the protein protocols handbook

Protein Protocols Edited by John M. Walker The Handbook SECOND EDITION Humana Press Protein Protocols Edited by John M. Walker The Handbook SECOND EDITION Humana Press The Protein Protocols Handbook HUMANA PRESS TOTOWA, NEW JERSEY Edited by John M. Walker University of Hertfordshire, Hatfield, UK The Protein Protocols Handbook SECOND EDITION © 2002 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 humanapress.com All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. The content and opinions expressed in this book are the sole work of the authors and editors, who have warranted due diligence in the creation and issuance of their work. 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Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 Library of Congress Cataloging in Publication Data The Protein Protocols Handbook: Second Edition / edited by John M. Walker. p. cm. ISBN 0-89603-940-4 (HB); 0-89603-941-2 (PB) Includes bibliographical references and index. 1. Proteins Analysis Laboratory manuals. I. Walker, John M., 1948- Qp551 .P697512 2002 572'.6 dc21 2001039829 Preface v 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 Biology™ 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 step- 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. Hence, for this second edition I have introduced 60 chapters/protocols not present in the first edition, significantly updated a number of chapters remaining from the first edition, and increased the overall length of the book from 144 to 164 chapters. The new chapters particularly reflect the considerable developments in the use of mass spectrometry in protein characterization. Recognition of the now well-established central role of 2-D PAGE in proteomics has resulted in an expansion of chapters on this subject, and I have also included a number of new techniques for staining and analyzing protein blots. The section on glycoprotein analysis has been significantly expanded, and aspects of single chain antibodies and phage-displayed antibodies have been introduced in the section on antibodies. We each, of course, have our own favorite, commonly used methods, be it a gel system, gel-staining method, blotting method, and so on; I’m sure you will find yours here. However, I have, as before, also described alternatives for some of these tech- niques; though they may not be superior to the methods you commonly use, they may nevertheless be more appropriate in a particular situation. Only by knowing the range of techniques that are available to you, and the strengths and limitations of these techniques, will you be able to choose the method that best suits your purpose. Good luck in your protein analysis! John M. Walker Contents Preface v Contributors xix PART I: QUANTITATION OF PROTEINS 1 Protein Determination by UV Absorption Alastair Aitken and Michèle P. Learmonth 3 2 The Lowry Method for Protein Quantitation Jakob H. Waterborg 7 3 The Bicinchoninic Acid (BCA) Assay for Protein Quantitation John M. Walker 11 4 The Bradford Method for Protein Quantitation Nicholas J. Kruger 15 5 Ultrafast Protein Determinations Using Microwave Enhancement Robert E. Akins and Rocky S. Tuan 23 6 The Nitric Acid Method for Protein Estimation in Biological Samples Scott A. Boerner, Yean Kit Lee, Scott H. Kaufmann, and Keith C. Bible 31 7 Quantitation of Tryptophan in Proteins Alastair Aitken and Michèle P. Learmonth 41 8 Flow Cytometric Quantitation of Cellular Proteins Thomas D. Friedrich, F. Andrew Ray, Judith A. Laffin, and John M. Lehman 45 9 Kinetic Silver Staining of Proteins Douglas D. Root and Kuan Wang 51 PART II: E LECTROPHORESIS OF P ROTEINS AND P EPTIDES AND D ETECTION IN G ELS 10 Nondenaturing Polyacrylamide Gel Electrophoresis of Proteins John M. Walker 57 11 