High-Resolution Imaging of Cellular Proteins: Methods and Protocols (Methods in Molecular Biology, 1474) 1493963503, 9781493963508

Citation preview

Methods in Molecular Biology 1474

Steven D. Schwartzbach Omar Skalli Thomas Schikorski Editors

High-Resolution Imaging of Cellular Proteins Methods and Protocols

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes: http://www.springer.com/series/7651

High-Resolution Imaging of Cellular Proteins Methods and Protocols

Edited by

Steven D. Schwartzbach Department of Biological Sciences, The University of Memphis, Memphis, TN, USA

Omar Skalli Department of Biological Sciences, The University of Memphis, Memphis, TN, USA

Thomas Schikorski Department of Anatomy, Universidad Central del Caribe, Bayamon, PR, USA

Editors Steven D. Schwartzbach Department of Biological Sciences The University of Memphis Memphis, TN, USA

Omar Skalli Department of Biological Sciences The University of Memphis Memphis, TN, USA

Thomas Schikorski Department of Anatomy Universidad Central del Caribe Bayamon, PR, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-6350-8 ISBN 978-1-4939-6352-2 (eBook) DOI 10.1007/978-1-4939-6352-2 Library of Congress Control Number: 2016946471 © Springer Science+Business Media New York 2016 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms 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 specific 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. Printed on acid-free paper This Humana Press imprint is published by Springer Nature The registered company is Springer Science+Business Media LLC New York

Preface Cell biology is the study of the structure and function of cells, cellular organelles, and subcellular structures. Originally, cellular functions were studied without a detailed knowledge of the structures involved and structures were described without an understanding of their function. Imaging techniques that simultaneously studied structure and function or at least correlated function with structure eventually closed this disparity. Several technologies have been greatly responsible for this progress by providing high-resolution images rich in functional information. Over the past decades, these technologies and their combinations have provided possibilities previously unthinkable. This book contains a collection of timely techniques and methods that have been instrumental in the evolution of microscopy from a purely descriptive technique to one of four-dimensional imaging in living organisms. The biochemist and molecular biologist determines the functions of the molecules and macromolecular complexes found within cellular structures. They isolate individual cellular constituents and reconstruct vital cellular processes. These in vitro experiments provide a valuable understanding of cellular function. But, biochemistry lacks the potential to place this knowledge into the cellular context of cell types and cellular compartments. Electron microscopy (EM) was the first technique to bridge the gap between biochemistry, molecular biology, and cellular context by localizing macromolecules to cellular structures. EM collected valuable information about proteins contained within a structure and their spatial relationships with other proteins and structures. The combination of immunolabeling with EM made it even possible to localize specific proteins of known function to subcellular structures. Immunoelectron microscopy can be used to study virtually every unicellular and multicellular organism. The only requirements are suitable protocols and the availability of an antibody to the molecule whose structural location is to be determined. Although introduced decades ago, this technology is far from obsolete because of its nanometer precision in localizing proteins in cells and tissues. More recently, labeling methods for scanning EM and for serial sections and electron tomography were developed and used to visualize specific biomolecules within the three-dimensional structure of organelles and subcellular compartments. Computer-assisted image acquisition and analysis greatly contributed to this development. Advances in light microscopy soon made it a competitive alternative to EM for studies correlating structure and function. The introduction of video cameras marked a breakthrough adding a new dimension, time, to microscopic observations of structure in a living cell. The development of fluorescent dyes that could be conjugated to antibodies and dyes localized to specific subcellular compartments further advanced live cell imaging. To fully utilize the potential of these probes confocal and two-photon microscopes were designed. These microscopes overcame the limitations of standard fluorescent microscopes by increasing the localization accuracy in tissue and the resolution in the z-axis. The next innovation that boosted functional live cell imaging was the discovery of the green fluorescent protein (GFP). The ability to use DNA cloning methods to create constructs encoding a protein of interest fused to GFP opened the door to using live cells for studying the function of specific proteins. The development of a diverse color pallet of fluorescent proteins

v

vi

Preface

and of methods to make fluorescence dependent upon the interaction of two proteins as well as photocontrollable fluorescent tags and the constant advances in designing ever faster and more sensitive cameras have greatly expanded the structure function information that can be obtained from live cell imaging. It did not take long before correlative methods were developed in which the distribution of specific proteins was examined first by confocal microscopy and then by EM. An example from our own work demonstrates how confocal and immunoelectron microscopy provide unexpected insights into structure-function relationships. Thus, immunoelectron microscopy first demonstrated that the Euglena light harvesting chlorophyll a/b binding protein of photosystem II (LHCPII) is present in the Golgi apparatus prior to its presence in the chloroplast. This finding was the impetus for detailed biochemical studies that elucidated a new mechanism for chloroplast protein import, namely transport from the ER to the Golgi apparatus to the chloroplast. This volume takes into account the increasingly multidisciplinary nature of microscopy by presenting three toolboxes. The molecular toolbox focuses on the development of molecular tools for microscopy. It will present methods for the expression of epitope-tagged proteins in animal cells. Methods for the production of antipeptide and polyclonal antibodies and how to conjugate colloidal gold to these proteins will also be presented. A molecular toolbox would be incomplete without the discussion of genetic tools that exploit viral vectors to optimize the transfer of genes into living cells. This technology is also addressed in the following toolboxes together with light and electron microscopic imaging. From the fluorescent microscopy toolbox, this section presents methods that span diverse applications based on the use of conventional fluorophores and expressed fluorescent proteins such as GFP in plants, parasites, and animal cells. Fluorescence microscopy also enables monitoring protein-protein interactions in real time and bimolecular complementation methods enabling this feat will be presented. A pH-sensitive GFP variant is used to monitor exocytosis and endocytosis of synaptic vesicles in real time. How the trafficking of proteins or organelles can be monitored by Fluorescence Recovery after Photobleaching and Fluorescence Redistribution after Photoactivation is presented. Finally one chapter presents the labeling of brain cells and the imaging of these cells in the living brain. From the EM toolbox, this section details methods for cryo-ultramicrotomy and rapid freeze-replacement fixation which have the advantage of retaining protein antigenicity but at the expense of ultrastructural integrity as well as chemical fixation methods that maintain structural integrity while sacrificing protein antigenicity. The toolbox also includes a protocol for immunogold labeling of freeze-fracture replicas. This technique is known for its high sensitivity and its capability of localizing proteins to nanoscale protein assemblies like ion channels. Plants and algae contain cell walls, vacuoles, and other structures which present barriers to antibody penetration and complicate fixation. Due to these problems, separate chapters will discuss fixation and immunolabeling protocols for animals, plants, and yeast. Pre- and post-embedding immunogold labeling protocols will be presented. Preembedding methods perform immunogold labeling before ultrathin sections are prepared from resin-embedded samples resulting in greater sensitivity and better microstructure preservation. Post-embedding methods perform immunolabeling after ultrathin sections are prepared from resin-embedded samples resulting in decreased antigenicity. The detailed methods and notes will facilitate choosing the best method for the antibody and biological material to be studied. Extending these approaches, methods will be presented for immunogold labeling of two antigens for protein colocalization studies, for glycan localization, for nanogold enhancement allowing immunogold labeling using smaller gold particles

Preface

vii

which more easily enter cells, and for immunogold scanning EM. For many years, the advances in genetics and functional imaging were not used to advance EM. Recently however, these advances have been used to develop powerful EM techniques. Reporter genes suitable for EM and fixation techniques that capture structure at a defined time point have been developed. Furthermore, these techniques are suitable for correlative light-electron microscopy. The volume presents two examples of these advances; the use of genetically engineered horseradish peroxidase as a genetically encoded label for electron microscopy and superfast fixation for monitoring cellular processes second by second. It is our hope that the toolboxes created by this volume will be used by cell biologists interested in understanding structure-function relationships at the fundamental level as well as by cancer biologists, toxicologists, and microbiologists interested in understanding disease mechanisms as a foundation to developing new therapies. Memphis, TN, USA Memphis, TN, USA Bayamon, PR, USA

Steven D. Schwartzbach Omar Skalli Thomas Schikorski

Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

PART I

MOLECULAR TOOLBOX

1 Expression of Epitope-Tagged Proteins in Mammalian Cells in Culture . . . . . . Jay M. Bhatt, Melanie L. Styers, and Elizabeth Sztul 2 Antibody Production with Synthetic Peptides . . . . . . . . . . . . . . . . . . . . . . . . . Bao-Shiang Lee, Jin-Sheng Huang, Lasanthi P. Jayathilaka, Jenny Lee, and Shalini Gupta 3 Production and Purification of Polyclonal Antibodies . . . . . . . . . . . . . . . . . . . Masami Nakazawa, Mari Mukumoto, and Kazutaka Miyatake 4 Preparation of Colloidal Gold Particles and Conjugation to Protein A/G/L, IgG, F(ab′)2, and Streptavidin . . . . . . . . . . . . . . . . . . . . . Sadaki Yokota 5 Helper-Dependent Adenoviral Vectors and Their Use for Neuroscience Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mónica S. Montesinos, Rachel Satterfield, and Samuel M. Young Jr.

PART II

v xi

3 25

49

61

73

FLUORESCENT MICROSCOPY TOOLBOX

6 Localizing Proteins in Fixed Giardia lamblia and Live Cultured Mammalian Cells by Confocal Fluorescence Microscopy . . . . . . . . . . . . . . . . . Lilian Nyindodo-Ogari, Steven D. Schwartzbach, Omar Skalli, and Carlos E. Estraño 7 Using Fluorescent Protein Fusions to Study Protein Subcellular Localization and Dynamics in Plant Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yong Cui, Caiji Gao, Qiong Zhao, and Liwen Jiang 8 Using FRAP or FRAPA to Visualize the Movement of Fluorescently Labeled Proteins or Cellular Organelles in Live Cultured Neurons Transformed with Adeno-Associated Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . Yaara Tevet and Daniel Gitler 9 Bimolecular Fluorescence Complementation (BiFc) Analysis of Protein–Protein Interactions and Assessment of Subcellular Localization in Live Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evan P.S. Pratt, Jake L. Owens, Gregory H. Hockerman, and Chang-Deng Hu 10 Viral Injection and Cranial Window Implantation for In Vivo Two-Photon Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gordon B. Smith and David Fitzpatrick

ix

93

113

125

153

171

x

Contents

11 Imaging Synaptic Vesicle Exocytosis-Endocytosis with pH-Sensitive Fluorescent Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Olusoji A.T. Afuwape and Ege T. Kavalali

PART III

187

ELECTRON MICROSCOPY TOOLBOX

12 Immunogold Protein Localization on Grid-Glued Freeze-Fracture Replicas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Harumi Harada and Ryuichi Shigemoto 13 Pre-embedding Double-Label Immunoelectron Microscopy of Chemically Fixed Tissue Culture Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lou G. Boykins, Jonathan C.R. Jones, Carlos E. Estraño, Steven D. Schwartzbach, and Omar Skalli 14 Immunoelectron Microscopy of Cryofixed and Freeze-Substituted Plant Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Miyuki Takeuchi, Keiji Takabe, and Yoshinobu Mineyuki 15 Immunoelectron Microscopy of Cryofixed Freeze-Substituted Yeast Saccharomyces cerevisiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jindřiška Fišerová, Christine Richardson, and Martin W. Goldberg 16 Pre-embedding Method of Electron Microscopy for Glycan Localization in Mammalian Tissues and Cells Using Lectin Probes . . . . . . . . . . . . . . . . . . . Yoshihiro Akimoto, Kuniaki Takata, and Hayato Kawakami 17 Pre-embedding Nanogold Silver and Gold Intensification . . . . . . . . . . . . . . . . Akitsugu Yamamoto and Ryuichi Masaki 18 Post-embedding Mammalian Tissue for Immunoelectron Microscopy: A Standardized Procedure Based on Heat-Induced Antigen Retrieval . . . . . . . Shuji Yamashita 19 Pre- and Post-embedding Immunogold Labeling of Tissue Sections . . . . . . . . Jonathan C.R. Jones 20 Immunogold Labeling for Scanning Electron Microscopy . . . . . . . . . . . . . . . . Martin W. Goldberg and Jindřiška Fišerová 21 Monitoring Synaptic Vesicle Protein Sorting with Enhanced Horseradish Peroxidase in the Electron Microscope. . . . . . . . . . . . . . . . . . . . . Thomas Schikorski 22 Functional Nanoscale Imaging of Synaptic Vesicle Cycling with Superfast Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thomas Schikorski Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

203

217

233

243

259 269

279 291 309

327

343 359

Contributors OLUSOJI A.T. AFUWAPE • Department of Neuroscience, UT Southwestern Medical Center, Dallas, TX, USA YOSHIHIRO AKIMOTO • Department of Anatomy, Kyorin University School of Medicine, Mitaka, Tokyo, Japan JAY M. BHATT • Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, USA LOU G. BOYKINS • Department of Biological Sciences, The University of Memphis, Memphis, TN, USA YONG CUI • Centre for Cell and Developmental Biology and State Key Laboratory of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China CARLOS E. ESTRAÑO • Department of Biological Sciences, The University of Memphis, Memphis, TN, USA JINDRˇ IŠKA FIŠEROVÁ • Department of Biology of the Cell Nucleus, Institute of Molecular Genetics AS CR, v.v.i., Prague, Czech Republic DAVID FITZPATRICK • Department of Functional Architecture and Development of Cerebral Cortex, Max Planck Florida Institute for Neuroscience, Jupiter, FL, USA CAIJI GAO • Centre for Cell and Developmental Biology and State Key Laboratory of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China DANIEL GITLER • Department of Physiology and Cell Biology, Faculty of Health Sciences and Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva, Israel MARTIN W. GOLDBERG • School of Biological and Biomedical Sciences, Durham University, Durham, UK SHALINI GUPTA • Protein Research Laboratory, Research Resources Center, University of Illinois at Chicago, Chicago, IL, USA HARUMI HARADA • IST Austria, Klosterneuburg, Austria GREGORY H. HOCKERMAN • Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN, USA CHANG-DENG HU • Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN, USA JIN-SHENG HUANG • Protein Research Laboratory, Research Resources Center, University of Illinois at Chicago, Chicago, IL, USA LASANTHI P. JAYATHILAKA • Protein Research Laboratory, Research Resources Center, University of Illinois at Chicago, Chicago, IL, USA LIWEN JIANG • Centre for Cell and Developmental Biology and State Key Laboratory of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China; CUHK Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China

xi

xii

Contributors

JONATHAN C.R. JONES • School of Molecular Biosciences, Washington State University, Pullman, WA, USA EGE T. KAVALALI • Department of Neuroscience, UT Southwestern Medical Center, Dallas, TX, USA; Department of Physiology, UT Southwestern Medical Center, Dallas, TX, USA HAYATO KAWAKAMI • Department of Anatomy, Kyorin University School of Medicine, Mitaka, Tokyo, Japan BAO-SHIANG LEE • Protein Research Laboratory, Research Resources Center, University of Illinois at Chicago, Chicago, IL, USA JENNY LEE • Department of Chemical Engineering, Columbia University, New York, NY, USA RYUICHI MASAKI • Department of Animal Bio-Science, Faculty of Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, Japan YOSHINOBU MINEYUKI • Department of Picobiology, Graduate School of Life Science, University of Hyogo, Himeji, Hyogo, Japan KAZUTAKA MIYATAKE • Department of Applied Biological Chemistry, Osaka Prefecture University, Osaka, Japan MÓNICA S. MONTESINOS • Research Group Molecular Mechanisms of Synaptic Function, Max Planck Florida Institute for Neuroscience, Jupiter, FL, USA MARI MUKUMOTO • Department of Applied Biological Chemistry, Osaka Prefecture University, Osaka, Japan MASAMI NAKAZAWA • Department of Applied Biological Chemistry, Osaka Prefecture University, Osaka, Japan LILIAN NYINDODO-OGARI • Baptist College of Health Sciences, Memphis, TN, USA; Department of Biological Sciences, The University of Memphis, Memphis, TN, USA JAKE L. OWENS • Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN, USA EVAN P.S. PRATT • Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN, USA CHRISTINE RICHARDSON • School of Biological and Biomedical Sciences, Durham University, Durham, UK RACHEL SATTERFIELD • Research Group Molecular Mechanisms of Synaptic Function, Max Planck Florida Institute for Neuroscience, Jupiter, FL, USA THOMAS SCHIKORSKI • Department of Anatomy, Universidad Central del Caribe, Bayamon, PR, USA STEVEN D. SCHWARTZBACH • Department of Biological Sciences, The University of Memphis, Memphis, TN, USA RYUICHI SHIGEMOTO • IST Austria, Klosterneuburg, Austria OMAR SKALLI • Department of Biological Sciences, The University of Memphis, Memphis, TN, USA GORDON B. SMITH • Department of Functional Architecture and Development of Cerebral Cortex, Max Planck Florida Institute for Neuroscience, Jupiter, FL, USA MELANIE L. STYERS • Department of Biology, Birmingham-Southern College, Birmingham, AL, USA ELIZABETH SZTUL • Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, USA KEIJI TAKABE • Division of Forest and Biomaterials Science, Graduate School of Agriculture, Kyoto University, Kyoto, Japan

