M ETHODS IN MOLECULAR BIOLOGY

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

Detection of Blotted Proteins

Methods and Protocols

Edited by Biji T. Kurien

Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA; Arthritis and Clinical Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA; Department of Veterans Affairs Medical Center, Oklahoma City, OK, USA R. Hal Scofield

Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA; Arthritis and Clinical Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA; Department of Veterans Affairs Medical Center, Oklahoma City, OK, USA Editors Biji T. Kurien R. Hal Scofi eld 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 Arthritis and Clinical Immunology Program Arthritis and Clinical Immunology Program Oklahoma Medical Research Foundation Oklahoma Medical Research Foundation 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 978-1-4939-2717-3 ISBN 978-1-4939-2718-0 (eBook) DOI 10.1007/978-1-4939-2718-0

Library of Congress Control Number: 2015941322

Springer New York Heidelberg Dordrecht London © Springer Science+Business Media New York 2015 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: Detection of two ribosomal proteins (L7 and L12 from E. coli) by horseradish peroxidase- and fl uorescein-conjugated antibodies on a western blot developed with goat serum against the purifi ed proteins or pre- immune serum. 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) Preface

In a previous edition we combined western blotting techniques with other methods of detecting blotted molecular species. Because of the continuing advance of new techniques, we have chosen to produce two separate volumes in 2015—one compiling western blot techniques and the other compiling methods of detecting blotted proteins. As it is for west- ern blotting, there is a wide variety of techniques for detection after blotting. Some of these are minor variations on the originally reported procedure, while others are only tangentially connected. We hope in this volume to give readers practical methods by which the different procedures can be performed. That is, we hope our readers will be able to institute a new procedure in their research laboratories by reading this book, or a downloaded chapter. We imagine that the book or chapter will be open on a high lab bench. We know from experi- ence that doing this by use of the methods in most papers is diffi cult. If our readers are in fact able to do this, then we will have succeeded in our editing of this work.

Oklahoma City, OK, USA Biji T. Kurien, Ph.D. R. Hal Scofield, M.D.

v

Contents

Preface...... v Contributors ...... xi

1 Western Blotting: Origin and Ascent of the Species ...... 1 W. Neal Burnette 2 Methods to Concentrate Proteins for Protein Isolation, Proteomic, and Peptidomic Evaluation ...... 5 J. P. Dean Goldring 3 Measuring Protein Concentration on Nitrocellulose and After the Electrophoretic Transfer of Protein to Nitrocellulose ...... 19 J. P. Dean Goldring 4 Detection of Blotted Proteins: Not All Blockers Are Created Equal ...... 27 Vishal Kothari and Suresh T. Mathews 5 Protein Stains to Detect Antigen on Membranes ...... 33 Anil Dsouza and R. Hal Scofield 6 Fluorescent Labeling of Proteins and Its Application to SDS-PAGE and Western Blotting ...... 41 F. Javier Alba , Salvador Bartolomé , Antonio Bermúdez , and Joan-Ramon Daban 7 Rapid, Antibody-Free Detection of Recombinant Proteins on Blots Using Enzyme Fragment Complementation ...... 51 Neil W. Charter , Joe Horecka , Chin-Yee Loh , Albert Doan , Tom Wehrman , and Keith R. Olson 8 Use of Nonradioactive Detection Method for North- and South-Western Blot ...... 63 Claudia Franke , Daniel Gräfe , Holger Bartsch , and Michael P. Bachmann 9 Immunoblotting Using Radiolabeled Reagents for Detection ...... 73 Holger Bartsch , Claudia Franke , and Michael P. Bachmann 10 Immunoblotting of Antigens: Whole, Strip, and New-Line Nitrocellulose Membrane Immunoblotting Using the Chemiluminescence Technique ...... 79 Yaser Dorri 11 Detection of Protein Carbonyls by Means of Biotin Hydrazide–Streptavidin Affinity Methods ...... 95 Kenneth Hensley 12 Direct Immunodetection of Antigens Within the Precast Polyacrylamide Gel ...... 101 Surbhi Desai , Boguslawa R. Dworecki , and Marie C. Nlend