SDS Polyacrylamide Gel Electrophoresis of Proteins John M. Walker 61 12 Gradient SDS Polyacrylamide Gel Electrophoresis of Proteins John M. Walker 69 13 SDS-Polyacrylamide Gel Electrophoresis of Peptides Ralph C. Judd 73 vii viii Contents 14 Identification of Nucleic Acid Binding Proteins Using Nondenaturing Sodium Decyl Sulfate Polyacrylamide Gel Electrophoresis (SDecS-Page) Robert E. Akins and Rocky S. Tuan 81 15 Cetyltrimethylammonium Bromide Discontinuous Gel Electrophoresis of Proteins: M r -Based Separation of Proteins with Retained Native Activity Robert E. Akins and Rocky S. Tuan 87 16 Acetic–Acid–Urea Polyacrylamide Gel Electrophoresis of Basic Proteins Jakob H. Waterborg 103 17 Acid–Urea–Triton Polyacrylamide Gel Electrophoresis of Histones Jakob H. Waterborg 113 18 Isoelectric Focusing of Proteins in Ultra-Thin Polyacrylamide Gels John M. Walker 125 19 Protein Solubility in Two-Dimensional Electrophoresis: Basic Principles and Issues Thierry Rabilloud 131 20 Preparation of Protein Samples from Mouse and Human Tissues for 2-D Electrophoresis Joachim Klose 141 21 Radiolabeling of Eukaryotic Cells and Subsequent Preparation for 2-D Electrophoresis Nick Bizios 159 22 Two-Dimensional Polyacrylamide Gel Electrophoresis Using Carrier Ampholyte pH Gradients in the First Dimension Patricia Gravel 163 23 Casting Immobilized pH Gradients (IPGs) Elisabetta Gianazza 169 24 Nonequilibrium pH Gel Electrophoresis (NEPHGE) Mary F. Lopez 181 25 Difference Gel Electrophoresis Mustafa Ünlü and Jonathan Minden 185 26 Comparing 2-D Electrophoretic Gels Across Internet Databases Peter F. Lemkin and Gregory C. Thornwall 197 27 Immunoblotting of 2-D Electrophoresis Separated Proteins Barbara Magi, Luca Bini, Sabrina Liberatori, Roberto Raggiaschi, and Vitaliano Pallini 215 28 Quantification of Radiolabeled Proteins in Polyacrylamide Gels Wayne R. Springer 231 29 Quantification of Proteins on Polyacrylamide Gels Bryan John Smith 237 Contents ix 30 Rapid and Sensitive Staining of Unfixed Proteins in Polyacrylamide Gels with Nile Red Joan-Ramon Daban, Salvador Bartolomé, Antonio Bermúdez, and F. Javier Alba 243 31 Zinc-Reverse Staining Technique Carlos Fernandez-Patron 251 32 Protein Staining with Calconcarboxylic Acid in Polyacrylamide Gels Jung-Kap Choi, Hee-Youn Hong, and Gyurng-Soo Yoo 259 33 Detection of Proteins in Polyacrylamide Gels by Silver Staining Michael J. Dunn 265 34 Background-Free Protein Detection in Polyacrylamide Gels and on Electroblots Using Transition Metal Chelate Stains Wayne F. Patton 273 35 Detection of Proteins in Polyacrylamide Gels by Fluorescent Staining Michael J. Dunn 287 36 Detection of Proteins and Sialoglycoproteins in Polyacrylamide Gels Using Eosin Y Stain Fan Lin and Gary E. Wise 295 37 Electroelution of Proteins from Polyacrylamide Gels Paul Jenö and Martin Horst 299 38 Autoradiography and Fluorography of Acrylamide Gels Antonella Circolo and Sunita Gulati 307 PART III: BLOTTING AND DETECTION METHODS 39 Protein Blotting by Electroblotting Mark Page and Robin Thorpe 317 40 Protein Blotting by the Semidry Method Patricia Gravel 321 41 Protein Blotting by the Capillary Method John M. Walker 335 42 Protein Blotting of Basic Proteins Resolved on Acid-Urea-Trinton-Polyacrylamide Gels Geneviève P. Delcuve and James R. Davie 337 43 Alkaline Phosphatase Labeling of IgG Antibody G. Brian Wisdom 343 44 β-Galactosidase Labeling of IgG Antibody G. Brian Wisdom 345 45 Horseradish Peroxidase Labeling of IgG Antibody G. Brian Wisdom 347 46 Digoxigenin (DIG) Labeling of IgG Antibody G. Brian Wisdom 349 47 Conjugation of Fluorochromes to Antibodies Su-Yau Mao 351 48 Coupling of Antibodies with Biotin Rosaria P. Haugland and Wendy W. You 355 49 Preparation of Avidin Conjugates Rosaria P. Haugland and Mahesh K. Bhalgat 365 50 MDPF