Contributors

xiii

KUNIAKI TAKATA • Gunma Prefectural College of Health Sciences, Maebashi, Gunma, Japan MIYUKI TAKEUCHI • Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyou-ku, Tokyo, Japan YAARA TEVET • Department of Physiology and Cell Biology, Faculty of Health Sciences and Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva, Israel AKITSUGU YAMAMOTO • Department of Animal Bio-Science, Faculty of Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, Japan SHUJI YAMASHITA • Department of Pathology, School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan SADAKI YOKOTA • Section of Functional Morphology, Faculty of Pharmaceutical Sciences, Nagasaki International University, Sasebo, Nagasaki, Japan SAMUEL M. YOUNG JR. • Research Group Molecular Mechanisms of Synaptic Function, Max Planck Florida Institute for Neuroscience, Jupiter, FL, USA QIONG ZHAO • Centre for Cell and Developmental Biology and State Key Laboratory of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China

Part I Molecular Toolbox

Chapter 1 Expression of Epitope-Tagged Proteins in Mammalian Cells in Culture Jay M. Bhatt, Melanie L. Styers, and Elizabeth Sztul Abstract Before the advent of molecular methods to tag proteins, visualization of proteins within cells required the use of antibodies directed against the protein of interest. Thus, only proteins for which antibodies were available could be visualized. Epitope tagging allows the detection of all proteins with existing sequence information, irrespective of the availability of antibodies directed against them. This technique involves the generation of DNA constructs that express the protein of interest tagged with an epitope that can be recognized by a commercially available antibody. Proteins can be tagged with a wide variety of epitopes using commercially available vectors that allow expression in mammalian cells. Epitope-tagged proteins are easily transfected into mammalian cell lines and, in most cases, tightly mimic the behavior of the endogenous protein. Tagged proteins exogenously expressed in cells provide different types of information depending on the subsequent detection approaches. Using immunofluorescence and immunoelectron microscopy with anti-tag antibodies, relative to known markers of cellular organelles, can provide information on the subcellular localization of the tagged protein and may provide clues regarding the protein’s function. Immunofluorescence with anti-tag antibodies can also be utilized to assess the tagged protein’s responses to cellular signals and pharmacological treatments. Immunoprecipitations with anti-tag antibodies can recover protein complexes containing the protein of interest, resulting in the identification of interacting proteins. Recovery of tagged proteins on affinity matrices allows their purification for use in biochemical assays. In addition, specialized fluorescent tags, such as the green fluorescent protein (GFP) allow the analysis of cellular dynamics in live cells in real time. Key words Transfection, Western blot, Immunofluorescence microscopy, Immunoprecipitation, Epitope tag, Green fluorescent protein (GFP)

1

Introduction In the past, approaches to study protein localization within cells required the generation of highly specific antibodies directed against the protein of interest. In addition to their specificity, these antibodies also had to recognize the desired antigen subsequent to the fixation conditions necessary to preserve cells and tissues for microscopy. However, with the advent of molecular cloning and the mapping of the human genome, scientists can now introduce

Steven D. Schwartzbach et al. (eds.), High-Resolution Imaging of Cellular Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 1474, DOI 10.1007/978-1-4939-6352-2_1, © Springer Science+Business Media New York 2016

3

4

Jay M. Bhatt et al.

tags into any protein of interest to facilitate visualization of proteins for which no antibodies are currently available. A wide variety of DNA vectors that tag proteins for expression in mammalian cells are commercially available (see Note 1). The tags range in size from a few amino acids, such as the (His)6 tag; to small peptide epitopes, such as the hemagglutinin (HA), FLAG, V-5 and myc tags; and small proteins, such as green fluorescent protein (GFP) and glutathione-S-transferase (GST). Some vectors allow tagging with dual tags, one at the N- and one at the C-terminus (e.g., the pcDNA3.1/myc-his vectors). A wide variety of well-characterized monoclonal and polyclonal antibodies, useful for most applications, are readily available for all common tags from commercial sources such as Abcam, Thermo Scientific, Roche, and BD Biosciences. Thus, the wide variety of vectors allowing the insertion of distinct tags and the spectrum of available anti-tag antibodies allow for the unprecedented level of analysis of proteins for which antibodies are currently unavailable. 1.1 Major Considerations for Epitope-Tagging a Protein

The optimal result of tagging is to generate a tagged protein that behaves in a manner analogous to the untagged endogenous protein. Thus, the type and the placement of a tag have to be carefully considered, since inappropriate tagging may negatively influence the behavior of the protein. There are a number of considerations and alternative approaches that can be taken to generate a correctly folded and functional tagged protein. Below, we describe the most common ways to decide on the optimal tagging strategy.

1.1.1 Selecting an Optimal Tag

Epitope tags are added to a protein of interest for a variety of reasons, including means to detect the protein’s localization and behavior in cells, increase the protein’s solubilization and stability, allow purification, and alter enzymatic activity when attached to a protein of interest. Some tags may have dual roles. For example, the GST tag enhances solubility of proteins and also can be used for purification. Choosing an epitope tag mainly depends on whether the purpose of the experiment is the detection or the purification of the protein. Tags such as myc, FLAG, and GFP are preferred if the main goal of the study is to detect the proteins within cells. Tags such as (His)6, glutathione S-transferase (GST), and streptavidin binding protein (SBP) are needed when the goal is to purify the protein of interest. It is possible that the folding or functionality of a protein might be altered by the size of the tag. In that case, one should consider changing the tag. For example, instead of the GFP tag, myc could be used to localize the protein in cells via immunofluorescence; instead of the GST tag, (His)6 could be used to purify the protein using nickel beads. A number of other factors described below also influence which tag should be used for generating a useful recombinant protein.

Expression of Epitope-Tagged Proteins in Mammalian Cells in Culture

5

1.1.2 Choosing the Placement of the Tag at the N- or C-Terminus of a Protein

The majority of available tagging vectors are designed to insert the tag either at the N- or the C-terminus of the protein. It is important to consider that any kind of tag can potentially alter the physiological function of a protein by altering its folding, sterically hindering protein–protein interactions, or making it insoluble. Hence, it becomes critical to consider which end of the protein should be tagged. As a general rule of thumb, one should avoid tagging the protein near its functional domains, since the tag might hinder the protein’s function. In the case of proteins with informative domain structure (for example, containing a kinase domain, SH3 domain, Walker motif, etc.), place the tag at the end opposite the predicted functional domain. If the protein sequence is not informative, then it would be wise to tag the protein at both ends and test the localization and if possible, the function of both recombinant versions. For example, Zhou and Graham found that when Drs2p (a lipid translocating ATPase at the Golgi) was tagged with TAP at the C-terminus, the resulting recombinant protein was catalytically inactive [1]. However, when Drs2p was tagged at the N-terminus with TAP, the ATPase activity of Drs2p was unaffected. Proteins that contain signal peptides at their N-termini must be tagged at their C-termini, because their N-terminal signal peptides will be removed. Similarly, proteins that are lipid modified on their N-termini, such as the Rab and Arf GTPases of the Ras superfamily, must be tagged at the C-terminus, because their N-termini are cleaved after lipid addition.

1.2 Key Parameters that Must Be Characterized for Every Epitope-Tagged Protein

There is no a priori way to know whether a tagged protein will mimic the behavior of the endogenous protein. However, a number of increasingly stringent assays can be used to test the tagged protein’s behavior and compare it with the known or implied behavior of the endogenous protein. Any deviation from the known or expected behavior should be further investigated by using an alternative tag or placing the tag at a different position within the protein. It is usually true that observing similar behaviors of a particular protein tagged with different tags reflects normal functionality.

1.2.1 Correct Molecular Weight of the Recombinant Protein

Western blotting of cell lysates from cells expressing the tagged protein must be performed to ensure that a protein of the appropriate size is produced. It is important to keep in mind that large tags such as GFP or GST add significantly (~30 kDa) to the molecular weight of a protein, and this must be taken into consideration in calculating the expected molecular weight of the recombinant protein. For example, the endogenous untagged guanine nucleotide exchange factor GBF1 that facilitates ER-Golgi traffic migrates on SDS-PAGE gels as a ~200 kDa protein. Similarly, when GBF1 is tagged with the small myc-tag, the resultant tagged protein migrates at ~200 kDa (Fig. 1a). However, when GBF1 is tagged with the large GFP-tag, the resultant protein migrates at ~230 kDa on an SDS-PAGE gel (Fig. 1b).

6

Jay M. Bhatt et al.

Fig. 1 Western blotting can be used to test expression of epitope-tagged proteins. HeLa cells were either mock transfected (Control), transfected with myctagged GBF1 (GBF1-myc), or transfected with GFP-tagged GBF1 (GBF1-GFP). Cells were lysed 24 h later. Equal amounts of each lysate were processed for SDS-PAGE. Gels were transferred to nitrocellulose, and the nitrocellulose filters were cut at a molecular weight of 90 kDa according to the migration of prestained molecular markers. The upper parts of the blots were Western blotted using either (a) anti-myc antibodies (Covance, Princeton, NJ) or (b) anti-GFP antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The lower part of each blot was probed using an anti-actin antibody (Sigma Aldrich) to demonstrate equal loading of cell lysates in each lane. Bands corresponding to the expected molecular weights of myc-tagged GBF1 (~204 kDa) and the larger GFP-tagged GBF1 (~230 kDa) were evident in transfected cell lysates, but not in controls. Image previously published in ref. 2

1.2.2 Expression Levels

Western blotting is used for a semi-quantitative determination of the expression levels of the epitope-tagged protein and allows for comparisons between different samples or between different experiments. This technique can also be used to monitor the response of the tagged protein to physiological and pharmacological treatment. For example, induction of autophagy may correlate with a rapid degradation of the tagged protein.

1.2.3 Transfection Efficiency

Immunofluorescence is used to assess the expression efficiency by measuring the relative proportion of transfected versus untransfected cells within a field of cells. It is recommended to do a time course of transfection efficiency to monitor the optimum time of expression. In addition, extremely low apparent transfection efficiency may reflect rapid degradation of the tagged protein or a cytotoxic effect of expression of the protein. Defining a time course of expression (see Subheading 1.2.4) can give insight into whether the tagged protein is unstable or whether it is cytotoxic.

1.2.4 Time Course of Expression

Most vectors utilized for expression of epitope-tagged proteins in mammalian cells drive expression using the strong cytomegalovirus (CMV) promoter. Such strong expression can mask the true localization of a protein and also can lead to dominant negative phenotypes, as observed in the case of overexpression of GBF1 [3]. Thus, it is advisable to perform preliminary experiments to monitor the expression and localization of the tagged protein beginning approximately

Expression of Epitope-Tagged Proteins in Mammalian Cells in Culture

7

6 h after transfection and continuing until 72 h post transfection to assess the level of expression and to note any changes in localization as the protein levels increase in cells. For proteins affected by high overexpression, tetracycline inducible or repressible vectors are commercially available and allow for tight control of protein expression levels. However, use of these vectors requires the selection of stable cell lines (reviewed in https://www.mirusbio.com/tech-resources/ tips/generate-stable-cell-lines and http://www.lifetechnologies. com/us/en/home/references/gibco-cell-culture-basics/transfection-basics/transfection-methods/stable-transfection.html). 1.2.5 Functionality of the Tagged Protein

One of the key issues associated with expression of any epitopetagged protein is the question of how well the tagged protein reflects the behavior of the endogenous untagged protein. In cases in which the localization of the endogenous protein is known or can be predicted, localization of the tagged protein to the appropriate compartment provides a level of confidence that the tagged protein is correctly folded and might be functional. In most cases, epitopetagged proteins faithfully mimic the localization and function of their endogenous counterparts. For example, staining untransfected cells with anti-GBF1 antibodies detects endogenous GBF1 in a perinuclear crescent that is characteristic of the architecture of the Golgi complex (Fig. 2a). Similarly, GBF1 tagged with myc and detected in transfected cells using anti-myc antibodies shows a similar perinuclear staining pattern (Fig. 2b). Thus, myc-tagging GBF1 does not affect its ability to localize correctly in cells, suggesting that myctagged GBF1 might be functional. However, tagging can lead to the misfolding of the tagged protein and often results in its aggregation

Fig. 2 The localization of tagged proteins often mimics the localization of endogenous proteins. (a) HeLa cells were processed for immunofluorescence with antibodies to GBF1 to detect the localization of the endogenous GBF1. GBF1 is observed in a perinuclear staining pattern that is characteristic of Golgi complex staining. (b) HeLa cells were transfected with myc-tagged GBF1 and 24 h later processed for immunofluorescence with antibodies to myc. The myc-tagged GBF1 localizes to a perinuclear Golgi structure that is analogous to the localization of the endogenous GBF1 in panel a

8

Jay M. Bhatt et al.

and loss of function. Aggregation can be assessed by immunofluorescence, with the aggregated proteins accumulating in large cytoplasmic aggresomes close to the nucleus and the microtubule organizing center [4, 5]. Detection of large aggregates within the transfected cells has to be viewed with extreme caution, as it almost always suggests misfolding of the tagged protein. Loss of function is often difficult to assess, but sometimes can be tested by expressing the tagged protein in a null cell line (if one exists) to determine whether the tagged protein can rescue the cellular function of the endogenous protein. Another way to test function is to express the tagged protein in cells in which the endogenous protein is depleted by siRNAi (see [6]) or deleted by CRISPR-Cas9 (reviewed in ref. 7). In some cases, a pharmacological replacement assay can be used. In this approach, the endogenous protein is inhibited with a drug, while the exogenously expressed tagged protein is engineered to be resistant to the drug, and when expressed in cells, represents the sole functional species within the cell. For example, GBF1 function is essential for the biogenesis of the Golgi complex in cells [2, 8]. GBF1 is an enzyme that is inhibited by the fungal metabolite Brefeldin A (BFA), and when cells are treated with BFA, the drug inhibits GBF1 and causes Golgi disassembly into punctate structures (Fig. 3a, b). However, an amino acid substitution at residue 795 of GBF1 (GBF1/795) confers resistance to BFA. Thus, the function of an exogenously expressed GBF1 can be tested by its ability to maintain Golgi architecture in cells in which the endogenous GBF1 is inactivated with BFA. This approach is exemplified in Fig. 3c–e that shows an experiment in which cells were transfected with GFP-tagged GBF1/795 and subsequently treated with BFA. The expression of GFP-GBF1/795 was detected with anti-GFP antibodies while the architecture of the Golgi was detected with antibodies to the p115 tethering protein that represents a Golgi marker. As shown in Fig. 3c, a single cell in a field of cells is expressing GBF1/795. Importantly, the same cell shows an intact Golgi stained with the p115 marker (Fig. 3d), while cells that do not express GBF1/795 have dispersed Golgi as shown by the dispersed localization of the p115 marker (Fig. 3d). The merged image (Fig. 3e) clearly shows that only the cell expressing GFP-GBF1/795 contains intact Golgi complex. This implies that the exogenously expressed GFP-GBF1/795 is functional since it supports Golgi biogenesis. Whenever possible, expression of the tagged protein should be compared to the known localization and/or function of the endogenous protein. Considering the wide variety of small and large tags and the possibility of tagging at different positions within a protein of interest, varying the size and/or type of epitope and the localization within the protein can often eliminate folding problems and maintain a functional protein.

