The Protein Protocols Handbook

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.