Staining of Proteins on Western Blots F. Javier Alba and Joan-Ramon Daban 375 51 Copper Iodide Staining of Proteins and Its Silver Enhancement Douglas D. Root and Kuan Wang 381 52 Detection of Proteins on Blots Using Direct Blue 71 Hee-Youn Hong, Gyurng-Soo Yoo, and Jung-Kap Choi 387 53 Protein Staining and Immunodetection Using Immunogold Susan J. Fowler 393 54 Detection of Polypeptides on Immunoblots Using Enzyme-Conjugated or Radiolabeled Secondary Ligands Nicholas J. Kruger 405 55 Utilization of Avidin- or Streptavidin-Biotin as a Highly Sensitive Method to Stain Total Proteins on Membranes Kenneth E. Santora, Stefanie A. Nelson, Kristi A. Lewis, and William J. LaRochelle 415 56 Detection of Protein on Western Blots Using Chemifluorescence Catherine Copse and Susan J. Fowler 421 57 Quantification of Proteins on Western Blots using ECL Joanne Dickinson and Susan J. Fowler 429 58 Reutilization of Western Blots After Chemiluminescent Detection or Autoradiography Scott H. Kaufmann 439 PART IV: CHEMICAL MODIFICATION OF PROTEINS AND PEPTIDE PRODUCTION AND PURIFICATION 59 Carboxymethylation of Cysteine Using Iodoacetamide/Iodoacetic Acid Alastair Aitken and Michèle P. Learmonth 455 60 Performic Acid Oxidation Alastair Aitken and Michèle P. Learmonth 457 61 Succinylation of Proteins Alastair Aitken and Michèle P. Learmonth 459 62 Pyridylethylation of Cysteine Residues Malcolm Ward 461 x Contents 63 Side Chain Selective Chemical Modifications of Proteins Dan S. Tawfik 465 64 Nitration of Tyrosines Dan S. Tawfik 469 65 Ethoxyformylation of Histidine Dan S. Tawfik 473 66 Modification of Arginine Side Chains with p -Hydroxyphenylglyoxal Dan S. Tawfik 475 67 Amidation of Carboxyl Groups Dan S. Tawfik 477 68 Amidination of Lysine Side Chains Dan S. Tawfik 479 69 Modification of Tryptophan with 2-Hydroxy-5-Nitrobenzylbromide Dan S. Tawfik 481 70 Modification of Sulfhydryl Groups with DTNB Dan S. Tawfik 483 71 Chemical Cleavage of Proteins at Methionyl-X Peptide Bonds Bryan John Smith 485 72 Chemical Cleavage of Proteins at Tryptophanyl-X Peptide Bonds Bryan John Smith 493 73 Chemical Cleavage of Proteins at Aspartyl-X Peptide Bonds Bryan John Smith 499 74 Chemical Cleavage of Proteins at Cysteinyl-X Peptide Bonds Bryan John Smith 503 75 Chemical Cleavage of Proteins at Asparaginyl-Glycyl Peptide Bonds Bryan John Smith 507 76 Enzymatic Digestion of Proteins in Solution and in SDS Polyacrylamide Gels Kathryn L. Stone and Kenneth R. Williams 511 77 Enzymatic Digestion of Membrane-Bound Proteins for Peptide Mapping and Internal Sequence Analysis Joseph Fernandez and Sheenah Mische 523 78 Reverse Phase HPLC Separation of Enzymatic Digests of Proteins Kathryn L. Stone and Kenneth R. Williams 533 PART V: PROTEIN/PEPTIDE CHARACTERIZATION 79 Peptide Mapping by Two-Dimensional Thin-Layer Electrophoresis–Thin-Layer Chromatography Ralph C. Judd 543 Contents xi [...]... similarly 3 Following the heating step, the color developed is stable for at least 1 h 4 Note, that like the Lowry assay, response to the BCA assay is dependent on the amino acid composition of the protein, and therefore an absolute concentration of protein cannot be determined The BSA standard curve can only therefore be used to compare the relative protein concentration of similar protein solutions 5... the protein content by removing the contribution to absorbance by nucleotides at 280 nm, by measuring the A260 which is largely owing to the latter (6) Protein (mg/mL) = 1.55 A280 0.76 A260 (5) Other formulae (using similar principles of absorbance differences) employed to determine protein in the possible presence of nucleic acids are the following (7,8): Protein (mg/mL) = (A235 A280)/2.51 (6) Protein. .. estimated by determining the amount of dye in the blue ionic form This is usually achieved