Expression of Epitope-Tagged Proteins in Mammalian Cells in Culture

9

Fig. 3 Tagged proteins often retain their functionality. (a, b) HeLa cells were mock treated (i.e., incubated in media containing 0.1 % DMSO) or treated with the GBF1 inhibitor BFA (i.e., incubated in media containing 0.5 μg/mL BFA in DMSO) for 30 min. Cells were then fixed and processed for immunofluorescence with antibodies to the GM130 Golgi marker. In mock-treated cells, the Golgi complex is intact and observed in a perinuclear staining pattern that is characteristic of Golgi complex architecture in mammalian cells (a). However, in BFA-treated cells, the Golgi is dispersed as shown by the localization of GM130 in small puncta dispersed throughout the cell (b). (c–e) HeLa cells were transfected with GFP-tagged GBF1 containing an amino acid substitution at residue 795 (GBF1/795) that makes GBF1 resistant to BFA. After 24 h, cells were treated with 0.5 μg/mL BFA for 30 min to inactivate the endogenous GBF1. Cells were then fixed and processed for double label immunofluorescence with antibodies to GFP to detect cells expressing the GFP-tagged GBF1/795 (green) and with antibodies to the p115 marker to monitor Golgi architecture in all cells (red). A single cell expresses the BFA-resistant GFP-tagged GBF1 (c), and it alone maintains intact Golgi (d, arrow), while cells not expressing the BFA-resistant GFP-tagged GBF1 have dispersed Golgi. The merged image (e, yellow staining) clearly shows that only the cell expressing the BFA-resistant GFP-tagged GBF1 maintains the architecture of the Golgi complex. This indicates that the BFA-resistant GFP-tagged GBF1 is functional. In all images, the nuclei are stained blue with Hoescht 1.3 Applications of Epitope-Tagging Technology 1.3.1 Defining Protein Subcellular Localization and Behavior

Epitope tagging allows the in situ localization of the tagged protein to a morphologically recognizable cellular structure and facilitates co-localization of the tagged protein with markers for specific cellular organelles. Detailed protocols for double-label colocalization are provided in Chapter 6 of this volume. This technique also allows the analysis of changes in the localization of the tagged protein after particular stimuli or pharmacological treatments. For co-localization double-staining studies, a mixture two of different antibodies or organelle-specific dyes are utilized simultaneously to visualize the protein of interest versus an organelle marker (see Note 2).

10

Jay M. Bhatt et al.

One has to be cautious when selecting antibodies for localization, as the two primary antibodies that are used must originate from different species or must represent different IgG isotypes. For example, rabbit anti-(target protein 1) and mouse anti-(target protein 2) antibodies could be effectively used for a co-localization experiment. 1.3.2 Probing Interactions of the Tagged Protein with Other Proteins

To assess an interaction between tagged recombinant proteins, two or more proteins of interest are tagged with different tags and are co-transfected into mammalian cells. The cells are lysed under non-denaturing conditions to preserve any pre-formed complexes, and immunoprecipitated with antibodies to one of the used tags. The interacting partners within the protein complex are then detected by Western blotting with antibodies to the other tags. To probe an interaction between a tagged recombinant protein and endogenous untagged proteins, a single tagged protein is transfected into cells, and the cell lysates are precipitated with anti-tag antibodies. The interacting partners within the protein complex are detected either by Western blotting with an antibody against a candidate protein (assuming antibodies are available) or by mass spectrometry. Precipitations are routinely performed in parallel from cells transfected with an empty vector (control) and from cells transfected with the tagged protein (experimental).

1.3.3 Generating Protein for In Vitro Reconstituted Biochemical Assays

Mammalian cells are used to express and purify proteins that require posttranslational modifications, such as glycosylation, phosphorylation, or addition of myristate or geranyl-geranyl groups to be functional. Expression of recombinant proteins in mammalian cells is usually at relatively low levels, and high affinity capture tags such as HaloTag or Biotin are best suited for such experiments, since they allow efficient purification even at low levels of expression. However, for proteins that do not require extensive posttranslational modifications, bacterial expression systems such as E. coli are usually preferred due to their ease of handling and higher levels of protein expression.

1.3.4 Defining Dynamics of the Tagged Protein

Mammalian cells expressing fluorescently tagged proteins can be used to visualize protein dynamics in living cells in real time. The importance of GFP tagging was recognized by the awarding of the 2008 Nobel Prize in Chemistry to Osamu Shimomura, Martin Chalfie, and Roger Y. Tsien for their transformative applications of GFP to modern science. Approaches in GFP usage are discussed in Chapters 6, 7, and 8 of this volume. With careful control, expression of epitope-tagged constructs in mammalian cells can be a highly useful technique for expressing the protein of interest for microscopy-based studies and for biochemical analyses. In this chapter, we discuss approaches and provide examples detailing how to transfect cells and how to assess the expression of

Expression of Epitope-Tagged Proteins in Mammalian Cells in Culture

11

tagged proteins by Western blotting (Fig. 1), how the expression and the localization of the tagged proteins can be detected by immunofluorescence (Fig. 2), and how the functionality of the tagged protein can be assessed by a cellular “functional replacement” assay (Fig. 3). The techniques described include mammalian cell culture on glass and plastic, transfection of DNA constructs into mammalian cells, Western blotting and immunofluorescence. A list of the materials required is followed by a detailed discussion of the protocols for these techniques.

2

Materials Solutions may be stored at room temperature unless noted otherwise.

2.1 Cell Culture and Transfection

1. HeLa cell stock stored at −80 °C (see Note 3). 2. PBS: Sterile 1× phosphate-buffered saline (Mediatech, Inc., Manassas, VA). 3. 10 % FBS MEM: Supplement Minimum Essential Media with glucose and l-glutamine (MEM, Mediatech, Inc., Manassas, VA) with 10 % fetal bovine serum (FBS, Life Technologies, Grand Island, NY), 1 mM sodium pyruvate, 0.075 % sodium bicarbonate, 100 units/mL penicillin and 100 μg/mL streptomycin using a concentrated sterile penicillin–streptomycin solution (Mediatech, Inc., Manassas, VA). Store at 4 °C. 4. Trypsin–EDTA: Sterile solution of 0.25 % trypsin and 1 mM ethylenediamine tetraacetic acid (EDTA) (Mediatech, Inc., Manassas, VA). Store at 4 °C. 5. 35 and 100 mm tissue culture dishes. 6. 24-well tissue culture plates. 7. 12 mm circular coverslips. 8. 1.5 mL sterile, nuclease-free microcentrifuge tubes. 9. Serum-free RPMI (Mediatech, Inc., Manassas, VA). Store at 4 °C. 10. TransIT-LT1 polyamine transfection reagent (Mirus Corporation, Madison, WI). Store at 4 °C (see Note 4). 11. Miniprep of transformation vector encoding epitope tagged protein in sterile nuclease-free water. The DNA concentration should be at least 50 μg/mL for transfection (see Notes 5 and 6). Store at −20 °C. 12. Miniprep of control transformation vector that does not contain an insert; empty vector. The DNA concentration should be at least 50 μg/mL for transfection (see Notes 5 and 6). Store at −20 °C. 13. 37 °C incubator containing 5 % CO2.

12

2.2

Jay M. Bhatt et al.

Cell Lysis

1. 10× PBS (phosphate-buffered saline, not sterile): 137 mM NaCl, 27 mM KCl, 43 mM KH2PO4, 14 mM Na2HPO4, pH 7.4. Dilute to 1× PBS by combining 100 mL 10× PBS with 900 mL H2O for final use. 2. 50× cOmplete protease inhibitors (Roche Diagnostics, Indianapolis, IN) stock solution: Dissolve one pellet of cOmplete protease inhibitors in 1 mL of deionized water to make a 50× stock solution. Store stock solution at −20 °C. 3. RIPA buffer: 150 mM NaCl, 1 % NP-40, 0.5 % sodium deoxycholate, 0.1 % SDS, 50 mM Tris–HCl, pH 8.0. Store at 4 °C. Just prior to use, add protease inhibitors to a final concentration of 1× from the 50× cOmplete protease inhibitors stock solution. 4. Teflon cell scrapers. 5. 1.5 mL microcentrifuge tubes. 6. Microcentrifuge. 7. Pierce BCA (bicinchoninic acid) Protein Assay kit (Thermo Scientific, Rockford, IL). 8. Visible wavelength spectrophotometer. 9. Cuvettes. 10. 4× Laemmli sample buffer: 8 % sodium dodecyl sulfate (SDS) (see Note 7), 40 % glycerol, 20 % 2-mercaptoethanol, 0.008 % bromophenol blue, 0.25 M Tris–HCl, pH 6.8. Store at 4 °C. 11. Deionized water.

2.3 SDSPolyacrylamide Gel Electrophoresis and Western Blotting

1. 30 % acrylamide–bisacrylamide Hercules, CA) (see Note 8).

solution

29:1

(Bio-Rad,

2. 1.5 M Tris–HCl, pH 8.8. 3. 1.0 M Tris–HCl, pH 6.8. 4. 10 % SDS: Make a 10 % solution in H2O (see Note 7). 5. 10 % APS: 10 % ammonium persulfate in H2O. Solution should be freshly made every 2–4 weeks and stored at 4 °C in order to promote rapid gel polymerization. 6. TEMED: N,N,N,N′-Tetramethyl-ethylenediamine (Bio-Rad, Hercules, CA). 7. Isopropanol. 8. Kimwipes (Kimberly Clarke, Neenah, WI). 9. Tris/Glycine/SDS running buffer (10×): 250 mM Tris base, 1.9 M glycine, 1 % SDS, pH 8.3 (see Note 7). Dilute to 1× Tris/ Glycine/SDS running buffer by combining 100 mL 10× Tris/ Glycine/SDS running buffer with 900 mL H2O for final use. 10. Kaleidoscope prestained molecular weight markers (Bio-Rad, Hercules, CA).

13

Expression of Epitope-Tagged Proteins in Mammalian Cells in Culture

11. Gel loading tips. 12. Mini-PROTEAN Hercules, CA).

gel

electrophoresis

system

(Bio-Rad,

13. Nitropure nitrocellulose membrane (Cole Parmer, Vernon Hills, IL). 14. Whatman paper (Fisher Scientific, Pittsburgh, PA). 15. Sponge (Bio-Rad, Hercules, CA): Fits transfer cassette. 16. Methanol. 17. 10× Tris-Glycine Transfer Buffer: 20 mM Tris base, 150 mM glycine. For 1× solution, combine 100 mL of 10× buffer, 800 mL of H2O, and 100 mL of methanol. 1× buffer should be prepared just prior to use to prevent evaporation of methanol. 18. Ice container apparatus.

(Bio-Rad,

Hercules,

CA):

Fits

transfer

19. Tupperware or other small plastic container. 20. 10× TBS (Tris-buffered saline): 1.5 M NaCl, 0.1 M Tris–HCl pH 8.0. Combine 100 mL of 10× TBS and 900 mL of H2O to make a 1× solution. 21. TBST (Tris-buffered Saline containing 0.1 % Tween 20): Add 1 mL of Tween 20 to 1 L of 1× TBS. 22. Blocking solution: 5 % Carnation nonfat evaporated milk (can be obtained from your local grocer) in TBST. Store at 4 °C for up to 1 week. 23. Primary antibody solution: 5 % bovine serum albumin (BSA) in TBST containing the primary antibody to the epitope tag diluted at the manufacturer’s recommended concentration for Western blotting. 0.05 % sodium azide (NaN3) may be added to the primary antibody solution and the solution stored at 4 °C. This allows the solution to be reused for 3–4 months. 24. Secondary antibody solution: Dilute HRP-conjugated secondary antibody (Invitrogen, Carlsbad, CA) into blocking solution at the manufacturer’s recommended dilution for Western blotting (see Note 9). 25. Super Signal West Pico Chemiluminescent substrate (Thermo Scientific, Rockford, IL). Store solution at 4 °C and use according to manufacturer’s instructions. 26. Mini-Trans Blot tank transfer apparatus (Bio-Rad, Hercules, CA). 2.4 Immunofluorescence

1. Millipore 0.22 micron mixed cellulose ester membranes for. Should be sized appropriately to fit Büchner funnel (see below). 2. Filter apparatus: Side-arm Erlenmeyer flask capped with a Büchner funnel with glass frit. Place Millipore 0.22 micron mixed cellulose ester membrane in funnel prior to filtration. Connect apparatus to a vacuum line for efficient filtration.

14

Jay M. Bhatt et al.

3. 10× PBSf (phosphate-buffered saline): 137 mM NaCl, 27 mM KCl, 43 mM KH2PO4, 14 mM Na2HPO4, pH 7.4. Vacuumfilter solution using filter apparatus and 0.22 micron mixed cellulose ester membrane to remove particulate matter. Dilute to 1× PBSf by combining 100 mL 10× PBSf with 900 mL H2O for final use. 4. 3 % PFA: 3 % paraformaldehyde in 1× PBSf. Dissolve paraformaldehyde in 1× PBSf by heating at 60 °C for 20–30 min. The final solution will remain slightly cloudy and must be vacuumfiltered as described above. Aliquot filtered solution in 10 mL aliquots and store at −20 °C (see Note 10). 5. 10 mM NH4Cl in 1× PBSf. Vacuum-filter solution as described above to remove particulate matter. Store solution at −20 °C. 6. 0.01 % Triton X-100 in 1× PBSf: Solution should be prepared immediately prior to use. 7. PBST (phosphate-buffered saline containing 0.01 % Tween 20): 1× PBSf + 0.01 % Tween 20. Vacuum-filter solution as described above to remove particulate matter. 8. 2.5 % goat serum in PBST: Vacuum-filter solution as described above to remove particulate matter, aliquot, and store at −20 °C. 9. 0.4 % fish skin gelatin in PBST: 0.4 % gelatin from cold water fish skin (Sigma Aldrich, St. Louis, MO) in PBST. Vacuum-filter solution as described above to remove particulate matter, aliquot, and store at −20 °C. 10. Secondary antibody: Goat α-mouse or Goat α-rabbit Alexa 488 (Molecular Probes/Invitrogen, Eugene, OR) (see Note 11). 11. Hoescht 33258 (Molecular Probes/Invitrogen, Eugene, OR) solution: Dilute stock solution (1 mg/mL in H2O, store at 4 °C) in PBS to a final concentration of 0.1 μg/mL just prior to use. 12. Mounting media: 20 mM Tris–HCl, pH 8.0, 0.5 % N-propyl gallate (Sigma Aldrich, St. Louis, MO), 50 % glycerol. Dissolve N-propyl gallate in Tris–HCl, pH 8.0 prior to the addition of glycerol. Mix solution by gentle rocking overnight at 4 °C to minimize introduction of bubbles into the media. Aliquot mounting media in 50 μL aliquots and store at −80 °C. 13. Glass slides. 14. Clear or colored nail polish.

3

Methods

3.1 Cell Culture and Transfection

1. Rapidly thaw a vial containing 1 mL of HeLa cells preserved in liquid nitrogen in a water bath at 37 °C. 2. Immediately following thawing, dilute cells into 10 mL of 10 % FBS MEM in order to prevent toxicity from concentrated DMSO present in the freezing media.

