The Protein Protocols Handbook

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ISBN: 9780896033399 | 0896033392
Cover: Paperback
Copyright: 1/1/1996

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-- 145 concise, step-by-step protocols for the analysis of proteins and peptides
-- Provides more than three times as many protocols than the competitors' manuals -- in the incomparable Methods in Molecular Biology "TM" style -- at an affordable price
-- Combound in a large 7 x l0 inch format for easy benchtop use
-- An indispensable reference for industry-based researchers, professors, and their students

Preface

Contributors

Part I: Quantitation of Proteins

Protein Determination by UV Absorption

(4)

Alastair Aitken

Michele Learmonth

The Lowry Method for Protein Quantitation

(4)

Jakob H. Waterborg

Harry R. Matthews

The Bicinchoninic Acid (BCA) Assay for Protein Quantitation

(4)

John M. Walker

The Bradford Method for Protein Quantitation

(6)

Nicholas J. Kruger

Ultrafast Protein Determinations Using Microwave Enhancement

(8)

Robert E. Akins

Rocky S. Tuan

Quantitation of Tryptophan in Proteins

(4)

Alastair Aitken

Michele Learmonth

Protein Quantitation Using Flow Cytometry

(6)

F. Andrew Ray

Thomas D. Friedrich

Judith Laffin

John M. Lehman

Copper Iodide Staining of Proteins on Solid Phases

(6)

Douglas D. Root

Kuan Wang

Kinetic Silver Staining of Proteins Adsorbed to Microtiter Plates

(6)

Douglas D. Root

Kuan Wang

Part II: electrophoresis of Proteins and Peptides and Detection in Gels

Nondenaturing Polyacrylamide Gel Electrophoresis of Proteins

(4)

John M. Walker

SDS Polyacrylamide Gel Electrophoresis of Proteins

(8)

John M. Walker

Gradient SDS Polyacrylamide Gel Electrophoresis of Proteins

(4)

John M. Walker

Cetyltrimethylammonium Bromide Discontinuous Gel Electrophoresis of Proteins: M-Based Separation of Proteins with Retained Native Activity

(16)

Robert E. Akins

Rocky S. Tuan

Acetic Acid-Urea Polyacrylamide Gel Electrophoresis of Basic Proteins

(8)

Jakob H. Waterborg

Acid-Urea-Triton Polyacryamide Gels for Histones

(10)

Jakob H. Waterborg

SDS-Polyacrylamide Gel Electrophoresis of Peptides

101

(8)

Ralph C. Judd

Isoelectric Focusing of Proteins in Ultra-Thin Polyacrylamide Gels

109

(6)

John M. Walker

Isoelectric Focusing Under Denaturing Conditions

115

(6)

Christopher F. Thurston

Lucy F. Henley

Radioisotopic Labeling of Proteins for Polyacrylamide Gel Electrophoresis

121

(6)

Jeffrey W. Pollard

Two-Dimensional Page Using Carrier Ampholyte pH Gradients in the First Dimension

127

(6)

Patricia Gravel

Olivier Golaz

Two-Dimensional Page Using Immobilized pH Gradients

133

(6)

Graeme R. Guy

Robin J. Philp

Two-Dimensional Page Using Flat-Bed IEF in the First Dimension

139

(4)

Robin J. Philp

Free Zone Capillary Electrophoresis

143

(12)

David J. Begley

Capillary Isoelectric Focusing with Electro-Osmotic Flow Mobilization

155

(6)

Thomas J. Pritchett

Quantification of Radiolabeled Proteins in Polyacrylamide Gels

161

(6)

Wayne R. Springer

Quantification of Proteins by Staining in Polyacrylamide Gels

167

(6)

Bryan John Smith

Quantitation of Proteins Separated by Electrophoresis Using Coomassie Brilliant Blue

173

(6)

Ulf Neumann

Rapid Staining of Proteins in Polyacrylamide Gels with Nile Red

179

(8)

Joan-Ramon Daban

Salvador Bartolome

Antonio Bermudez

Zn2+-Reverse Staining of Proteins in Polyacrylamide Gels

187

(10)

Juan M. Garcia-Segura

Mercedes Ferreras

Protein Staining with Calconcarboxylic Acid in Polyacrylamide Gels

197

(6)

Jung-Kap Choi

Hee-Youn Hong

Gyurng-Soo Yoo

Detection of Proteins and Sialoglycoproteins in Polyacrylamide Gels Using Eosin Y Stain

203

(4)

Fan Lin

Gary E. Wise

Electroelution of Proteins from Polyacrylamide Gels

207

(8)

Paul Jeno

Martin Horst

High-Performance Electrophoresis Chromatography

215

(8)

