page 571
Diagonal Electrophoresis for Detecting Disulfide Bridges\r......Page 575
Estimation of Disulfide Bonds Using Ellman\'s Reagent\r......Page 580
Quantitation of Cys Residues and Disulfide Bonds by Electrophoresis\r......Page 582
Analyzing Protein Phosphorylation\r......Page 588
Mass Spectrometric Analysis of Protein Phosphorylation\r......Page 594
Identification of Proteins Modified by Protein (D-Aspartyl/L-Isoaspartyl) Carboxyl Methyltransferases\r......Page 608
Analysis of Protein Palmitoylation\r......Page 617
Incorporation of Radiolabeled Prenyl Alchohols and Their Analogs into Mammalian Cell Proteins\r......Page 624
The Metabolic Labeling and Analysis of Isoprenylated Proteins\r......Page 640
2-D Phosphopeptide Mapping\r......Page 655
Detection and Characterization of Mutations by Mass Spectrometry\r......Page 663
Peptide Sequencing by Nanoelectrospray Tandem Mass Spectrometry\r......Page 675
MALDI-MS for Protein Identification Using Peptide and Fragment Ion Masses\r......Page 693
Protein Ladder Sequencing\r......Page 715
Sequence Analysis with WinGene/WinPep\r......Page 722
Isolation of Proteins Cross-Linked to DNA with Cisplatin\r......Page 728
Isolation of Proteins Cross-Linked to DNA with Formaldehyde\r......Page 733
Part 6:  Glycoproteins\r......Page 738
Detection of Glycoproteins in Gels and Blots\r......Page 739
Staining of Glycoproteins in SDS Gels\r......Page 751
Identification of Glycoproteins on Nitrocellulose Membranes Using Lectin Binding\r......Page 756
A Lectin-Binding Assay for Rapid Characteriztion of the Glycosylation of Purified Glycoproteins\r......Page 772
Chemical Methods of Analysis of Glycoproteins\r......Page 780
Monosaccharide Analysis by HPAEC\r......Page 782
Monosaccharide Analysis by Gas Chromatography (GC)\r......Page 785
Determination of Monosaccharide Linkage and Substitution Patterns by GC-MS Methylation Analysis\r......Page 787
Sialic Acid Analysis by HPAEC-PAD\r......Page 790
Chemical Release of O-Linked Oligosaccharide Chains\r......Page 792
O-Linked Oligosaccharide Profiling by HPLC\r......Page 794
O-Linked Oligosaccharide Profiling by HPAEC-PAD\r......Page 796
Release of N-Linked Oligosaccharide Chains by Hydrazinolysis\r......Page 798
Enzymatic Release of O- and N-Linked Oligosaccharide Chains\r......Page 802
N-Linked Oligosaccharide Profiling by HPLC on Porous Graphitized Carbon (PGC)\r......Page 804
N-Linked Oligosaccharide Profiling by HPAEC-PAD\r......Page 806
HPAEC-PAD Analysis of Monosaccharides Released by Exoglycosidase Digestion Using the CarboPac MA1 Column\r......Page 808
Microassay Analyses of Protein Glycosylation\r......Page 815
Polyacrylamide Gel Electrophoresis of Fluorophore-Labeled Carbohydrates from Glycoproteins\r......Page 825
HPLC Analysis of Fluorescently Labeled Glycans\r......Page 838
Glycoprofiling Purified Glyocproteins Using Surface Plasmon Resonance\r......Page 857
Sequencing Heparan Sulfate Saccharides\r......Page 864
Analysis of Glycoprotein Heterogeneity by Capillary Electrophoresis and Mass Spectrometry\r......Page 876
Affinity Chromatography of Oligosaccharides and Glycopeptides with Immobilized Lectins\r......Page 887
Part 7:  Antibody Technologies\r......Page 902
Antibody Production\r......Page 903
Production of Antbodies Using Proteins in Gel Bands\r......Page 909
Raising Highly Specific Polyclonal Antibodies Using Biocompatible Support-Bound Antigens\r......Page 913
Production of Antisera Using Peptide Conjugates\r......Page 921
The Chloramine T Method for Radiolabeling Protein\r......Page 930
The Lactoperoxidase Method for Radiolabeling Protein\r......Page 933
The Bolton and Hunter Method for Radiolabeling Protein\r......Page 935