Expression of Epitope-Tagged Proteins in Mammalian Cells in Culture

15

3. Pellet cells at 800 × g for 5 min in a clinical centrifuge. Aspirate the media and resuspend in 10 mL of fresh 10 % FBS MEM. 4. Add the cell suspension to a 100 mm tissue culture dish. After cells have attached to the substratum (approximately 6–8 h), change the media using 10 mL of fresh 10 % FBS MEM. 5. Feed 100 mm stock culture dishes of HeLa cells every 2–3 days by changing the media to 10 mL of fresh 10 % FBS MEM. Passage cells when they reach confluence (approximately 5 days after a 1:7 dilution—see Note 12). 6. Passage HeLa cells from a confluent 100 mm stock culture dishes by first washing cells with 10 mL of sterile PBS, and then incubating cells in 1 mL trypsin/EDTA for approximately 5 min at room temperature. 7. Rinse cells off of the plate using 6 mL of 10 % FBS MEM, and add 1 mL of cell suspension to a 100 mm plate containing 9 mL of 10 % FBS MEM. 8. One day before transfection (see Note 13), seed 3 mL of cell suspension (diluted 1:7 as described above) to each of two 35 mm dishes or 1 mL of cell suspension to each of two wells of a 24-well plate containing a glass coverslip sterilized by autoclaving or with a flame (see Note 14). Duplicate dishes/coverslips allow for transfection of both the epitopetagged DNA construct and the control empty vector (see Note 15). A 1:7 split of cells will generally provide experimental cultures appropriate for transfection the following day, in addition to a stock culture in a 100 mm dish. 9. For each DNA construct to be transfected (empty vector and vector containing the epitope-tagged protein), add 125 μL of serum-free RPMI to two 1.5 mL sterile, nuclease-free microcentrifuge tubes (50 μL for 24-well plate—see Note 16). 10. For each construct, to one of the tubes containing RPMI, add 3 μL of TransIT LT1 (1.5 μL for 24-well plate) (see Note 17). To the second tube, add 1 μg of plasmid DNA (0.5 μg for a 24-well plate). The total volume of the plasmid DNA should be less than 20 μL (10 μL for a 24-well plate). 11. Incubate the two solutions for 5 min at room temperature. 12. Following the incubation, combine the two solutions and mix by pipetting or inversion. Incubate mixture for 20 min at room temperature to allow the DNA and transfection reagent to complex. The mixed solutions should not be incubated for longer than 45 min before addition to cells. 13. Add each transfection mixture (containing either the empty vector or the vector containing the epitope-tagged protein) to a single plate of cells containing 2.25 mL of fresh media (0.9 mL for 24-well plate).

16

Jay M. Bhatt et al.

14. Incubate cells in the transfection media for 4–6 h in a standard incubator in 5 % CO2 at 37 °C. At this time, change the media to fresh 10 % FBS MEM. This step decreases toxicity caused by the transfection reagent. Cell lysis or fixation for immunoelectron microscopy or immunofluorescence should be performed 24–48 h after transfection, dependent upon the protein being expressed. 3.2

Cell Lysis

1. Wash transfected cells three times with 2 mL of PBS in order to remove media and cellular debris. After aspirating the final wash, add 0.5 mL of RIPA buffer containing protease inhibitors to the cells (see Note 18). 2. Lift cells from the tissue culture dish by scraping the bottom of the dish with a Teflon cell scraper. Collect cell lysates in a 1.5 mL microcentrifuge tube and incubate for 30 min at 4 °C before passing the lysate through 23G and 27G needles 20 times each to ensure efficient lysis (see Note 19). 3. Remove cellular debris and unbroken cells by centrifugation at 16,100 × g for 30 min at 4 °C in a standard microcentrifuge. Transfer soluble lysates to a fresh tube to be maintained at 4 °C. At this point, lysates may be stored at −80 °C for later use. 4. Quantify the protein concentrations of each cell lysate using the Pierce BCA assay kit (see Note 20) according to the manufacturer’s instructions. Formation of the colored product is assessed using a standard visible wavelength spectrophotometer and cuvettes. 5. To equal amounts of protein from each lysate (15–25 μg), add 10 μL of 4× Laemmli sample buffer and sufficient deionized water to make a final volume of 40 μL. Store samples at 4 °C while SDS-PAGE gel is being prepared. Samples may also be stored at −20 °C or at −80 °C for extended storage.

3.3 SDS-PAGE Electrophoresis and Western Blotting

1. Assemble the gel using glass plates and 1.5 mM spacers freshly cleaned with 70 % ethanol. 2. Combine 4.0 mL H2O, 3.3 mL 30 % Acrylamide/Bisacrylamide Solution 29:1 (see Notes 8 and 21), 2.5 mL 1.5 Tris–HCl, pH 8.8, 0.1 mL 10 % SDS, 0.1 mL 10 % APS, and 0.004 mL of TEMED to make the resolving (lower) gel solution. Immediately, pipette the lower gel solution into the apparatus using disposable pipettes to a level approximately 5 mm below where the bottom of the sample wells will be located. Pipette a small volume (1–2 mL) of isopropanol on top of the resolving gel solution in order to form a flat surface. Polymerization generally occurs within 30 min. 3. After the gel has polymerized, pour off the isopropanol and remove the excess using a Kimwipes. The stacking (upper) gel solution is prepared by combining 3.4 mL H2O, 0.83 mL 30 % Acrylamide/Bisacrylamide Solution 29:1 (see Note 8),

Expression of Epitope-Tagged Proteins in Mammalian Cells in Culture

17

0.63 mL 1.0 M Tris–HCl, pH 6.8, 0.05 mL 10 % SDS, 0.05 mL 10 % APS, and 0.005 mL TEMED. Immediately pipette the stacking solution into the apparatus and insert the comb slowly, making sure to avoid bubbles. Polymerization will occur in less than 30 min. 4. After polymerization, transfer the gel to the running setup, and fill the tank with 1× Tris/Glycine/SDS running buffer. 5. Prior to loading, boil samples prepared in step 5 of the Cell Lysis protocol for 5 min to denature proteins. 6. Load 10 μL of Kaleidoscope prestained molecular weight markers in the first well using gel loading tips. (Note that molecular weight markers should not be boiled prior to loading.) Boiled control and experimental samples should be loaded in adjacent wells (see Note 22). 7. Run the gel at approximately 80 V constant voltage until the proteins exit the stacking gel and enter the resolving gel. The gel can then be run at approximately 180 V until the dye front nears the bottom of the gel. 8. Turn off the power, disassemble the electrophoresis apparatus, and carefully cut off the stacking gel using a spacer. 9. Cut the Whatman paper and the Nitropure nitrocellulose membrane (see Note 23) to match the size of the sponges and the gel, respectively. Presoak the Whatman paper in transfer buffer and the Nitropure nitrocellulose membrane in water for 2 min prior to assembly of the transfer cassette. 10. Assemble the transfer cassette in the following order: sponge, Whatman paper, gel, Nitropure nitrocellulose membrane, Whatman paper, and sponge (see Note 24). Insert the cassette into the buffer-filled transfer apparatus with the membrane facing the positive pole. An ice container can be added to reduce heating. 11. Transfer the proteins to the nitrocellulose membrane by running the apparatus at low constant voltage (10 V) overnight or higher constant voltage (80 V) for 4 h at 4 °C. 12. Disassemble the transfer apparatus and place the nitrocellulose membrane in a small Tupperware container using forceps. To inhibit nonspecific antibody binding, incubate membrane in blocking solution for 45 min at room temperature. All incubation steps for Western blotting should be performed with agitation at room temperature unless otherwise specified. 13. Incubate the membrane in 10 mL of the primary antibody solution in a small plastic container for 2–4 h at room temperature (or overnight at 4 °C). Sealed plastic bags can be used for smaller volumes (see Note 25). 14. Wash the membrane in 15 mL TBST three times for 10 min each to remove unbound primary antibody.

18

Jay M. Bhatt et al.

15. Incubate the membrane in 10 mL of secondary antibody solution for 1–2 h (see Note 26). 16. Wash the membrane in 15 mL TBST three times for 10 min each to remove unbound secondary antibodies. 17. Detect bound antibodies using Super Signal West Pico Chemiluminescence substrate. Combine 1 mL of Luminol/ Enhancer solution and 1 mL of Stable Peroxide Buffer solution. Distribute the mix uniformly over the surface of the membrane, wrap the moist membrane in plastic wrap, and incubate for 5 min. 18. Transfer the membrane to a new piece of plastic wrap and wick off the excess solution using a Kimwipes. 19. Expose the blot to film. Exposure times will vary depending upon antibody affinity and level of expression (see Note 27). Figure 1a, b show Western blots of HeLa cells either mock transfected (Control), or transfected with epitope-tagged GBF1 (Golgi-specific Brefeldin A Resistant Guanine Nucleotide Exchange Factor 1) constructs. In Fig. 1a, GBF1 was tagged using the small myc epitope, and the blot was probed with anti-myc antibodies. In Fig. 1b, GBF1 was tagged with GFP, and the blot was probed with anti-GFP antibodies. Note that bands corresponding to the expected molecular weights of myc- and GFP-tagged GBF1 were evident in transfected cell lysates but not in controls. This evidence indicates that the tagged protein is expressed and that the antibody specifically recognizes only the tagged protein. Blots for the cellular housekeeping protein actin demonstrate equal protein loading of control and experimental samples. 3.4 Immunofluorescence

1. Wash cells growing on glass coverslips in 24-well dishes three times with 0.5 mL 1× PBSf (each) to remove media and cellular debris (see Note 28). 2. Fix cells in 0.5 mL of 3 % PFA for 10–15 min (see Note 29). 3. In order to stop the fixation reaction, aspirate the 3 % PFA and quench coverslips by adding 0.5 mL of 10 mM NH4Cl in 1× PBSf for 10 min. 4. Wash coverslips with 0.5 mL 1× PBS f (each) three times ( see Note 30 ). 5. Permeabilize cells by incubating in 0.5 mL 0.1 % Triton X-100 in 1× PBSf for 7–10 min. 6. Wash cells in 0.5 mL 1× PBSf three times for 2 min each. At this point, coverslips can be stored in 1 mL 1× PBSf at 4 °C for up to 2 weeks. 7. To block remaining reactive sites, incubate coverslips in 0.5 mL 2.5 % Goat Serum in PBST for 5 min.

Expression of Epitope-Tagged Proteins in Mammalian Cells in Culture

19

8. Incubate coverslips in primary antibody diluted as suggested by the manufacturer in 0.5 mL 0.4 % fish skin gelatin in PBST for 45 min to 1 h at 37 °C (see Note 31). Note that smaller volumes of primary antibody can be used by transferring the coverslips to a humidified chamber (see Note 32). For colocalization studies, two different antibodies (from different species or with different isotypes) may be utilized to visualize the tagged protein and the organelle of interest. 9. Wash coverslips five times with 0.5 mL PBST for 5 min each. 10. Block remaining reactive sites by incubating in 0.5 mL 0.4 % fish skin gelatin in PBST for 5 min. 11. Incubate coverslips in species-specific secondary antibody conjugated to Alexa 488 diluted into 0.5 mL 2.5 % Goat Serum in PBST for 30–45 min (see Notes 11 and 33). For co-localization studies, two secondary antibodies that correspond to the primary antibodies may be utilized. Each of the antibodies must be conjugated to a different fluor for independent visualization. 12. Incubate coverslips in Hoescht 33258 solution for 5 min to stain nuclei. 13. Wash coverslips five times with 0.5 mL PBST for 5 min each. 14. Mount coverslips on glass slides by gently placing coverslips cells facing down onto a small (5 μL) drop of mounting media. Aspirate excess media and seal the edges of the coverslip with nail polish. 15. Cells can be visualized by epifluorescence or confocal microscopy immediately after the nail polish dries (see Note 34). Figure 2 shows images of HeLa cells stained with antibodies directed against GBF1 (Golgi-specific Brefeldin A Resistant Guanine Nucleotide Exchange Factor 1) or images of cells expressing myc-tagged GBF1 and stained with anti-myc antibodies. Endogenous GBF1 localizes to the perinuclear Golgi apparatus (Fig. 2a). Staining of cells transfected with myc-tagged GBF1 shows that the myc-tagged protein exhibits a localization similar to the endogenous GBF1 (Fig. 2b).

4

Notes 1. Table 1: Commonly used tags for generating epitope-tagged proteins. Representative commercially available vectors that insert either a small or a large tag are listed. Using the vector name in a search will identify commercial vendors that supply the listed vector. 2. Table 2: Well-characterized markers for subcellular compartments. Antibodies to the listed protein markers or specific dyes can be used to define the localization of exogenously expressed tagged proteins in cells. All are commercially available.

20

Jay M. Bhatt et al.

Table 1 Commonly used tags for generating epitope-tagged proteins Name

Commercial vector

Small tags Polyhistidine (His6)

pcDNA 3.1/His A, B, and C

Myc

pCMV-myc

FLAG

pFLAG-CMV 3 and 4

Hemagglutinin antigen (HA)

pCMV-HA

S-

pSF-CMV-Puro-COOH-S-Tag*

V5

pcDNA 3.1/V5-His A, B, and C

Streptavidin binding protein (SBP)

pNTAP-B

Large tags GFP

pAcGFP1-N1

RFP

pDsRed2-C1

YFP

pZsYellow1-C1

Biotin ligase (Bir A*)

pcDNA3.1 mycBioID

Glutathione S-transferase (GST)

pDEST27

Maltose-binding protein (MBP)

pSF-CMV-Puro-NH2-MBP*

Halo tag

pFN28A-Halotag-CMV

Table 2 Well-characterized markers for subcellular compartments Cellular compartment

Marker

Plasma membrane

Cadherin, Sodium Potassium ATPase

Cytoskeleton

Vimentin, Desmin, Tubulin, Actin

Early endosomes

EEA1, Rab5, Rab4

Endoplasmic reticulum (ER)

Calnexin, BIP, Calreticulin

Golgi

GM130, Giantin, TGN38, Ceramide

Mitochondria

COX IV, TOMM20, AIF, Mitotracker

Lysosomes

LAMP1, M6PR, Lysotracker

Nuclear envelope

Lamin A, Nup98, BODIPY, Hoechst

Expression of Epitope-Tagged Proteins in Mammalian Cells in Culture

21

3. Epitope-tagged proteins can be expressed in many different types of tissue culture cells; however, expression varies significantly among different cell types. Two of the most commonly used cell lines are HeLa cells (ATCC number CCL-2) and Cos-7 cells (ATCC number CRL-1651), both easily transfectable and highly resilient cell models. However, other cell types, such as HEK293T or MDCK cells, can also be transfected to high efficiency. 4. Commonly used transfection reagents include salt-mediated reagents, such as CaCl2, and cationic lipid-based reagents, such as Mirus TransIT LT1 (the reagent utilized in this protocol), Lipofectamine 2000 (Thermo Scientific, Rockford, IL), and Fugene 6 (Roche Diagnostics, Indianapolis, IN). In all cases, the reagent is added to the DNA to form complexes which are then added to cells. Salt-based complexes are taken into the cells by endocytosis, while the lipid–DNA complexes can enter the cell either through endocytosis or through fusion with the plasma membrane. However, in the event that the cells utilized are not easily transfected, electroporation, which utilizes electric shock to force DNA plasmids into cells, can also be utilized to produce high levels of expression of epitope-tagged proteins. 5. DNA may be prepared from 1 to 5 mL of an overnight bacterial culture using any standard miniprep kit, such as the QIAprep spin miniprep kit (Qiagen, Valencia, CA), according to the manufacturer’s instructions. DNA should be resuspended in sterile nuclease-free water. 6. DNA concentration can be assessed either by determining the absorbance of the DNA solution at a wavelength of 260 nm using a standard spectrophotometer (1 absorbance unit = 50 μg/mL DNA) or by comparing 1 μL of plasmid DNA to quantitative DNA standards on an agarose gel. For most plasmids, a standard miniprep resuspended in 50 μL of sterile, nuclease-free water will yield approximately 200 ng DNA/ μL. If a sufficient concentration for transfection is not achieved, the DNA may be concentrated by ethanol or isopropanol precipitation, followed by resuspension in a smaller volume of sterile nuclease-free water. 7. A mask should be worn when weighing SDS, as it aerosolizes easily and can be an irritant. 8. Note that unpolymerized acrylamide is a potent neurotoxin and should be handled with care, using gloves and standard precautions. 9. A wide variety of HRP- and gold-conjugated secondary antibodies are commercially available from many companies, including Invitrogen (Carlsbad, CA). The secondary antibody should be specific for the species from which the primary antibody was isolated. Generally anti-mouse and anti-rabbit secondary antibodies are significantly more specific than anti-goat,