Serge Desnoyers

Sylvie Bourassa

Guy G. Poirier

Drying Polyacrylamide Gels

223

(6)

Bryan John Smith

Detection of Proteins in Polyacrylamide Gels by Silver Staining

229

(6)

Michael J. Dunn

Autoradiography and Fluorography of Acrylamide Gels

235

(10)

Antonella Circolo

Sunita Gulati

Part III: Blotting and Detection Methods

Protein Blotting by Electroblotting

245

(4)

Mark Page

Robin Thorpe

Protein Blotting by the Semidry Method

249

(12)

Patricia Gravel

Olivier Golaz

Protein Blotting by the Capillary Method

261

(2)

John M. Walker

Protein Blotting of Basic Proteins Resolved on Acid-Urea-Triton-Polyacrylamide Gels

263

(6)

Genevieve P. Delcuve

James R. Davie

Alkaline Phosphatase Labeling of Antibody Using Glutaraldehyde

269

(2)

G. Brian Wisdom

β-Galactosidase Labeling of Antibody Using MBS

271

(2)

G. Brian Wisdom

Horseradish Peroxidase Labeling of Antibody Using Periodate Oxidation

273

(2)

G. Brian Wisdom

Protein Staining and Immunodetection Using Colloidal Gold

275

(14)

Susan J. Fowler

Fluorescent Protein Staining on Nitrocellulose with Subsequent Immunodetection of Antigen

289

(4)

Donald F. Summers

Boguslaw Szewczyk

Coupling of Antibodies with Biotin

293

(10)

Rosaria P. Haugland

Wendy W. You

Preparation of Avidin Conjugates

303

(10)

Rosaria P. Haugland

Mahesh K. Bhalgat

Detection of Polypeptides on Blots Using Secondary Antibodies or Protein A

313

(10)

Nicholas J. Kruger

Detection of Proteins on Blots Using Avidin-or Streptavidin-Biotin

323

(6)

William J. LaRochelle

Detection of Proteins on Blots Using Chemiluminescent Systems

329

(10)

Graeme R. Guy

Part IV: Chemical Modification of Proteins and Peptide Production and Purification

Carboxymethylation of Cysteine Using lodoacetamide/lodoacetic Acid

339

(2)

Alastair Aitken

Michele Learmonth

Performic Acid Oxidation

341

(2)

Alastair Aitken

Michele Learmonth

Succinylation of Proteins

343

(2)

Alastair Aitken

Michele Learmonth

Pyridylethylation of Cysteine Residues

345

(4)

Malcolm Ward

Side-Chain Selective Chemical Modifications of Proteins

349

(4)

Dan S. Tawfik

Nitration of Tyrosines

353

(4)

Dan S. Tawfik

Ethoxyformylation of Histidine

357

(2)

Dan S. Tawfik

Modification of Arginine Side Chains with p-Hydroxyphenylglyoxal

359

(2)

Dan S. Tawfik

Amidation of Carboxyl Groups

361

(2)

Dan S. Tawfik

Amidination of Lysine Side Chains

363

(2)

Dan S. Tawfik

Modification of Tryptophan with 2-Hydroxy-5-Nitrobenzylbromide

365

(2)

Dan S. Tawfik

Modification of Sulfhydryl Groups with DTNB

367

(2)

Dan S. Tawfik

Chemical Cleavage of Proteins at Methionyl Residues

369

(6)

Bryan John Smith

Chemical Cleavage of Proteins at Tryptophan Residues

375

(6)

Bryan John Smith

Chemical Cleavage of Proteins at Aspartyl Residues

381

(4)

Bryan John Smith

Chemical Cleavage of Proteins at Cysteinyl Residues

385

(4)

Bryan John Smith

Chemical Cleavage of Proteins at Asparaginyl-Glycyl Peptide Bonds

389

(4)

Bryan John Smith

Preparation of Peptides for Microsequencing from Proteins in Polyacrylamide Gels

393

(6)

Robin J. Philp

In Situ Chemical and Enzymatic Digestions of Proteins Immobilized on Miniature Hydrophobic Columns

399

(6)

Malcolm Ward

Enzymatic Digestion of Membrane-Bound Proteins for Peptide Mapping and Internal Sequence Analysis

405

(10)

Joseph Fernandez

Sheenah M. Mische

Enzymatic Digestion of Proteins in Solution and in SDS Polyacrylamide Gels

415

(12)

Kathryn L. Stone

Kenneth R. Williams

Reverse-Phase HPLC Separation of Enzymatic Digests of Proteins

427

(10)

Kathryn L. Stone

Kenneth R. Williams

Part V: Protein/Peptide Characterization

Peptide Mapping by Two-Dimensional Thin-Layer Electrophoresis-Thin-Layer Chromatography

437

(10)