vii viii Contents

13 Quantitative Analysis of Signal Transduction with In-Cell Western Immunofluorescence Assays ...... 115 Vince Boveia and Amy Schutz-Geschwender 14 Ultrasensitive Protein Detection on Dot Blots and Western Blots with Semiconducting Polymer Dots ...... 131 Fangmao Ye , Polina B. Smith , and Daniel T. Chiu 15 Co-detection of Target and Total Protein by CyDye Labeling and Fluorescent ECL Plex Immunoblotting in a Standard Proteomics Workflow ...... 139 Caitriona Scaife , Ciara A. McManus , Pamela M. Donoghue , and Michael J. Dunn 16 Using Biotinylated Proteins to Demonstrate Immunodetection of Antigens via Western Blotting, Dot Blots, and Immunohistochemistry . . . . 151 Thomas Millar , Ronald Knighton , and Jo-Anne Chuck 17 Calcium Binding by Ro 60 Multiple Antigenic Peptides on PVDF Membrane...... 165 Biji T. Kurien and Michael P. Bachmann 18 Sequential Use of Immunoblots for Characterization of Autoantibody Specificities ...... 173 Holger Bartsch and Michael P. Bachmann 19 Nanogold Immunodetection Detection Systems for the Identification of Autoantigens by Western Blotting...... 179 Jacen S. Moore and R. Hal Scofield 20 Application of Intermittent Microwave Irradiation to Western Blot Analysis ...... 185 Yu-Ting Liu and Shinya Toyokuni 21 Visualization of Unstained Protein Bands on PVDF ...... 191 Jun Park , Masaharu Mabuchi , and Ajay Sharma 22 Multiplexed Fluorescent Immunodetection Using Low Autofluorescence Immobilon®-FL Membrane ...... 195 Jun Park , Masaharu Mabuchi , and Ajay Sharma 23 Cold Microwave-Enabled Protein Detection and Quantification ...... 207 Niels Grützner , Romy M. Heilmann , Aaron G. Smith , Carol B. Johnson , Stanislav Vitha , Jörg M. Steiner , and Andreas K. Holzenburg 24 TLC-Blot (Far-Eastern Blot) and Its Application to Functional Lipidomics . . . . . 219 Takao Taki 25 Analysis of Electroblotted Proteins by Mass Spectrometry ...... 243 Jose L. Luque-Garcia and Thomas A. Neubert 26 On-Membrane Renaturation of Recombinant Ro60 Autoantigen by Calcium Ions ...... 255 Biji T. Kurien and Michael P. Bachmann 27 Phosphoprotein Detection on Protein Electroblot Using a Phosphate-Specific Fluorophore ...... 263 Lee Broderick Bockus and R. Hal Scofield Contents ix

28 Purification of Tryptic Digests on Polyvinylidene Difluoride Membrane . . . . . 273 Biji T. Kurien and R. Hal Scofield 29 Detection of Blotted Proteins on Nitrocellulose/PVDF Membranes by Alta ...... 279 Jayanta K. Pal , Shilpa J. Rao , and Dhanashri J. Godbole 30 Nonstripping “Rainbow” and Multiple Antigen Detection (MAD) Western Blotting...... 287 Stan (Stanislaw) Krajewski , Michelle M. Tsukamoto , Xianshu Huang , and Sebastian B. Krajewski 31 Supported Molecular Matrix Electrophoresis...... 303 Yu-ki Matsuno and Akihiko Kameyama 32 Parafilm-M®, An Available Cost-Effective Alternative for Immuno-blot Pouches...... 313 Syed M. S. Quadri 33 Succinylation-Alcian Blue Staining of Mucins on Polyvinylidene Difluoride Membranes ...... 325 Akihiko Kameyama , Weijie Dong , and Yu-ki Matsuno 34 Comparison of Chemiluminescence vs. Infrared Techniques for Detection of Fetuin-A in Saliva ...... 333 Suresh T. Mathews , Emily Graff , Robert L. Judd , and Vishal Kothari 35 A Novel Methodology for Stripping and Reprobing of Western Blots Originally Developed with Colorimetric Substrate TMB ...... 349 Parmita Kar , Saurabh Kumar Agnihotri , Archana Sharma , Rekha Sachan , Madan Lal Bhatt , and Monika Sachdev 36 Other Notable Methods of Membrane Protein Detection: A Brief Review . . . . 357 Biji T. Kurien and R. Hal Scofield 37 Nitrocellulose Membrane: The New Canvas ...... 371 Jasmin R. Kurien and Bianca A. Kurien 38 Invisible Ink Marking in ECL Membrane Assays ...... 375 Biji T. Kurien