22

Jay M. Bhatt et al.

anti-sheep, or anti-chicken secondary antibodies. In addition, isotype specific secondary antibodies are also commercially available if the isotype of the primary antibody is known. 10. Paraformaldehyde is highly toxic. One should always wear a mask while weighing dry paraformaldehyde, and aerosolization should be avoided. 11. For use with other wavelengths of light or with other species of primary antibodies, secondary antibodies of varying species specificities attached to Alexa Fluor dyes that fluoresce at different wavelengths are available from Molecular Probes/ Invitrogen (Eugene, OR). 12. The notation “1:7” indicates that the cells from a single 100 mm tissue culture plate are diluted in such a way that they can be used to generate seven new 100 mm plates. 13. Cells should be transfected at least 1 day after seeding to achieve optimal survival. The confluency of the cells should be between 65 and 90 % for optimal transfection efficiency. 14. Some cell types do not adhere well when glass is used as a substratum. In this case, glass coverslips can be coated with fetal bovine serum, collagen, or fibronectin overnight at 37 °C to improve adherence. 15. Transfections are routinely performed by transfecting both an empty vector (control) and the same vector containing an insert encoding the tagged protein (experimental) in parallel. This method provides a negative control for Western blotting and immunofluorescence. 16. Volumes for transfections are described for 35 mm dishes, with volumes for 24-well plates following in parentheses. 17. Transfection efficiency varies greatly dependent upon the cell type and the protein being expressed. It may be necessary to optimize transfection efficiency by using varying amounts of transfection reagent and DNA to obtain optimal efficiency. Transfection efficiency can reach up to 80 %, but generally varies between 10 and 50 %. For some primary or difficult to transfect cell lines, other techniques, such as electroporation, in which a short electric shock is used to allow DNA penetration into cells, may be necessary to achieve sufficient levels of transfection. 18. Buffer A can be used instead of RIPA buffer to lyse cells. Buffer A composition: 10 mM Hepes–NaOH, pH 7.4; 150 mM NaCl; 1 mM EGTA; 0.1 mM MgCl2 supplemented with cOmplete protease inhibitors just prior to use. Store at 4 °C. 19. In the case of inefficient lysis or for solubilization of membranebound proteins, brief sonication or mechanical disruption using a Dounce homogenizer can increase solubilization. However, care should be taken to avoid the formation of bubbles, as this can lead to protein precipitation.

Expression of Epitope-Tagged Proteins in Mammalian Cells in Culture

23

20. It is imperative when using RIPA buffer as the lysis buffer to utilize the BCA assay, as the detergents present in RIPA buffer may interfere with the Bradford assay. 21. This protocol specifies a 10 % acrylamide gel, useful for most applications; however, the percentage of acrylamide may be adjusted for better separation of very large or very small proteins. 22. Western blotting is routinely performed on lysates from cells transfected with an empty vector (control) and with the same vector containing an insert encoding the tagged protein (experimental) loaded in adjacent lanes on SDS-PAGE. The lysates in the control lane contain only the tag, and the antibodies should detect either no bands (for small tags) or a single band corresponding to the molecular weight of the tag alone (for large tags) in that lane. The lysates in the experimental lanes should contain a single band of the molecular weight appropriate for the tagged protein. Large tags such as GFP (~30 kDa) add significantly to the molecular weight of a protein, and this must be taken into consideration when calculating the expected molecular weight. If bands other than the expected tagged protein are detected in both lanes, this indicates that the antibodies recognize additional cellular proteins. In this case, a more specific antibody or different protein tag should be used. 23. The Nitropure nitrocellulose membrane should be manipulated with forceps to avoid transfer of keratins and other proteins to the membrane prior to use. 24. Care should be taken to remove any air bubbles present between the gel and the membrane. Air bubbles block transfer of proteins to the membrane. Air bubbles can be removed by gently rolling a glass test tube or glass pipette over the assembled stack. 25. The optimal working dilution for primary anti-tag antibodies varies from 1:200 to 1:5000, but the concentration recommended by the manufacturer is usually a good starting point for analysis. 26. The optimal working dilution for HRP-conjugated secondary antibodies varies from 1:2000 to 1:10,000, but the concentration recommended by the manufacturer is usually a good starting point for analysis. 27. For low-affinity antibodies or low expressing proteins, stronger ECL detection kits can be utilized. The West Femto Maximum Sensitivity Chemiluminescent Substrate kit from Thermo Scientific is highly sensitive and can be used to detect low signals. 28. All steps should be performed at room temperature unless stated otherwise. Solutions should be removed from coverslips by aspiration, taking care not to aspirate directly from the center of the coverslip. Touching the aspirator directly to the coverslip

24

Jay M. Bhatt et al.

may result in shearing of cells. Coverslips should never be aspirated to the point of being completely dry, as this will lead to alterations in cellular morphology. 29. Some antibodies may require fixation in methanol. In these cases, cells should be incubated in methanol at −20 °C for 5 min, followed by 5–6 washes with PBS. Methanol causes both fixation and cell permeabilization, so steps 5 and 6 may be eliminated when using this method. 30. GFP-tagged proteins can be visualized without the use of antibodies, although antibodies may enhance signal. For GFPtagged proteins, fluorescence may be visualized directly in live cells or after fixation and washing. 31. Typically working concentrations for primary antibodies range from 1:100 to 1:1000 in FSG. 32. Volumes used for primary antibody incubation can be minimized by placing the coverslips in a chamber containing damp Whatman paper covered with Parafilm. The coverslips can be placed face up on the Parafilm and a 50 μL drop of primary antibody solution can be placed on top of the coverslip. However, care should be taken to minimize evaporation and prevent drying of the coverslips. 33. Typically working concentrations for secondary antibodies conjugated to Alexa Fluor dyes range from 1:100 to 1:500 in FSG. 34. The coverslips are usually good for visualization-based analyses for approximately 2 months. However, for experiments involving quantification of fluorescence intensities, imaging must be performed within the first week after staining. References 1. Szul T, Grabski R, Lyons S, Morohashi Y, Shestopal S, Lowe M, Sztul E (2007) Dissecting the role of the ARF guanine nucleotide exchange factor GBF1 in Golgi biogenesis and protein trafficking. J Cell Sci 120:3929–3940 2. Styers ML, Lowery J, Sztul E (2010) Transient expression of epitope-tagged proteins in mammalian cells. Methods Mol Biol 657:43–61 3. Claude A, Zhao BP, Melancon P (2003) Characterization of alternatively spliced and truncated forms of the Arf guanine nucleotide exchange factor GBF1 defines regions important for activity. Biochem Biophys Res Commun 303:160–169 4. Garcia-Mata R, Bebok Z, Sorscher EJ, Sztul ES (1999) Characterization and dynamics of aggresome formation by a cytosolic GFPchimera. J Cell Biol 146:1239–1254

5. Garcia-Mata R, Gao YS, Sztul E (2002) Hassles with taking out the garbage: aggravating aggresomes. Traffic 3:388–396 6. Shen X, Hong MS, Moss J, Vaughan M (2007) BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein, is required for correct glycosylation and function of integrin beta1. Proc Natl Acad Sci U S A 104:1230–1235 7. Doudna JA, Charpentier E (2014) Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346:1258096 8. Garcia-Mata R, Szul T, Alvarez C, Sztul E (2003) ADP-ribosylation factor/COPIdependent events at the endoplasmic reticulumGolgi interface are regulated by the guanine nucleotide exchange factor GBF1. Mol Biol Cell 14:2250–2261

Chapter 2 Antibody Production with Synthetic Peptides Bao-Shiang Lee, Jin-Sheng Huang, Lasanthi P. Jayathilaka, Jenny Lee, and Shalini Gupta Abstract Peptides (usually 10–20 amino acid residues in length) can be used as effectively as proteins in raising antibodies producing both polyclonal and monoclonal antibodies routinely with titers higher than 20,000. Peptide antigens do not function as immunogens unless they are conjugated to proteins. Production of high quality antipeptide antibodies is dependent upon peptide sequence selection, the success of peptide synthesis, peptide–carrier protein conjugation, the humoral immune response in the host animal, the adjuvant used, the peptide dose administered, the injection method, and the purification of the antibody. Peptide sequence selection is probably the most critical step in the production of antipeptide antibodies. Although the process for designing peptide antigens is not exact, several guidelines and computational B-cell epitope prediction methods can help maximize the likelihood of producing antipeptide antibodies that recognize the protein. Antibodies raised by peptides have become essential tools in life science research. Virtually all phospho-specific antibodies are now produced using phosphopeptides as antigens. Typically, 5–20 mg of peptide is enough for antipeptide antibody production. It takes 3 months to produce a polyclonal antipeptide antibody in rabbits that yields ~100 mL of serum which corresponds to ~8–10 mg of the specific antibody after affinity purification using a peptide column. Key words Antipeptide antibody, Immunogenicity, Antigenicity, Peptide synthesis, Peptide–carrier protein conjugation, Keyhole limpet hemocyanin (KLH), Polyclonal and monoclonal antibodies, Phospho-specific antibodies, Antibody titer

1

Introduction Antibodies are routinely used in a variety of biomedical fields including biotechnology, medicine, immunotherapy, and diagnosis, and antibodies are one of the most useful biomolecules for life science research. With their high specificity and binding ability (the typical equilibrium dissociation constant of an antibody–antigen complex is ~10−6–10−12 M), antibodies are mainly used for protein recognition [1–4]. Proteins, either prepared from biological specimens or made by recombinant methods, are traditionally used as immunogens to produce antibodies. Generating antibodies against a protein yields antibodies against numerous epitopes in the protein, which

Steven D. Schwartzbach et al. (eds.), High-Resolution Imaging of Cellular Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 1474, DOI 10.1007/978-1-4939-6352-2_2, © Springer Science+Business Media New York 2016

25

26

Bao-Shiang Lee et al.

maximizes the chance of an antibody recognizing the protein. However, this pool of antibodies does increase the possibility of cross-reactions with other proteins (see Note 1). Generating antibodies against a carefully selected synthetic peptide [1, 2, 5–7] (see Note 2), by contrast, will produce antibodies that are specific to the target protein. There are instances where using a peptide as antigen is advantageous over a protein, such as raising antibodies for a specific protein isoform, phosphorylated/glycosylated proteins [8–10] (see Note 3), and proteins that are not easily purified such as large membrane proteins. Peptide antigens are often used to generate polyclonal antibodies (mainly of IgG subclass) in either goat or rabbit that target unique epitopes, especially for protein families of high homology. The applications of antipeptide antibodies include gene product detection and identification, protein processing studies, diagnostic tests, protein localization, and determination of a protein active site. 1.1 Overall Strategy for Antipeptide Antibody Production

Low-cost, high-purity peptides can be obtained commercially (a list of companies which offer customer peptide synthesis can be found on the Peptide Resource Page [www.peptideresource.com]). There are many companies that also supply antipeptide antibodies (a list of companies which provide antibody services can be found on the Antibody Resource Page [www.antibodyresource.com]). However, not all antipeptide antibodies have a high titer and some cannot recognize native proteins. The potential drawback of choosing a peptide sequence that does not elicit a strong immune response and won’t correspond to an exposed region of the endogenous protein can be reduced substantially by carefully analyzing the protein sequence and structure using protein structure/antigenicity prediction websites such as IEDB (www.iedb.org). Additionally, co-immunization of several peptide antigens or immunization of a larger peptide antigen from a protein will statistically increase the chances of obtaining antibodies that will recognize the target protein (see Note 4). Peptides containing phosphorylated amino acids can also be used to produce phosphospecific antibodies [8, 9] (see Note 3). Although short peptides have been reported to generate good antibodies by themselves, a peptide is usually too small to induce an immune response and produce high titer antibodies. The minimum molecular weight needed to induce a sufficient immune response is ~5 kDa. A carrier protein which contains multiple epitopes helps elicit T helper- and B-cell responses and is therefore conjugated to the peptide. The most commonly used carrier protein is keyhole limpet hemocyanin (KLH, 4.5 × 105–1.3 × 107 Da), which has been shown to aid in the production of high titer antipeptide antibodies (see Note 5). KLH contains numerous exposed lysine residues, which allows for the covalent attachment of a large numbers of peptide molecules. With the advancements in peptide synthesis, peptide selection, and peptide–carrier protein conjugation, peptides are becoming the method of choice for antibody production .

Antipeptide Antibody

27

1.2 Analysis of Protein Sequence and Structure

Before the commencement of a new antipeptide antibody project, it is essential to know some basic features of the protein. The first step is ensuring that the correct species and protein sequence have been identified. Databases of protein structure (e.g., PDB [www. rcsb.org/pdb]) can aid in choosing epitopes that are readily accessible to the antibodies. Any potential cross-reactivity with other closely related proteins, e.g., domain structures, should be avoided. Obtaining the protein homology information at NCBI (www.ncbi. nlm.nih.gov), UniProtKB (www.uniprot.org), PIR (pir.georgetown.edu), ExPASy (us.expasy.org/tools), or HomoloGene (www. ncbi.nlm.nih.gov/homologene), predicting extracellular domains of transmembrane proteins using TMpred program (www. ch.embnet.org/software/TMPRED_form.html), determining the secondary structure and solvent accessibility/protein disorder/flexibility (IEDB and others), and predicting the 3D structure of a protein using I-Tasser (zhanglab.ccmb.med.umich.edu/I-TASSER) [11] is helpful. A list of commercially available antibodies can be obtained at www.antibodyresource.com/findantibody.html.

1.3 Selection of Peptide for Antibody Production

Although the exact peptide sequence to achieve the strongest immune response has to be determined empirically, many B-cell epitope databases/prediction servers (see Note 6) and selection guidelines/tips can help maximize the likelihood of success in producing antibodies of high quality. Other factors to be considered are the ease of peptide synthesis and peptide–carrier protein conjugation, peptide stability and solubility in buffers, and the specificity for the target protein. Since antibodies bind to epitopes on the surface of proteins, when examining a protein sequence for potential antigenic epitopes it is important to choose sequences that are found on the surface of the native protein, are flexible, contain both hydrophilic and hydrophobic amino acid residues, and preferably have antigenic amino acids (lysine, arginine, glutamic acid, aspartic acid, glutamine, asparagine, etc.). In addition, sequence regions with a β-turn or amphipathic helix character have been found to be antigenic. Peptide length, hydrophobicity, and a series of specific amino acids, which can render the peptide useless, should also be considered while designing the peptide antigen.