Ralph C. Judd

Peptide Mapping by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

447

(6)

Ralph C. Judd

Peptide Mapping by High-Performance Liquid Chromatography

453

(4)

Ralph C. Judd

Production of Protein Hydrolysates Using Enzymes

457

(4)

John M. Walker

Patricia J. Sweeney

Amino Acid Analysis Using Precolumn Derivatization with 6-Aminoquinolyl-n-Hydroxysuccinimidyl Carbamate

461

(6)

Malcolm Ward

Amino Acid Analysis Using Precolumn Derivatization with Phenylisothiocyanate

467

(6)

G. Brent Irvine

Molecular-Weight Estimation for Native Proteins Using Size-Exclusion High-Performance Liquid Chromatography

473

(6)

G. Brent Irvine

Detection of Disulfide-Linked Peptides by HPLC

479

(2)

Alastair Aitken

Michele Learmonth

Diagonal Electrophoresis for Detecting Disulfide Bridges

481

(6)

Alastair Aitken

Michele Learmonth

Estimation of Disulfide Bonds Using Ellman's Reagent

487

(2)

Alastair Aitken

Michele Learmonth

Quantitation of Cysteine Residues and Disulfide Bonds by Electrophoresis

489

(6)

Alastair Aitken

Michele Learmonth

Detection of Disulfide-Linked Peptides by Mass Spectrometry

495

(6)

Alastair Aitken

Michele Learmonth

Analyzing Protein Phosphorylation

501

(6)

John Colyer

Identification of Proteins Modified by Protein (D-Aspartyl/L-Isoaspartyl) Carboxyl Methyltransferase

507

(10)

Darin J. Weber

Philip N. McFadden

Analysis of Protein Palmitoylation

517

(8)

Morag A. Grassie

Graeme Milligan

Removal of Pyroglutamic Acid Residues from the N-Terminus of Peptides and Proteins

525

(4)

John M. Walker

Patricia J. Sweeney

The Dansyl Method for Identifying N-Terminal Amino Acids

529

(6)

John M. Walker

The Dansyl-Edman Method for Peptide Sequencing

535

(6)

John M. Walker

Matrix-Assisted Laser Desorption Ionization Mass Spectrometry as a Complement to Internal Protein Sequencing

541

(16)

Kenneth R. Williams

Suzy M. Samandar

Kathryn L. Stone

Melissa Saylor

John Rush

A Manual C-Terminal Sequencing Procedure for Peptides: The Thiocyanate Degradation Method

557

(12)

Franca Casagranda

John F. K. Wilshire

C-Terminal Sequence Analysis with Carboxypeptidase Y

569

(4)

John M. Walker

Julia S. Winder

Rapid Epitope Mapping by Carboxypeptidase Digestion and Immunoblotting

573

(8)

Philip S. Low

Jie Yuan

Epitope Mapping of a Protein Using the Geysen (PEPSCAN) Procedure

581

(14)

J. Mark Carter

Epitope Mapping of Protein Antigens by Competition ELISA

595

(8)

Glenn E. Morris

Part VI: Glycoproteins

Identification of Glycoproteins on Nitrocellulose Membranes Using Lectin Blotting

603

(16)

Patricia Gravel

Olivier Golaz

A Lectin-Binding Assay for the Rapid Characterization of the Glycosylation of Purified Glycoproteins

619

(8)

Mohammad T. Goodarzi

Graham A. Turner

Staining of Glycoproteins/Proteoglycans on SDS-Gels

627

(6)

Holger J. Moller

Jorgen H. Poulsen

Chemical Methods of Analysis of Glycoproteins

633

(2)

Elizabeth F. Hounsell

Michael J. Davies

Kevin D. Smith

Monosaccharide Analysis by HPAEC

635

(4)

Elizabeth F. Hounsell

Michael J. Davies

Kevin D. Smith

Monosaccharide Analysis by GC

639

(2)

Elizabeth F. Hounsell

Michael J. Davies

Kevin D. Smith

Determination of Monosaccharide Linkage and Substitution Patterns by GC-MS Methylation Analysis

641

(4)

Elizabeth F. Hounsell

Michael J. Davies

Kevin D. Smith

Sialic Acid Analysis by HPAEC-PAD

645

(2)

Elizabeth F. Hounsell

Michael J. Davies

Kevin D. Smith

Chemical Release of O-Linked Oligosaccharide Chains

647

(2)

Elizabeth F. Hounsell

Michael J. Davies

Kevin D. Smith

O-Linked Oligosaccharide Profiling by HPLC

649

(2)

Elizabeth F. Hounsell

Michael J. Davies

Kevin D. Smith

O-Linked Oligosaccharide Profiling by HPAEC-PAD

651

(2)

