Western Blotting: Methods and Protocols

525 Pages·2015·21.04 MB·English

by  Biji T. Kurien, R. Hal Scofield

Methods in
Molecular Biology 1312
Biji T. Kurien
R. Hal Scofi eld Editors
Western
Blotting
Methods and Protocols
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hat fi eld, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651

Western Blotting
Methods and Protocols
Edited by
Biji T. Kurien
Arthritis and Clinical Immunology, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA;
Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA;
Department of Veterans Affairs Medical Center, Oklahoma City, OK, USA
R. Hal Scofield
Arthritis and Clinical Immunology, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA;
Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA;
Department of Veterans Affairs Medical Center, Oklahoma City, OK, USA
Editors
Biji T. K urien R. H al Scofi eld
Arthritis and Clinical Immunology Arthritis and Clinical Immunology
Oklahoma Medical Research Foundation Oklahoma Medical Research Foundation
Oklahoma City, OK , USA Oklahoma City, OK, USA
Department of Medicine, Department of Medicine,
University of Oklahoma Health Sciences Center University of Oklahoma Health Sciences Center
Oklahoma City, OK , USA Oklahoma City, OK, USA
Department of Veterans Affairs Medical Center Department of Veterans Affairs Medical Center
Oklahoma City, OK , USA Oklahoma City, OK, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-2693-0 ISBN 978-1-4939-2694-7 (eBook)
DOI 10.1007/978-1-4939-2694-7
Library of Congress Control Number: 2015938704
Springer New York Heidelberg Dordrecht London
© Springer Science+Business Media New York 2 015
This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not
imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and
regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to
be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,
express or implied, with respect to the material contained herein or for any errors or omissions that may have been made.
Cover illustration: Coomassie stained urea gel and corresponding amido black stained blot of two-dimensional
separations of chicken liver ribosomal proteins. Rearranged and colorized photographs from Towbin, H., Staehelin, T.,
and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure
and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354.
Printed on acid-free paper
Humana Press is a brand of Springer
Springer Science+Business Media LLC New York is part of Springer Science+Business Media (www.springer.com)
Prefa ce
Western Blotting
As we noted in a previous volume of protein blotting, western immunoblotting is a funda-
mental procedure in the modern biomedical research laboratory, as well as in laboratories
doing research in other areas. The technique is routinely used now for research purposes,
for instance, clinical medical laboratories. The development of this technique occurred
within the memory of many of us, including the two of us. But, the variations on the theme
nearly 40 years later are quite remarkable, varying from small variations on the original
theme to techniques that barely resemble the original idea. But, why does the research
enterprise need a compilation of these vast and disparate techniques? It seems to us that
virtually everyone who reads these words will have tried to get a new technique started in
their laboratory by reading methods found in published papers. We imagine that virtually
everyone doing this has found it a daunting task. Similar to cooking recipes, most methods
section or even procedure protocols do not tell one exactly what to do. So, the goal of this
volume is to give practical methods such that you will be able to open this volume or down-
loaded chapter on your lab bench, follow the step-by-step instructions, and successfully
perform a new procedure. If investigators are able to do this, then we will consider our
efforts a success. Since the publication of the last volume, one of the chapter authors has
died. Rafael Pont-Lezica, Professor Emeritus of Plant Physiology, Biochemistry, and
Biotechnology at the Université de Toulouse, died on September 26, 2011. Despite his
emeritus status, he maintained an active engagement in research. He spent much of his
career studying plant cell wall. While a Visiting Professor at Washington University in St.
Louis, Missouri, USA, he developed the tissue printing technique, a creative alternative to
techniques such as in situ hybridization or immunocytochemistry.
Oklahoma City, OK, USA Biji T. Kurien, Ph.D.
R. Hal Scofield, M.D.
v

Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x i
1 The Early Days of Blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Edwin Southern
2 O rigins of Protein Blotting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Harry T owbin
3 W estern Blotting: Remembrance of Things Past . . . . . . . . . . . . . . . . . . . . . . . 9
W. N eal B urnette
4 S imian Virus 40 and Protein Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3
Jaime R enart
5 W estern Blotting: An Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 7
Biji T. K urien and R. H al S cofield
6 F rom Little Helpers to Automation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Biji T. K urien and Harry T owbin
7 S pectrophotometric Methods to Determine Protein Concentration. . . . . . . . . 4 1
J.P . Dean Goldring
8 Solubilization of Proteins: The Importance of Lysis Buffer Choice . . . . . . . . . 4 9
Mandy Peach , Noelle M arsh , Ewa I . M iskiewicz , and Daniel J. M acPhee
9 S imultaneous Immunoblotting Analysis with Activity Gel
Electrophoresis and 2-D Gel Electrophoresis. . . . . . . . . . . . . . . . . . . . . . . . . . 61
Der-Yen Lee and Geen-Dong Chang
10 Diffusion Blotting: A Rapid and Simple Method for Production
of Multiple Blots from a Single Gel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Ingrid O lsen and Harald G . W iker
11 M ultiple Immunoblots by Passive Diffusion of Proteins
from a Single SDS-PAGE Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 7
Biji T. K urien and R. Hal S cofield
12 S lice Blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 7
Graeme Lowe
1 3 Localizing Proteins by Tissue Printing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3
Rafael F . P ont-Lezica
14 D ot-Immunobinding Assay (Dot-Iba). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Sumi S urendran , Annamma M athai , and Vishnampet V enkataraman
Radhakrishnan
15 A nalysis of Antibody Clonotype by Affinity Immunoblotting. . . . . . . . . . . . . . 1 09
Biji T . K urien and R . H al S cofield
16 Glycosaminoglycan Blotting and Detection
After Electrophoresis Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Nicola Volpi and Francesca M accari
vii
viii Contents
17 A Well-Based Reverse-Phase Protein Array of Formalin-F ixed
Paraffin-Embedded Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 29
Joon-Yong C hung and S tephen M . H ewitt
18 Q uantitative Computerized Western Blotting in Detail . . . . . . . . . . . . . . . . . . 1 41
Dalit Talmi-Frank , C harles L . J affe , and G ad Baneth
19 Cationic Electrophoresis and Eastern Blotting. . . . . . . . . . . . . . . . . . . . . . . . . 1 51
Engelbert Buxbaum
20 A Miniaturized Blotting System for Simultaneous Detection
of Different Autoantibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 65
Ulrich C anzler , Holger B artsch , Kai G roßmann , Werner Lehmann ,
Karsten C onrad , B iji T . K urien , Yaser D orri , R. Hal Scofield ,
and Michael P . B achmann
21 P roteomic Expressional Profiling of a Paraffin-Embedded Tissue
by Multiplex Tissue Immunoblotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 75
Joon-Yong C hung and Stephen M . Hewitt
22 P ost-Staining Electroblotting for Efficient and Reliable Peptide Blotting. . . . . 1 85
Der-Yen Lee and G een-Dong C hang
23 Multistrip Western Blotting: A Tool for Comparative
Quantitative Analysis of Multiple Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Edita A ksamitiene , Jan B. Hoek , and Anatoly Kiyatkin
24 Western Blotting Using PVDF Membranes and Its Downstream Applications. 2 27
Setsuko Komatsu
25 Blotting from PhastGel to Membranes by Ultrasound. . . . . . . . . . . . . . . . . . . 2 37
Joseph Kost and A haron A zagury
26 W estern Blotting of High and Low Molecular Weight Proteins Using Heat. . . 2 47
Biji T. Kurien and R. Hal Scofield
27 Membrane Strip Affinity Purification of Autoantibodies. . . . . . . . . . . . . . . . . . 2 57
Biji T . Kurien
28 S trip Immunoblotting of Multiple Antigenic Peptides. . . . . . . . . . . . . . . . . . . 2 69
Biji T. K urien
29 Double-Blotting: A Solution to the Problem of Nonspecific Binding
of Secondary Antibodies in Immunoblotting Procedures. . . . . . . . . . . . . . . . . 277
Françoise Lasne
30 Method for Resolution and Western Blotting of Very Large Proteins
Using Agarose Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
Marion L. G reaser and Chad M . Warren
31 I mmunodetection of P-Selectin Using an Antibody to Its C-Terminal Tag . . . 2 93
Padmaja Mehta-D’souza
32 Improvements and Variants of the Multiple Antigen Blot Assay-MABA:
An Immunoenzymatic Technique for Simultaneous Antigen
and Antibody Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
Oscar N oya , S andra L osada , Marilyan T oledo ,
Adriana Gauna , M aría Angelita L orenzo , H enry Bermúdez ,
and B elkisyolé Alarcón d e Noya
Contents ix
33 Blotting from Immobilized pH Gradient Gels: Application to Total
Cell Lysates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 21
Harry T owbin
34 I mmunoprecipitation: Western Blot for Proteins of Low Abundance. . . . . . . . 3 27
Edward P. Trieu and I ra N . T argoff
35 N ative Electrophoresis and Western Blot Analysis (NEWeB):
Methods and Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Ioannis N . M anoussopoulos and M ina Tsagris
36 Shift-Western Blotting: Separate Analysis of Protein and DNA
from Protein–DNA Complexes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
Matthias H arbers
37 G rid-Immunoblotting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375
Olga Yeliosof and R. H al S cofield
38 Detection and Quantification of Protein–Protein Interactions
by Far-Western Blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 79
Joshua A. J adwin , Bruce J . Mayer , and Kazuya M achida
39 Western Blot Analysis of Adhesive Interactions Under Fluid Shear
Conditions: The Blot Rolling Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
Robert S ackstein and Robert Fuhlbrigge
40 Centrifuge Blotting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
Jinny P aul
41 Blotting of Coomassie Blue-Stained Proteins from PAGE
Gels to Transparencies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 15
Jinny Paul
42 B -Cell ELISPOT: For the Identification of Antigen-Specific
Antibody-Secreting Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419
Hemangi B . S hah and K risti A . K oelsch
43 T Cell ELISPOT: For the Identification of Specific
Cytokine-S ecreting T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 27
Kerry M. Leehan and Kristi A . K oelsch
44 Membrane Microplates for One- and Two-Color ELISPOT
and FLUOROSPOT Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 35
Alexander E. K alyuzhny
45 S DS-PAGE to Immunoblot in One Hour. . . . . . . . . . . . . . . . . . . . . . . . . . . . 449
Biji T. Kurien , D ebashish Danda , Michael P. Bachmann ,
and R. H al S cofield
46 S ingle-Cell Western Blotting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455
Syed M. S. Quadri
47 Protein Detection by Simple Western™ Analysis . . . . . . . . . . . . . . . . . . . . . . . 4 65
Valerie M. Harris
48 Western Blotting Using Microfluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 69
Pothur R . S rinivas