1.3.1 Database of Experimental B-Cell Epitope and B-Cell Epitope Prediction (See Note 6)

The majority of published research has focused on peptidic B-cell epitopes, for which the amino acid residues are the structural units in computational analyses. B-cell epitopes, protein antigens that are recognized by the B cells, are classified as either continuous (linear) or discontinuous (conformational) epitopes. A continuous epitope is a single continuous stretch of amino acids within a protein sequence, while a discontinuous epitope has amino acid residues that are distantly separated in the sequence and are brought into physical proximity by protein folding. The majority of B-cell epitopes are conformational. The first step of designing peptides for raising

28

Bao-Shiang Lee et al.

antipeptide antibodies is to rely on finding one suitable peptide in a database of experimental epitopes. If this is not successful, numerous algorithms are used to provide a collection of possible antigenic peptides. The common epitopes from all the predictions are considered. Many computational methods [12–15] (see Note 6) have been proposed for predicting either linear or conformational B-cell epitopes. Methods for predicting linear B-cell epitopes range from simple propensity scale profiling to state-of-the-art machine learning prediction servers whereas conformational B-cell epitopes are predicted utilizing structure and physicochemical features derived from antibody-antigen complexes that could be correlated with antigenicity. Despite the large number of B-cell epitope prediction methods proposed in the literature, it was a major challenge in immunoinformatics to locate B-cell epitopes in the protein. However, recent advances using multiple parameters (e.g., antigen preprocessing and mimotope analysis [16, 17]) has dramatically improved the accuracy of predictions for B-cell epitopes. A mimotope is a peptide mimic of an epitope. It elicits an antibody response similar to its corresponding epitope and binds to antibodies raised by its corresponding epitope. Mimotopes are commonly identified from phage display libraries. The following sections list the guidelines/tips for the selection of peptides for successful antibody production. 1.3.2 Peptide Hydrophobicity/Surface Accessibility/ Flexibility/Antigenicity Factors

There is no foolproof way to determine whether a selected peptide can produce high quality antibodies recognizing the native protein other than injecting the peptide and analyzing the antibodies produced. From 0 to 75 % of antipeptide antibodies has been reported in the literature to recognize the native protein. Antibodies only bind to epitopes found on the surface of proteins and tend to bind with higher affinity when those epitopes are flexible enough to move into accessible positions. In general, ideal antigenic epitopes are hydrophilic, surface orientated and flexible . This is because in most natural environments, hydrophilic regions tend to reside on the surface, whereas hydrophobic regions are found hidden in the protein interior. Proline acts as a structural disruptor in the middle of regular secondary structural elements and is commonly found as the first amino acid residue in an alpha helix, in turns, and loops and in the edge strands of beta sheets. This may account for the fact that proline is usually solvent exposed and often forms part of a known epitope. Algorithms for predicting protein characteristics such as hydrophilicity/hydrophobicity and secondary structural regions aid in selection of a potentially exposed, immunogenic internal sequence for antibody generation. Some guidelines/tips to increase the likelihood of successful peptide selection are as follows: 1. Use a BLAST protein search with Protein Lounge (database containing numerous antigenic peptide targets, http://www. proteinlounge.com.) to compare all recommended peptides for sequence homology to other proteins.

Antipeptide Antibody

29

2. Use the peptide antigen database (www.genscript.com/ peptide-antigen-database.html) to predict linear epitopes. 3. Choose peptides that are in the N- or C-terminal region of the protein because these regions are usually solvent accessible and unstructured. Antibodies developed against these peptides are also more likely to recognize the native protein. The N terminal capped with an acetyl group and the C terminal with an amide group make the peptide appear more like a native protein and reduce the degradation of the peptide in the animal. In addition, the orientation of the peptide is extremely critical in certain cases. 4. Preferably choose peptides lying in long loops connecting secondary structure motifs. Avoid peptides that are located in inaccessible helical and beta sheet regions. Antigenic peptides should be located in solvent accessible regions and contain both hydrophobic and hydrophilic amino acid residues. This will increase the odds of the antibody recognizing the native protein. For proteins with known 3D coordinates, secondary structure and solvent accessibility can be obtained from the sequence link of the relevant entry at the Brookhaven data bank (www.rcsb.org/ pdb) and can be calculated using a variety of programs such as DSSP (swift.cmbi.ru.nl/gv/dssp), NACESS (www.bioinf.manchester.ac.uk/naccess), WHATIF (swift.cmbi.kun.nl/whatif; swift.cmbi.ru.nl/servers/html/index.html), or PDBsum (www. ebi.ac.uk/pdbsum). When no structural information is available, secondary structure and accessibility predictions can be obtained from the following servers with 80 % accuracy in predicting α-helixes, β-strands, and loops [11, 18]: I-Tasser (zhanglab. ccmb.med.umich.edu/I-TASSER), PHD (www.predictprotein. org), JPRED (www.compbio.dundee.ac.uk/~www-jpred), PSIPRED (bioinf.cs.ucl.ac.uk/psipred), PredAcc (bioserv.rpbs.jussieu. fr/RPBS/html/fr/T0_Home.html), RPBS (http://bioserv.rpbs. univ-paris-diderot.fr/index.html), ACCpro (scratch.proteomics. ics.uci.edu), SSpro (scratch.proteomics.ics.uci.edu), SSPRED (coot.embl.de/~fmilpetz/SSPRED/sspred.html), PREDATOR (http://mobyle.pasteur.fr/cgi-bin/portal.py?#forms::predator). Websites such as PROFsec (www.predictprotein.org) predict secondary structure elements and solvent accessibility using evolutionary information from multiple sequence alignments and a multi-level system. Meta-Disorder (MD) predicts intrinsically disordered proteins from protein sequences. The prediction is based on a system of neural networks that combines the outputs from several original prediction methods (NORSnet, DISOPRED2, PROFbval, and Ucon), with the evolutionary profiles and sequence features that correlate with the protein disorder, such as predicted solvent accessibility and protein flexibility.

30

Bao-Shiang Lee et al.

5. Check the peptide sequence against a number of criteria to estimate its suitability for antipeptide antibody production using websites for antigenicity prediction such as www.innovagen.se, immunax. dfci.harvard.edu/Tools/antigenic.html, www.immuneepitope. org/home.do, www.innovagen.se/custom-peptide-synthesis/ peptide-property-calculator/peptide-property-calculator.asp, and ca.expasy.org/tools/protscale.html. Several software programs such as MacVector, DNAStar, and PC-Gene that incorporate one or more of several accepted algorithms for predicting peptide antigenicity are also useful. 6. Avoid areas of the protein, such as the transmembrane regions, that are not accessible to antibodies. 7. Generally, use 10–20 amino acid long peptides although longer peptides give a good immune response and may provide relevant secondary structure (see Note 7). Peptide sequences of this length minimize synthesis problems and are reasonably soluble in aqueous solution. Longer peptides increase the risk of losing specificity and shorter peptides may elicit antibodies that would not recognize the native protein with sufficient affinity. 8. Design peptide antigens with a preference for hydrophilic or charged amino acid residues and regions containing at least 30 % immunogenic amino acids such as lysine, arginine, glutamic acid, aspartic acid, glutamine, and asparagine. 9. Incorporate proline and tyrosine residues into the immunogen to confer some structural motifs that are likely to be found in the native protein. 10. For antipeptide antibody production, avoid: (1) internal cysteines (replace with serine), (2) long chains of hydrophobic amino acid residues, (3) any coupling method that binds to an internal amino acid residue, (4) numerous serine, glutamine, or proline residues in a peptide sequence, (5) N-terminal glutamine or aspargine and C-terminal proline or glycine in a peptide, (6) Arginine–glycine–aspartic acid motifs, (7) small molecule binding sites, (8) biologically active regions, and (9) posttranslational modification sites. If the chosen sequence does not contain cysteine, it is common to place a cysteine residue at the N- or C-terminus to obtain controlled linking of the peptide to the carrier protein. Undesirable amino acid residues should be replaced with conservative amino acid residues to improve peptide properties, e.g., alanine or valine can be used to replace internal cysteine. If possible, the use of more than one peptide, a carboxyl-terminal hydrophilic sequence, an amino-terminal hydrophilic sequence, internal hydrophilic regions, and peptides lying in long loops connecting secondary structure motifs are desirable. The antigen, injection conditions, or host can be modified to increase the immune response.

Antipeptide Antibody 1.3.3 Peptide Solubility and Stability Factors

31

Investigating a protein sequence to identify the best peptides that can be successfully synthesized with high solubility is an essential step in antipeptide antibody production. Several things to consider are as follows: 1. Peptide solubility is strongly influenced by amino acid composition. Peptides with a high content of hydrophobic amino acid residues, such as leucine, valine, isoleucine, methionine, phenylalanine, and tryptophan, have either limited solubility in aqueous solution or are completely insoluble. It is advisable to keep the hydrophobic amino acid content below 50 % and ensure that there is at least one charged amino acid residue for every five amino acids. A single conservative replacement, such as replacing alanine with glycine or adding a set of polar amino acid residues to the N- or C-terminus, may also improve solubility. 2. During synthesis, β-sheet formation causes incomplete solvation of the growing peptide and results in many deletion sequences in the final product. This problem can be avoided by choosing sequences that do not contain multiple and adjacent valine, isoleucine, tyrosine, phenylalanine, tryptophan, leucine, glutamine, and threonine amino acid residues. If sequences cannot be chosen to avoid stretches of these amino acid residues, it often helps to break the pattern by making conservative replacements such as inserting a glycine or proline at every third amino acid residue, replacing glutamine with asparagine, or replacing threonine with serine. 3. Peptides containing multiple cysteine, methionine, or tryptophan residues are also difficult to obtain in high purity because these amino acid residues are susceptible to oxidation and/or side reactions. 4. The following amino acids or sequences are best avoided: (a) A sequence starting or ending with proline. (b) A sequence containing an acid labile aspartic acid–glycine bond. (c) Alanine, valine, threonine, proline, or serine doublets and sequences ending in valine, isoleucine, tryptophan, tyrosine, and phenylalanine. (d) Extremely long repeats of the same amino acid (e.g., arginine–arginine–…) and glutamine or asparagine at the N-terminus. 5. A peptide having an overall charge close to neutral is desirable. 6. Sequences ending with hydrophilic amino acid residues or free alpha reactive groups are preferred as these side groups will promote solubility.

32

Bao-Shiang Lee et al.

7. Limiting the number of contiguous charged or hydrophobic amino acid residues is helpful as they can create solubility problems. 8. Due to the nature of glycine and its lack of a side group, it does not behave as a hydrophobic amino acid residue unless continuous stretches exist. 1.4 Peptide Synthesis

Usually peptides used for antipeptide antibody production contain 10–20 amino acid residues and are readily obtainable either from a core facility or commercial vendors [19–22]. A state-of -the-art peptide synthesizer would have little problem producing 50 mg of peptide with >90 % purity even with several phosphorylated amino acids economically. Peptides are routinely synthesized in our laboratory using stepwise Fmoc solid-phase synthesis chemistry starting from the C-terminus as follows (see Note 8): (1) the Fmoc group of the amino-acid-preloaded resin is removed by 20 % piperidine, (2) the Fmoc-amino acid (with or without modification) is coupled to the resin-bound peptide using 0.1 M 2-(1H-Benzotriazole-1-yl)1,1,3,3,-tetramethyluronium hexafluorophosphate (HBTU) in dimethylformamide (DMF) containing 0.4 M 4-methylmorpholine for 30–60 min, (3) steps 1 and 2 are repeated until the last amino acid is added, (4) the Fmoc group of the resin-bound peptide is removed by 20 % piperidine, (5) the peptide is then deprotected and cleaved from the resin using trifluoroacetic acid (TFA), (6) ethyl ether is added to precipitate the peptide from the TFA solution and the precipitated peptide is lyophilized, (7) the crude peptide is purified on a reversed-phase C18 column using a preparative high-performance liquid chromatography system. A flow rate of 20 mL/min with solvent A (0.1 % TFA in deionized water) and solvent B (0.1 % TFA in acetonitrile) is used. The column is equilibrated with 5 % solvent B. After sample loading, the column is eluted with a linear gradient from 5 % solvent B to 100 % solvent B in 60 min, and (8) the pure peptide fraction is identified by matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) or electrospray ionization mass spectrometry (ESI MS). Dissolving peptides in aqueous solutions is not trivial. In practice, the peptide sequence should contain at least 20 % charged amino acid residues to facilitate solubilization. Hydrophilic peptides containing >25 % charged amino acid residues (glutamic acid, aspartic acid, lysine, arginine, and histidine) and peptides containing lysine + arginine + histidine residues) and basic peptides (lysine + arginine + histidine residues > glutamic acid + aspartic acid residues) are more soluble at neutral pH. Hydrophobic peptides containing 50–75 % hydrophobic amino acid residues may be insoluble or only partially soluble in aqueous solutions even if the sequence

Antipeptide Antibody

33

contains 25 % charged amino acid residues. It is best to first dissolve these peptides in a minimal amount of stronger solvents (such as DMF, acetonitrile, isopropyl alcohol, ethanol, 4–8 M guanidine HCl, 2–8 M urea, DMSO, ammonium bicarbonate, or acetic acid) and then slowly add the solution to a stirred aqueous buffer solution until the desired concentration is obtained. Very hydrophobic peptides containing >75 % hydrophobic amino acid residues will generally not dissolve in aqueous solutions and require initial solubilization in very strong solvents (such as TFA and formic acid) and may precipitate when added into an aqueous buffered solution. Peptide sequences containing a very high (>75 %) proportion of serine, threonine, glutamic acid, aspartic acid, lysine, arginine, histidine, asparagine, glutamine or tyrosine are capable of forming extensive intermolecular hydrogen bond networks and have a tendency to form gels in concentrated aqueous solutions. Sonication may help dissolve peptides to a small degree. Acetic acid (10 %) in the solvent will help dissolve basic peptides, whereas 10 % ammonium bicarbonate will help dissolve acidic peptides. Most of the peptides used for generating custom antibodies are reasonably hydrophilic and will dissolve readily in PBS, water, or saline solutions. If a small test batch of peptide solubilizes easily in PBS, then dissolving at a 1 mg/mL concentration to create a working stock of the peptide is advisable. Peptide sequences containing cysteine, methionine, or tryptophan are prone to air oxidation. It is recommended to purge the air out of the vial and replace it with a blanket of nitrogen or argon and the peptide should be dissolved in the absence of oxidant. Lyophilized peptides are extremely stable and can be stored with Drierite at −20 °C for years. Storing all peptides in their lyophilized state is recommended. 1.5 Animal Immunization

Once the antigen has been selected, the production of the antibody is dependent on the animal’s immune system. Despite decades of trying to create a comparable protein detection method, no other system (phage display, aptamers, etc.) has ever come close to mimicking the specificity of antibodies raised from an animal’s immune system. A number of species of animal are suitable hosts for antipeptide antibody production, including mice, guinea pigs, rats, hamsters, rabbits, chickens, pigs, goats, sheep, bovines, donkeys, and horses (see Note 9). Selection of the appropriate animal species is dependent on several factors (NIH animal care guidelines are generally followed): the presence of a homologous protein in the species being immunized, the amount of antibody required, the amount of antigen available for immunization, the necessity of monoclonal antibodies, the time required to obtain an antibody response, and the cost. In order to achieve maximum immune response, choosing an animal that is genetically very different from the source of the immunogen is important to avoid self-recognition of the immunogen by the host animal. As an example, it is more suitable to use a rabbit or mouse than a monkey for human proteins. For highly conserved mammalian

34

Bao-Shiang Lee et al.

proteins, raising antibodies in chickens is often a preferred alternative. Although the egg yolks can produce high levels of antibody, IgY purification is required before the antibody can be used in assays. Information on emulsion of adjuvant mixed with antigen, immunization routes, bleed, and antiserum preparation are widely available [1, 2]. Typically, a single sample bleed will yield 25 mL of serum from a rabbit, 200 μL from a mouse, and 2 mL from a rat, hamster, or guinea pig. Beside mice/rats, the most common host animal for polyclonal antipeptide antibody production is the New Zealand white rabbit (female, 8 weeks of age, used 95 % of the time). It has the ability to respond to broad classes of antigens, is easy to maintain and handle, can be safely bled repeatedly, and produces well characterized and easily purified antibodies in 77 days. In rabbit, the titers remain relatively level after the second booster and additional immunizations are used to maintain antibody titers rather than to increase them. For rabbit immunization, Freund’s adjuvant (see Note 10) is used, which should be emulsified aseptically using syringes or sonication. Solubility of antigen does not play an important role here. Twenty milliliter of serum (prebleed) is collected prior to the initial immunizations. The rabbits are then immunized by injection at four separate subcutaneous sites (two inguinal, two axillary) with 0.25 mg peptide-KLH emulsified together with Freund’s complete adjuvant (FCA). Injected material will drain quickly into the local lymphatic system and will become concentrated in the lymph nodes closest to the injection sites. A booster injection with 0.25 mg peptide-KLH emulsified with Freund’s incomplete adjuvant (FIA) is given 14 days after the initial immunization. Subsequent booster injections with 0.25 mg peptideKLH emulsified with Freund’s incomplete adjuvant (FIA) are given every 4 weeks. Twenty milliliter of serum is collected 10 days after each booster injection. At the conclusion of the animal immunization at day 77, a large-volume terminal bleed (~100 mL) is collected from the rabbit by exsanguination. Animals from various species reach immune maturation at different times. Generally, mice at 6 week of age and rabbits at 8 week of age are used. Even in genetically identical animals, the same antigen will elicit different immune responses. To combat this, in the case of the rabbit, at least two animals should be used, with three to four being preferable. For mice or other rodents, six animals are often used.