Index ...... 383

Contributors

SAURABH KUMAR AGNIHOTRI • Division of Endocrinology, Central Drug Research Institute , Lucknow, India F. JAVIER ALBA • Departament de Bioquímica i Biologia Molecular, Facultat de Biociències, Universitat Autònoma de Barcelona , Barcelona , Spain MICHAEL P. BACHMANN • Institut für Immunologie, Carl Gustav Carus Technische Universität Dresden , Dresden, Germany; Tumorimmunology, University Cancer Center (UCC) Carl Gustav Carus TU-Dresden, Dresden, Germany SALVADOR BARTOLOMÉ • Departament de Bioquímica i Biologia Molecular, Facultat de Biociències, Universitat Autònoma de Barcelona , Barcelona, Spain HOLGER BARTSCH • Institut für Immunologie, Carl Gustav Carus Technische Universität Dresden , Dresden , Germany ANTONIO BERMÚDEZ • Departament de Bioquímica i Biologia Molecular, Facultat de Biociències, Universitat Autònoma de Barcelona , Barcelona , Spain MADAN LAL BHATT • King George Medical University , Lucknow , India LEE BRODERICK BOCKUS • Arthritis and Immunology Program , Oklahoma Medical Research Foundation, Oklahoma City , OK, USA; Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA VINCE BOVEIA • LI-COR Biosciences , Lincoln, NE , USA W. NEAL BURNETTE • Molecular Pharmaceutics , Chapel Hill, NC , USA NEIL W. CHARTER • DiscoveRx Corporation , Fremont, CA , USA DANIEL T. CHIU • Department of Chemistry, University of Washington , Seattle, WA , USA JO-ANNE CHUCK • School of Science and Health, University of Western Sydney , Penrith South, NSW , Australia JOAN-RAMON DABAN • Departament de Bioquímica i Biologia Molecular, Facultat de Biociències, Universitat Autònoma de Barcelona , Barcelona , Spain SURBHI DESAI • Thermo Fisher Scientifi c , Rockford , IL , USA ALBERT DOAN • DiscoveRx Corporation , Fremont , CA , USA WEIJIE DONG • Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST) , Tsukuba , Japan PAMELA M. DONOGHUE • Proteome Research Centre, UCD Conway Institute of Biomolecular and Biomedical Research , Dublin , Ireland YASER DORRI • University of Oklahoma , Norman , OK , USA; Arthritis and Immunology Department, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA ANIL DSOUZA • Arthritis and Clinical Immunology Program, Oklahoma Medical Research Foundation , Oklahoma City , OK, USA MICHAEL J. DUNN • Proteome Research Centre, UCD Conway Institute of Biomolecular and Biomedical Research , Dublin , Ireland BOGUSLAWA R. DWORECKI • Thermo Fisher Scientifi c , Rockford, IL , USA CLAUDIA FRANKE • Institut für Immunologie, Carl Gustav Carus Technische Universität Dresden , Dresden , Germany DHANASHRI J. GODBOLE • Department of Biotechnology, Fergusson College , Pune , India J. P. DEAN GOLDRING • Biochemistry, University of KwaZulu-Natal , Scottsville, South Africa

xi xii Contributors

DANIEL GRÄFE • Institut für Immunologie, Carl Gustav Carus Technische Universität Dresden , Dresden , Germany EMILY GRAFF • Department of Anatomy, Physiology & Pharmacology, Boshell Diabetes and Metabolic Diseases Research Program, Auburn University , Auburn , AL , USA NIELS GRÜTZNER • Gastrointestinal Laboratory, Department Small Animal Clinic, Texas A&M University , College Station, TX, USA; Department of Clinical Veterinary Medicine, Swine Clinic, Vetsuisse University Bern, Bern, Switzerland ROMY M. HEILMANN • Gastrointestinal Laboratory, Department Small Animal Clinic, Texas A&M University , College Station, TX, USA KENNETH HENSLEY • Department of Pathology, University of Toledo Health Sciences Campus , Toledo, OH , USA ANDREAS K. HOLZENBURG • Microscopy and Imaging Center, Texas A&M University , College Station, TX, USA; Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, USA; Department of Biology, Texas A&M University, College Station, TX, USA JOE HORECKA • Stanford Genome Technology Center , Stanford , CA , USA XIANSHU HUANG • Sanford-Burnham Medical Research Institute , La Jolla, CA , USA CAROL B. JOHNSON • Microscopy and Imaging Center, Texas A&M University , College Station, TX , USA; Department of Biology, Texas A&M University, College Station, TX, USA ROBERT L. JUDD • Department of Anatomy, Physiology & Pharmacology, Boshell Diabetes and Metabolic Diseases Research Program, Auburn University , Auburn , AL , USA AKIHIKO KAMEYAMA • Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST) , Tsukuba, Japan PARMITA KAR • Division of Endocrinology, Central Drug Research Institute , Lucknow , India RONALD KNIGHTON • School of Science and Health, University of Western Sydney , Penrith South, NSW , Australia VISHAL KOTHARI • Department of Nutrition, Dietetics, and Hospitality Management, Auburn University , Auburn , AL, USA; Boshell Diabetes and Metabolic Diseases Research Program, Auburn University, Auburn, AL, USA SEBASTIAN B. KRAJEWSKI • Cellestan-ImmunoQuant (CIQ), Inc. , Oceanside , CA , USA STAN (STANISLAW) KRAJEWSKI • Sanford-Burnham Medical Research Institute , La Jolla, CA , USA; Cellestan-ImmunoQuant (CIQ), Inc., Oceanside, CA, USA BIANCA A. KURIEN • Mustang High School , Mustang , OK , USA BIJI T. KURIEN • Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA; Arthritis and Clinical Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA; Department of Veterans Affairs Medical Center, Oklahoma City, OK, USA JASMIN R. KURIEN • Mustang High School , Mustang , OK , USA YU-TING LIU • Nagoya University Graduate School of Medicine , Nagoya , Japan CHIN-YEE LOH • DiscoveRx Corporation , Fremont , CA , USA JOSE L. LUQUE-GARCIA • Department of Biochemistry and Molecular Pharmacology and Kimmel Center for Biology and Medicine at the Skirball Institute, New York University School of Medicine , New York , NY , USA; Department of Analytical Chemistry, Faculty of Chemical Sciences, Complutense University of Madrid, Madrid, Spain MASAHARU MABUCHI • Millipore Corporation , Danvers, MA , USA Contributors xiii