Elizabeth F. Hounsell

Michael J. Davies

Kevin D. Smith

Release of N-Linked Oligosaccharide Chains by Hydrazinolysis

653

(4)

Tsuguo Mizuochi

Elizabeth F. Hounsell

Enzymatic Release of O-and N-Linked Oligosaccharide Chains

657

(2)

Elizabeth F. Hounsell

Michael J. Devies

Kevin D. Smith

N-Linked Oligosaccharide Profiling by HPLC on Porous Graphitized Carbon (PGC)

659

(2)

Elizabeth F. Hounsell

Michael J. Davies

Kevin D. Smith

N-Linked Oligosaccharide Profiling by HPAEC-PAD

661

(4)

Elizabeth F. Hounsell

Michael J. Davies

Kevin D. Smith

Part VII: Immunochemical Techniques

The Chloramine T Method for Radiolabeling Protein

665

(4)

Graham S. Bailey

The Lactoperoxidase Method for Radiolabeling Protein

669

(2)

Graham S. Bailey

The Bolton and Hunter Method for Radiolabeling Protein

671

(2)

Graham S. Bailey

The Iodogen Method for Radiolabeling Protein

673

(2)

Graham S. Bailey

Purification and Assessment of Quality of Radioiodinated Protein

675

(4)

Graham S. Bailey

Conjugation of Peptides to Carrier Proteins via Glutaraldehyde

679

(10)

J. Mark Carter

Conjugation of Peptide to Carrier Proteins via m-Maleimidobenzoyl-N-Hydroxysuccinimide Ester (MBS)

689

(4)

J. Mark Carter

Conjugation of Peptides to Carrier Protein via Carbodiimide

693

(2)

J. Mark Carter

Raising of Polyclonal Antisera

695

(4)

Graham S. Bailey

Elution of SDS-PAGE Separated Proteins from Immobilon Membranes for Use as Antigens

699

(4)

Donald F. Summers

Boguslaw Szewczyk

Production of Highly Specific Polyclonal Antibodies Using a Combination of 2D Electrophoresis and Nitrocellulose-Bound Antigen

703

(8)

Monique Diano

Andre Le Bivic

Production and Characterization of Antibodies Against Peptides

711

(6)

J. Mark Carter

Production of Antibodies Using Proteins in Gel Bands

717

(4)

Sally Ann Amero

Tharappel C. James

Sarah C. R. Elgin

Purification of IgG by Precipitation with Sodium Sulfate or Ammonium Sulfate

721

(2)

Mark Page

Robin Thorpe

Purification of IgG Using Caprylic Acid

723

(2)

Mark Page

Robin Thorpe

Purification of IgG Using DEAE-Sepharose Chromatography

725

(2)

Mark Page

Robin Thorpe

Purification of IgG Using Ion-Exchange HPLC/FPLC

727

(4)

Mark Page

Robin Thorpe

Purification of IgG by Precipitation with Polyethylene Glycol (PEG)

731

(2)

Mark Page

Robin Thorpe

Purification of IgG Using Protein A or Protein G

733

(2)

Mark Page

Robin Thorpe

Purification of IgG Using Gel-Filtration Chromatography

735

(4)

Mark Page

Robin Thorpe

Purification of IgG Using Affinity Chromatography on Antigen-Ligand Columns

739

(4)

Mark Page

Robin Thorpe

Purification of IgG Using Thiophilic Chromatography

743

(2)

Mark Page

Robin Thorpe

Analysis of IgG Fractions by Electrophoresis

745

(4)

Mark Page

Robin Thorpe

Ouchterlony Double Immunodiffusion

749

(4)

Graham S. Bailey

Single Radial Immunodiffusion

753

(4)

Graham S. Bailey

Rocket Immunoelectrophoresis

757

(6)

John M. Walker

Two-Dimensional Immunoelectrophoresis

763

(8)

John M. Walker

Part VIII: Monoclonal Antibodies

Immunogen Preparation and Immunization Procedures for Rats and Mice

771

(2)

Mark Page

Robin Thorpe

Hybridoma Production

773

(4)

Mark Page

Robin Thorpe

Screening Hybridoma Culture Supernatants Using Solid-Phase Radiobinding Assay

777

(2)

Mark Page

Robin Thorpe

Screening Hybridoma Culture Supernatants Using ELISA

779

(2)

Mark Page

Robin Thorpe

Growth and Purification of Murine Monoclonal Antibodies

781

(2)

Mark Page

Robin Thorpe

A Rapid Method for Generating Large Numbers of High-Affinity Monoclonal Antibodies from a Single Mouse

783

(10)

Nguyen thi Man

Glenn E. Morris

Index

793

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The Protein Protocols Handbook

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Table of Contents

Supplemental Materials

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