2

Materials All solutions should be filtered through a 0.2 μm filter.

2.1 Peptide–Carrier Protein Conjugation

1. 0.01 M phosphate buffer pH 7.0: Use 0.01 M Na2HPO4 to adjust the pH of a 0.01 M solution of NaH2PO4 to pH 7.0. 2. 0.05 M phosphate buffer pH 6.0: Use 0.05 M Na2HPO4 to adjust the pH of a 0.05 M solution of NaH2PO4 to pH 6.0.

Antipeptide Antibody

35

3. 0.5 N HCl. 4. 2 N NaOH. 5. 0.1 M ammonium bicarbonate. 6. Dimethylformamide (DMF). 7. M-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS). 8. Keyhole limpet hemocyanin (KLH). 9. Bovine serum albumin (BSA). 10. pH paper (wide range: 1–14). 11. PD-10 column (Pharmacia Bioscience, Piscataway, NJ). 12. Lyophilizer. 2.2 Antipeptide Antibody Titer Determination by ELISA

1. Carbonate Buffer: 15 mM Na2CO3, 35 mM NaHCO3, 0.02 % NaN3. Adjust pH to 9.6 with 1 N NaOH. 2. Synthetic peptide (0.2–2.5 μM) in carbonate buffer. 3. Phosphate-buffered saline (PBS): 140 mM NaCl, 27 mM KCl, 7.2 mM Na2HPO4, 14.7 mM K2HPO4, pH 7.2. 4. PBS containing 0.05 % Tween 20 (PBST). 5. Blocking solution: 10 mg/mL BSA in PBST. 6. Secondary antibody: Goat anti-rabbit globulin conjugated to alkaline phosphatase. 7. Enzyme substrate: 1 mg/mL p-nitrophenyl phosphate, 0.2 M Tris buffer, 5 mM MgCl2, pH 8.0. 8. Stopping solution: 0.01 M ethylenediaminetetraacetic acid or 3 N NaOH. 9. 96-well flat bottom microtiter plate. 10. Microtiter plate reader with 405 nm filter.

2.3 Peptide Affinity Purification

1. CNBr-activated Sepharose 4 B (Pharmacia Bioscience, Piscataway, NJ). 2. Centrifuge. 3. 1 mM HCl. 4. 0.1 M Tris–HCl. 5. 1 M Tris–HCl buffer, pH 9.0. 6. 0.1 M acetate buffer containing 0.5 M NaCl, pH 4.0. 7. 0.1 M Tris–HCl buffer containing 0.5 M NaCl, pH 8.0. 8. Synthetic lyophilized peptide. 9. Coupling buffer: 0.1 M NaHCO3, pH 8.3, 0.5 M NaCl. 10. Washing buffer 1: 50 mM Tris–HCl, pH 8.0, 0.1 % Triton X-100, 0.5 M NaCl. 11. Washing buffer 2: 50 mM Tris–HCl, pH 9.0, 0.1 % Triton X-100, 0.5 M NaCl.

36

Bao-Shiang Lee et al.

12. Washing buffer 3: 50 mM sodium phosphate, pH 6.3, 0.1 % Triton X-100, 0.5 M NaCl. Use 50 mM Na2HPO4 containing 0.1 % Triton X-100 and 0.5 M NaCl to adjust the pH of a 50 mM solution of NaH2PO4 containing 0.1 % Triton X-100 and 0.5 M NaCl to pH 6.3. 13. Elution buffer: 50 mM glycine–HCl, pH 2.5, 0.1 % Triton X-100, 0.15 M NaCl. 14. Phosphate-buffered saline (PBS): 140 mM NaCl, 27 mM KCl, 7.2 mM Na2HPO4, 14.7 mM K2HPO4, pH 7.2. 15. 50 mM Tris–glycine, 0.15 M NaCl, 40 % glycerol, 0.02 % sodium azide, 0.05 % BSA, pH 7.4. 16. PD-10 column (Pharmacia Bioscience, Piscataway, NJ). 17. 1 × 10 cm chromatography column. 18. 0.2 μm Syringe filter. 19. UV spectrophotometer.

3

Methods

3.1 Peptide–Carrier Protein Conjugation

To stimulate antibody responses for smaller peptides, the peptides need to be covalently conjugated to a larger immunogenic carrier protein (KLH, BSA, etc.) prior to immunization. Poor conjugation of the peptide to the carrier protein is one of the reasons for antibody production failure. It is critical that the peptide to carrier protein molar ratio is high (one mole of peptide per 50 amino acids of carrier is a reasonable coupling ratio), and that all epitopes on the peptides are properly oriented in order to induce a high titer specific immune response (see Note 11). The method of coupling the peptide to the carrier protein is an often overlooked factor while designing the synthetic peptide. It is important to ensure that the peptide is presented to the immune system in a manner similar to that of the native protein. For example, N-terminal sequences should be coupled through the C-terminal amino acid and vice versa for C-terminal sequences. Internal sequences can be coupled at either end. Another consideration for internal sequences is to acetylate or amidate the unconjugated end, because the sequence in the native protein molecule would not contain a charged terminus. The sequence chosen should not have multiple amino acid residues that can react with the chosen chemistry, otherwise, shortening the peptide or choosing the sequence such that they are all localized at either the amino or the carboxyl terminus of the peptide is helpful. For internal sequences, peptides which end furthest from the predicted epitope are normally favored as this circumvents potential masking problems by the carrier. Several popular coupling methods which couple through sulfhydryl (cysteine), amino (α-amino or lysine), carboxylic acid groups (aspartic

Antipeptide Antibody

37

acid, glutamic acid or σ-carboxyl), or hydroxyl groups are available using coupling reagents such as glutaraldehyde, 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide hydrochloride (EDC), and m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) [5]. However, most peptides contain several amino, carboxyl, or hydroxyl groups in side chains of amino acid residues which result in multipoint attachment. It is preferable to attach a carrier protein through a sulfhydryl group in a cysteine residue present at either the N- or C-terminus of a peptide. The peptide–carrier protein ratio can be determined by MALDI-TOF MS or ESI MS. The EDC method is used for coupling the peptide and the carrier protein via the amino or carboxyl group of the peptide sequence, depending on the activation strategy. Carbodiimides can activate the side chain carboxyl groups of aspartic and glutamic acid as well as the carboxyl terminal group to make them reactive sites for coupling with primary amines. The activated peptides are mixed with the carrier protein to produce the final conjugate. If the carrier protein is activated first, the EDC method will couple the carrier protein through the N-terminal α-amine and possibly through the amine in the side-chain of lysine. The MBS is a heterobifunctional reagent that can be used to link peptides to carrier proteins via cysteine. The coupling takes place with the sulfhydryl group of the cysteine residue. A terminal cysteine may be added to the peptide sequence away from the epitope location to allow peptide conjugation to carrier proteins. If the peptide is derived from the N-terminus of the protein, the cysteine should be added to the C-terminus of the peptide and vice-versa. Our procedure, which is a popular protocol [23, 24], for coupling peptides to KLH or BSA through a cysteine is as follows [25]: 1. Dissolve 5 mg of KLH or BSA in 0.5 mL of 0.01 M phosphate buffer, pH 7. 2. Dissolve 3 mg of MBS in 200 μL DMF. 3. Add 70 μL of MBS solution to 0.5 mL of KLH or BSA solution and stir for 30 min at room temperature. Add 2 mL of 0.05 M phosphate buffer, pH 6. 4. Equilibrate a PD-10 column using approximately 25 mL of 0.05 M phosphate buffer, pH 6. Add the 2.5 mL of the MBS/ KLH or BSA/MBS solution to the column and elute with 3.5 mL of 0.05 M phosphate buffer, pH 6. Add 0.5 mL of deionized water to the 3.5 mL of purified KLH/MBS or BSA/MBS. 5. Dissolve 5 mg of peptide in 100 μL of DMF. Rapidly add 1 mL of purified KLH/MBS or BSA/MBS. Shake rapidly and immediately add 11 μL of 2 N NaOH. 6. Check the pH with pH paper. It should be 7.0–7.2. Too high a pH or too low a pH will stop the reaction between KLH/MBS or BSA/MBS and peptide. If needed, immediately add an appropriate amount of 0.5 N HCl or 2 N NaOH to change the pH.

38

Bao-Shiang Lee et al.

7. Stir or rotate the solution for 3 h or overnight at 4 °C. Finally, add 3 mL of ammonium bicarbonate (0.1 M) before lyophilizing the coupled peptide. 3.2 Antipeptide Antibody Titer Determination by ELISA

An antibody titer is defined as the highest antibody dilution that will yield a positive reactivity of a particular epitope in an assay system such as ELISA. This value gives an indication of the quality of an antibody preparation. This section describes an assay used to determine the titer of antipeptide antibodies in serum raised against a peptide or a protein containing the peptide sequence. 1. Coat the wells of a mictrotiter plate with 300 μL of 0.2–2.5 μM synthetic peptide, leaving wells at the end as blanks. Incubate overnight at 4 °C (see Note 12). 2. Discard the unbound synthetic peptide. 3. Wash the wells three times with PBST. 4. Block the unoccupied sites with 300 μL/well of blocking solution. 5. Wash the wells three times with PBST. 6. Prepare serial dilutions of antiserum with PBST ranging from 1:300 to 1:300,000. 7. Add 300 μL/well of the antiserum serial dilutions to the wells and incubate for 2 h at 37 °C. 8. Wash the wells three times with PBST. 9. Dilute the secondary antibody 1:7000 with PBST. 10. Add 300 μL/well of the secondary antibody to the wells and incubate at 37 °C for 2 h. 11. Wash the wells three times with PBST. 12. Add 50 μL enzyme substrate. Incubate for 10–30 min at 37 °C. 13. Terminate color development by addition of 100 μL of stopping solution. 14. Measure absorbance at 405 nm with a microtiter plate reader. 15. In our laboratory, each animal showed exceptional titers of greater than 1:500,000 against the peptide sequence and confirmed that a strong and specific immune response had occurred.

3.3 Peptide Affinity Purification of Antipeptide Antibody

Among many antibody purification methods [1–6], peptide affinity purification is the most effective technique for purification of the antipeptide antibody. The peptide affinity purification is used for isolating antibodies that recognize a specific epitope with the same specificity as that of monoclonal antibodies. 1. Swell 1 g dried CNBr-activated Sepharose 4 B in 50 mL of 1 mM HCl for 30 min. 2. Centrifuge for 5 min at 1000 × g and discard the supernatant.

Antipeptide Antibody

39

3. Wash the CNBr-activated Sepharose 4 B by swelling the resin in 50 mL of 1 mM HCl and after 15 min centrifuge at 1000 × g discarding the supernatant. Repeat this process twice. 4. Dissolve 10 mg of synthetic peptide in 5 mL of coupling buffer. 5. Mix the synthetic peptide solution with the swollen gel. Stir gently for 1 h. 6. Centrifuge for 5 min at 1000 × g and discard the supernatant. 7. Remove excess uncoupled synthetic peptide by washing the resin with 20 mL coupling buffer. Centrifuge for 5 min at 1000 × g and discard the supernatant. 8. Block remaining active groups by transferring the resin to 0.1 M Tris–HCl, pH 8.0, stand for 2 h. 9. Wash the resin with 0.1 M acetate buffer containing 0.5 M NaCl, pH 4.0. Centrifuge for 5 min at 1000 × g and discard the supernatant. 10. Wash the resin with 0.1 M Tris–HCl buffer containing 0.5 M NaCl, pH 8.0. Centrifuge for 5 min at 1000 × g and discard the supernatant. 11. Transfer the resin into PBS. 12. Pack the peptide affinity column by pouring the resin into a vertically held column. 13. Wash the column with 100 bed volumes of PBS. 14. Filter 15 mL rabbit serum through a 0.2 μm filter. 15. Dilute the serum with PBS to 50 mL. 16. Load the filtered serum onto the peptide affinity column. 17. Wash the column with 20 mL PBS. 18. Wash the column with 20 mL washing buffer 1. 19. Wash the column with 20 mL washing buffer 2. 20. Wash the column with 20 mL washing buffer 3. 21. Elute the antibodies from the column with 20 mL elution buffer and collect in a tube containing 4 mL of 1 M Tris–HCl buffer, pH 9.0. 22. Wash the column with 20 mL PBS (see Note 13). 23. Use PD-10 column to desalt the antibody by loading 2.5 mL antibody solution onto the column and eluting with 3.5 mL PBS as the desalting buffer. Aliquot and store purified antibody at −20 °C in 50 mM Tris–Glycine, 0.15 M NaCl, 40 % glycerol, 0.02 % sodium azide, 0.05 % BSA, pH 7.4 (see Note 14). 24. Determine the antibody concentration by measuring the absorbance at 280 nm of a 1 mL solution and multiply by 0.7 to determine the antibody concentration in mg/mL.

40

Bao-Shiang Lee et al.

25. Measure the titer of the purified antibody with the synthetic peptide using ELISA. 26. Evaluate the purified antibody with a purified protein/cellular protein extract using ELISA/western blotting/flow cytometry (see Note 15).