by measuring the absorbance of the solution at 595 nm (see Note 2) The dye appears to bind most readily to arginyl and lysyl residues of proteins (3,4) This specificity can lead to variation in the response of the assay to different proteins, which is the main drawback of the method (see Note 3) The original Bradford... of protein, and the microassay, which detects between 1 and 10 àg of protein The latter, although more sensitive, is also more prone to interference from other compounds because of the greater amount of sample relative to dye reagent in this form of the assay From: The Protein Protocols Handbook, 2nd Edition Edited by: J M Walker â Humana Press Inc., Totowa, NJ 15 16 Kruger 2 Materials 1 Reagent: The. .. 50% (9) 12 The amount of color produced in this assay by any given protein (or mixture of proteins) is dependent on the amino acid composition of the protein( s) (see Introduction) Therefore, two different proteins, each for example at concentrations of 1 mg/mL, can give different color yields in this assay It must be appreciated, therefore, that using bovine serum albumin (BSA) (or any other protein for... Analyt Biochem 82, 362371 The Lowry Method 7 2 The Lowry Method for Protein Quantitation Jakob H Waterborg 1 Introduction The most accurate method of determining protein concentration is probably acid hydrolysis followed by amino acid analysis Most other methods are sensitive to the amino acid composition of the protein, and absolute concentrations cannot be obtained (1) The procedure of Lowry et al... determined The extinction of nucleic acid in the 280-nm region may be as much as 10 times that of protein at their same wavelength, and hence, a few percent of nucleic acid can greatly influence the absorption 1.2 Far UV Absorbance The peptide bond absorbs strongly in the far UV with a maximum at about 190 nm This very strong absorption of proteins at these wavelengths has been used in protein determination... other protein for that matter) as a standard gives only an approximate measure of the protein concentration The only time when this method gives an absolute value for protein concentration is when the protein being analyzed is also used to construct the standard curve The most accurate way to determine the concentration of any protein solution is amino acid analysis 13 A means of speeding up this assay... contributions to the A205 (3) The advantages of this method include simplicity and sensitivity As in the method outlined in Subheading 3.1 the sample is recoverable and in addition there is little variation in response between different proteins, permitting near-absolute determination of protein Disadvantages of this method include the necessity for accurate calibration of the spectrophotometer in the far UV... comparison of the BCA, Lowry and Bradford assays for analyzing gylcosylated and non-glycosylated proteins have been made (6) Significant differences wee observed between the assays for non-glycosylated proteins with the BCA assay giving results closest to those from amino acid analysis Glycosylated proteins were underestimated by the Bradford the method and overestimated by the BCA and Lowry methods The results . Protein Protocols Edited by John M. Walker The Handbook SECOND EDITION Humana Press Protein Protocols Edited by John M. Walker The Handbook SECOND EDITION Humana Press The Protein Protocols Handbook HUMANA. or otherwise without written permission from the Publisher. The content and opinions expressed in this book are the sole work of the authors and editors, who have warranted due diligence in the. for the first time. All chapters are written in the same format as that used in the Methods in Molecular Biology™ series. Each chapter opens with a description of the basic theory behind the method