SURESH T. MATHEWS • Department of Nutrition, Dietetics, and Hospitality Management, Auburn University , Auburn, AL, USA; Boshell Diabetes and Metabolic Diseases Research Program, Auburn University, Auburn, AL, USA YU-KI MATSUNO • Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST) , Tsukuba, Japan CIARA A. MCMANUS • GlycoScience Group, NIBRT—National Institute for Bioprocessing Research and Training , Co Dublin, Ireland THOMAS MILLAR • School of Science and Health, University of Western Sydney , Penrith South, NSW, Australia JACEN S. MOORE • Department of Clinical Laboratory Sciences, University of Texas at El Paso, College of Health Sciences and School of Nursing , El Paso , TX, USA THOMAS A. NEUBERT • Department of Biochemistry and Molecular Pharmacology and Kimmel Center for Biology and Medicine at the Skirball Institute, New York University School of Medicine , New York , NY , USA MARIE C. NLEND • Thermo Fisher Scientifi c , Rockford , IL , USA KEITH R. OLSON • DiscoveRx Corporation , Fremont , CA , USA JAYANTA K. PAL • Cell and Molecular Biology Laboratory, Department of Biotechnology, Savitribai Phule Pune University , Pune , India JUN PARK • Millipore Corporation , Danvers, MA , USA SYED M. S. QUADRI • Arthritis and Clinical Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA; Department of Pathology, University of Oklahoma Health Sciences Center , Oklahoma City , OK , USA SHILPA J. RAO • Cell and Molecular Biology Laboratory, Department of Biotechnology, Savitribai Phule Pune University , Pune , India MONIKA SACHDEV • Division of Endocrinology, Central Drug Research Institute , Lucknow, India REKHA SACHAN • King George Medical University , Lucknow , India CAITRIONA SCAIFE • Proteome Research Centre, UCD Conway Institute of Biomolecular and Biomedical Research , Dublin, Ireland AMY SCHUTZ-GESCHWENDER • LI-COR Biosciences , Lincoln, NE, USA R. HAL SCOFIELD • Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA; Arthritis and Clinical Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA; Department of Veterans Affairs Medical Center, Oklahoma City, OK, USA AJAY SHARMA • Millipore Corporation , Danvers, MA , USA ARCHANA SHARMA • Division of Endocrinology, Central Drug Research Institute , Lucknow, India AARON G. SMITH • Microscopy and Imaging Center, Texas A&M University , College Station, TX, USA; Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, USA POLINA B. SMITH • Department of Chemistry, University of Washington , Seattle , WA , USA JÖRG M. STEINER • Gastrointestinal Laboratory, Department Small Animal Clinic, Texas A&M University , College Station, TX, USA TAKAO TAKI • Niigata University of Pharmacy and Applied Life Sciences , Tokushima , Japan HARRY TOWBIN • Institute of Pharmaceutical Sciences , Zurich , Switzerland xiv Contributors

SHINYA TOYOKUNI • Nagoya University Graduate School of Medicine , Nagoya, Japan; Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya, Japan MICHELLE M. TSUKAMOTO • Sanford-Burnham Medical Research Institute , La Jolla, CA , USA; Kaiser Permanente, Panorama City Hospital Bldg, Panorama City, CA, USA STANISLAV VITHA • Microscopy and Imaging Center, Texas A&M University , College Station, TX, USA TOM WEHRMAN • DiscoveRx Corporation , Fremont , CA , USA FANGMAO YE • Department of Chemistry, University of Washington , Seattle, WA , USA