4

Notes 1. Although large quantities of monoclonal antibodies (typically rat or mouse) with their high specificity and lower background noise can be produced with batch to batch homogeneity, they generally have a lower affinity, less utility, a longer response time for production, are less effective in immunoprecipitation/ chromatin immunoprecipitation, are less tolerant of minor changes in the antigen, and are more expensive than polyclonal antibodies. Polyclonal antibodies, though prone to batch-tobatch variability, are the preferred choice for detection of denatured proteins on western blots and in ELISA (often, antigens are partially denatured) and provide complete affinity maturation in a short time period. 2. All synthetic peptides may contain low amounts of contaminants such as peptides (missing amino acid residues) other than the target peptide and peptides with modified or still protected side-chains. Nowadays peptide synthesis is extremely successful for short peptides (< ~20 amino acid residues). It is common for long peptides to have failed sequences with missing amino acids, but this is not critical for antibody production. It is always a good idea to purify the peptides by reversed-phase high-performance liquid chromatography. Although pure peptides are always better, it is not crucial as even ~70 % pure peptides can be used to generate antibodies successfully. Antibodies raised by highly immunogenic impurities are seldom a concern. However, if affinity purification using a peptide column is required, the purer the peptide, the higher the specificity of the purified antibodies. 3. For raising antibodies against phosphopeptides, there are three critical factors for success: (1) the stability of the phosphorylation, (2) the purity of the peptide, and (3) purification of the phospho-specific antibody. The generation of a phosphospecific antibody includes the synthesis of phosphorylated and non-phosphorylated peptides, immunization with the phosphorylated peptide, and construction of phosphorylated and non-phosphorylated peptide affinity columns for successive purification. The antigen design choices are constrained by the sequence directly surrounding the phosphorylated amino acid residue of interest. Antigen sequences used for generating these

Antipeptide Antibody

41

phospho-specific antibodies are generally short. This forces the phosphorylated amino acid residue into the epitope recognized by the antibody. Co-immunization with either different lengths of peptides (in the case of a known phosphorylation site) or peptides containing predicted phosphorylation sites (in the case of unknown phosphorylation sites) may be used to increase the chance of success. The serum or purified IgG is passed through the non-phosphopeptide column first. The flow-through from the non-phosphopeptide column is applied to the phosphopeptide column to extract the phospho-specific antibody. This cycle can be repeated if resulting antibodies are still recognizing the nonphospho-peptide. Milk often contains high concentration of phospho-tyrosine and thus can give an abnormally high background if used as a blocking agent with phospho-tyrosine antibodies, so BSA is used instead. 4. A good antigen has three chemical features: (1) an epitope capable of being bound to a B cell antibody, (2) a site recognized simultaneously by a MHC class II molecule and the T cell receptor, and (3) it must be degradable by T-cells. In order to improve the chances of producing high titer antibodies, it is beneficial to use a few peptide sequences from a protein as antigens in a co-immunization protocol. However, a peptide that contains several known immunogenic epitopes may produce a lesser immune response than the individual epitopes. If mixed populations of antigens (individual KLH conjugates mixed in equimolar ratios) are used, an antibody response to several components is expected with antibodies to some of the compounds dominating the response. Peptide affinity columns can be used to isolate an antibody against each peptide. 5. Most proteins with an adequate number of side chains for coupling and minimal cross-reactivity with other proteins can serve as carriers. The resultant peptide–carrier protein complex is able to stimulate the immune system to produce antibodies against both the peptide and the carrier proteins. Thus, it is important that ELISA analysis be performed using either peptides or peptides conjugated to a different carrier protein. In addition, there is some evidence that antibodies that react to the cross-linker used to couple the peptide to carrier proteins can be produced. Invertebrate keyhole limpet hemocyanin (KLH, 4.5 × 105–1.3 × 107 Da), deactivated bacterial toxin tetanus toxoid (TT, 150,000 Da), bovine gamma globulin (BGG, 150,000 Da), bovine serum albumin (BSA, 69,000 Da), ovalbumin (OVA, 44,000 Da), flagellin (FLA, monomer, 40,000 Da), horse heart myoglobin (hhMb, 16,951 Da), and hen egg-white lysozyme (HEL, 15,000 Da) are the commonly used protein carriers. KLH is preferred since its large size and numerous epitopes generate a substantial immune response in

42

Bao-Shiang Lee et al.

the majority of animals, but it does not cause an adverse immune response in humans. KLH has an abundance of lysine residues for coupling peptides allowing for a high peptide–carrier protein ratio and increasing the likelihood of generating peptide-specific antibodies. In addition, because KLH is derived from the limpet, a gastropod, it is phylogenetically distant from mammalian proteins, thus reducing false positives in immunologically based research techniques in mammalian model organisms. However, a high-quality KLH should be used because it is susceptible to aggregation and precipitation, which limits its ability to conjugate peptides. Since BSA is often a component in many tissue culture media and assays, an antipeptide antibody raised against a BSA conjugated peptide should be avoided. Rabbits immunized with rabbit serum albumin (RSA) conjugate are less likely to raise antibodies to the carrier, as the RSA is recognized as self. Multiple Antigenic Peptide (MAP, juxtaposition of four or eight peptide molecules on a cross-linked lysine core) [26, 27] is simply an alternative peptide antigen. Its advantage is that the carrier protein conjugation step is not necessary and the core of lysines has superior adsorption characteristics for the detection of antibodies in solid-phase ELISA procedures. It is recommended for short (10–15 amino acid residues) or insoluble peptides. Difficulty in coupling amino acids occurs with longer peptides and the juxtaposition of more than 16 peptide molecules. Peptides located near the C-terminus of a protein require indirect MAP synthesis to have a preferable orientation. However, the major disadvantages of MAP is that it can bypass the immune response system in some hosts and MAP antibodies do not always react with the cognate protein. 6. B-cell epitope databases/prediction servers: UniProt knowledgebase; www.uniprot.org, Protein data bank (PDB); www.rcsb.org/pdb, Immune epitope database (IEDB); www.iedb.org, Bcipep B-cell epitope prediction server (Bcipep); www.imtech.res.in/raghava/bcipep, Conformational epitope database (CED); immunet.cn/ced, Epitome database of structurally inferred antigenic epitopes in proteins (Epitome); www.rostlab.org/services/epitome, AntiJen database of published experimentally determined conformational B-cell epitopes (AntiJen); www.ddg-pharmfac.net/antijen/AntiJen/ aj_bcell.htm, HIV molecular immunology database; www.hiv. lanl.gov/content/immunology/index, Peptide antigen database predicted by OptimumAntigen design tool; www.genscript.com/peptide-antigen-database.html, Epitopia bound dataset of 66 nonredundant complex structures (Epitopia); epitopia.tau.ac.il/trainData, Liang’s unbound dataset including 48 complexes and the unbound structures of antigens; sysbio.

Antipeptide Antibody

43

unl.edu/services, Benchmark sequence datasets; www.imtech. res.in/raghava/cbtope, MimoDB mimotope database (MimoDB); immunet.cn/mimodb. Finding common results from the above databases is desirable. Linear B-cell epitope databases/prediction servers: Immune epitope database (IEDB); www.iedb.org, ABCpred B-cell epitope prediction server (ABCpred); www.imtech.res.in/raghava/abcpred, BepiPred B-cell epitope prediction server (BepiPred); www. cbs.dtu.dk/services/BepiPred, LEP-LP B-cell epitope prediction server (LEP-LP); biotools.cs.ntou.edu.tw/lepd_antigenicity.php, BCPred B-cell epitope prediction server (BCpred); ailab.cs.iastate.edu/bcpreds, FBCPred B-cell epitope prediction server (FBCPred); ailab.cs.iastate.edu/bcpreds, Epitopia B-cell epitope prediction server (Epitopia); epitopia.tau.ac.il, BayesB B-cell epitope prediction server (BayesB); www.immunopred.org/bayesb, BROracle B-cell epitope prediction server (BROracle); sites.google.com/site/oracleclassifiers, LEPS B-cell epitope prediction server (LEPS); leps.cs.ntou.edu.tw, SVMTriP B-cell epitope prediction server (SVMTriP); sysbio. unl.edu/SVMTriP, LBtope B-cell epitope prediction server (LBtope); crdd.osdd.net/raghava/lbtope, JalView B-cell epitope prediction server (JalView); www.jalview.org, ScanProsite B-cell epitope prediction server (ScanProsite); prosite.expasy. org/scanprosite, PRATT B-cell epitope prediction server (PRATT); www.ebi.ac.uk/Tools/pfa/pratt, MimoScan B-cell epitope prediction server (MimoScan); immunet.cn/sarotup/ cgi-bin/MimoScan.pl. Finding common results from the above predictions is advantageous. Conformational B-cell epitope databases/prediction servers: COBEpro B-cell epitope prediction server (COBEpro); scratch.proteomics.ics.uci.edu, CBTOPE B-cell epitope prediction server (CBTOPE); www. imtech.res.in/raghava/cbtope, BEST B-cell epitope prediction server (BEST); biomine.ece.ualberta.ca/BEST, Bprediction B-cell epitope prediction server (Bprediction); http://bcell.whu.edu.cn, DiscoTope B-cell epitope prediction server (DiscoTope); www.cbs.dtu.dk/services/DiscoTope, Epitopia B-cell epitope prediction server (Epitopia); epitopia. tau.ac.il, EPCES B-cell epitope prediction server (EPCES); sysbio.unl.edu/services/EPCES, EPSVR B-cell epitope prediction server (EPSVR); sysbio.unl.edu/EPSVR, EPMeta B-cell epitope prediction server (EPMeta); sysbio.unl.edu/ EPMeta, SEPPA B-cell epitope prediction server (SEPPA); badd.tongji.edu.cn/seppa, EpiSearch B-cell epitope prediction server (EpiSearch); curie.utmb.edu/episearch.html, Pepitope B-cell epitope prediction server (Pepitope); pepitope.tau.ac.il, PepMapper B-cell epitope prediction server (PepMapper); informatics.nenu.edu.cn/PepMapper. Common results from the above predictions are located if possible.

44

Bao-Shiang Lee et al.

7. Many short peptides are able to elicit a strong immune response. Antibodies against human angiotensin II Asp-ArgVal-Tyr-Ile-His-Pro-Phe and bovine angiotensin II Asp-ArgVal-Tyr-Val-His-Pro-Phe with a titer of 100,000 and 800,000, respectively, have been successfully produced. Other examples of shorter epitopes are the Flag epitope (DYKDDDDK) and His6 tag epitope (HHHHHH), which are widely used in the purification, identification, and functional analysis of proteins. 8. All reagents are of highest purity to ensure superb quality in synthesis. Water content is minimized in solvents and all containers are purged with nitrogen and sealed to ensure the longest possible shelf life. 9. There is little need to prescreen animals prior to immunization for antibodies reacting with the peptide in most instances, except in studies of certain organisms such as yeast. All animals used for antibody production are certified specific pathogenfree, which provides a cleaner basis for antibody production. However, animals may already have generated closely related antibodies to something in their environment or their feed which may be similar to the antigen of choice. It is therefore a good idea to immunize more than one animal using a standard protocol. At the conclusion, the animal(s) with the best antibody response are continued on extended protocols. 10. Nonspecific stimulators of the immune response are known as adjuvants. Freund’s Complete Adjuvant (FCA) should be used for the primary injection (first immunization) only. Freund’s Incomplete Adjuvant (FIA) should be used for subsequence immunizations to prevent lesions at the sites of injection. Data shows positive and negative aspects about using FCA which was developed in the 1930s. It contains heat-killed Mycobacterium tuberculosis, paraffin oil, and non-metabolizable mannide monooleate and elicits a delayed hypersensitivity reaction. The water-in-oil emulsion used in FCA is stable, provides a slow release of antigen, and protects the antigen from degradation. The drawback with FCA is that it may cause granulomas and inflammation at the injection site with an intradermal injection. Note: FCA should be avoided for studies of Mycobacterium. Though FCA has been a mainstay and has shown consistently superior results than alternative adjuvants in antibody production, some animal care and use committees reject the use of FCA due to its toxicity to the host animal. Therefore, other adjuvants such as Ribi, TiterMax, and Adjuvax [1] should be used in this case. 11. Immunogenicity is influenced by multiple characteristics of an antigen including stability, foreignness, molecular size, chemical composition and heterogeneity, susceptibility to antigen processing and presentation, and modifications. Due to slow release and

Antipeptide Antibody

45

rapid phagocytosis, particulate antigens are usually much better immunogens than soluble molecules. Polymeric antigens like Multiple antigen peptide (MAP) can also have a strong effect on immunogenicity. Peptide orientation is extremely important in certain cases. An antigen’s immunogenicity can be improved in the following ways: (1) antigen modification with dinitrophenol or arsanilic acid, (2) denaturation of the antigen, (3) selection of a different carrier, (4) including t-cell receptor class II protein binding sites, (5) coupling antigen to sheep red blood cells, and (6) coupling antigen to beads. Alternate animal species should be tried and the dose of antigen increased. The optimum dose to achieve the strongest immune response has to be determined empirically. In general, 0.5–1 mg for rabbit and 50–100 μg for mouse are recommended. 12. Most peptides can be coated on ELISA plates using carbonate buffer at pH 9.6. If the peptide does not adsorb completely, other buffers in the pH 4 to 8 range can be tried. The coupling of peptides to BSA to facilitate coating is usually not necessary. 13. A typical peptide affinity purification of 20 mL of serum yields approximately 2 mg of specific antibody. Although the capacity does decrease slightly with each use, the column can be reused many times depending on the stability of the immobilized peptide and should be stored at 4 °C in PBS with sodium azide (0.015–0.1 M). The columns should not be frozen. 14. Antibodies (purified, serum, plasma, ascetic fluid) are relatively stable proteins that retain their activity in a wide range of biological conditions due to their compact and stable protein domains. Although the storage of antibody solution is straightforward, precautions such as avoiding repeated freeze–thaw cycles, excessive protein concentration dilution (particularly of purified antibody), and bacterial or fungal growth should be taken into account. Serum, plasma, and ascites fluid that contain sodium azide (0.015–0.1 M) can be stored at 4 °C for a few months. Serum, plasma, and ascites fluid without sodium azide, however, should be stored in aliquots at −20 °C in order to avoid bacterial or fungal growth. Purified antibodies that contain sodium azide can also be stored at 4 °C for a few months to allow for use without repeated freeze–thaw cycles and at −20 °C for longer-term storage. Purified antibodies should be stored at relatively high concentrations (e.g., 10 mg/ mL) at neutral pH. Sodium azide is poisonous and known to interfere with certain assays. Filter sterilization (0.2–0.45 μm pore size) is an alternative to sodium azide. The addition of 50 % glycerol can help avoid freeze–thaw cycles while keeping antibody solutions at −20 °C. In addition, addition of BSA, if it does not interfere with the downstream applications, can increase protein concentration and improve stability of the

46

Bao-Shiang Lee et al.

antibody. However, it is not required if the above guidelines and aliquot strategies are followed. 15. In cases where an antibody has a high titer as measured by ELISA against the peptide but a low titer against the native protein, likely explanations are as follows: (1) the peptide sequence corresponds to a nonexposed region of the native protein (using a different extraction buffer such as 85 % formic acid may help expose this region), (2) the protein’s conformation in the peptide region differs from the peptide enough that the antibody has trouble recognizing the native protein, and (3) the target protein is not present in the sample. We have successfully raised an antibody that detects protein (vascular endothelial growth factor receptor) by using a larger peptide (9 kDa) without a carrier when an antibody raised by a short peptide with a carrier fails to detect the protein. Antipeptide antibodies raised with linear epitopes work well in western blots but may not work for flow cytometry because the majority of B-cell epitopes are conformational. The advantage of using western blotting (one or two dimensional native or sodium dodecyl sulfate polyacrylamide gel electrophoresis) [2, 3, 28, 29] over the ELISA method to evaluate the antibody is that in the western blot, proteins are separated and probed individually by antibody. It is not uncommon to see multiple bands in protein western blots even when affinity-purified antibody is used. This is not indicative of a problem with the antibody’s specificity and typically occurs for one of the following reasons: (1) the antipeptide antibody recognizes a homologous protein in the sample that shares one or more epitopes with the peptide sequence, (2) the protein has a different molecular weight than previously predicted, and (3) the antibody recognizes either cleaved fragments of the protein at lower molecular weights or aggregated multimers of the native protein at higher molecular weights.

Acknowledgments We thank the support of the Research Resources Center at the University of Illinois at Chicago. References 1. Howard GC, Kaser MR (eds) (2007) Making and using antibodies: a practical handbook. CRC Press, Taylor & Francis, Boca Raton, FL 2. Harlow DL (1988) Antibodies: a laboratory manual. Cold Spring Harbor Laboratory, New York

3. Rosenberg IM (1996) Protein analysis and purification benchtop techniques. Birkhauser, Boston 4. Walker JM (ed) (2009) The protein protocols handbook. Humana Press, Totowa, NJ

Antipeptide Antibody 5. Coligan JE, Kruisbeek AM, Margulies DH et al (1996) Current protocols in immunology, vol 2. John Wiley & Sons, New York, pp 9.0.1–9.8.15 6. Wisdom GB (ed) (1993) Peptide antigen: a practical approach. Oxford University Press, Oxford 7. Delves PJ (1997) Antibody production essential techniques. John Wiley & Sons, New York 8. Archuleta AJ, Stutzke CA, Nixon KM et al (2011) Optimized protocol to make phosphospecific antibodies that work. Methods Mol Biol 717:69–88 9. Arur S, Schedl T (2014) Generation and purification of highly specific antibodies for detecting post-translationally modified proteins in vivo. Nat Protoc 9:375–395 10. Teo CF, Ingale S, Wolfert MA et al (2010) Glycopeptide-specific monoclonal antibodies suggest new roles for O-GlcNAc. Nat Chem Biol 6:338–343 11. Yang J, Yan R, Roy A et al (2015) The I-TASSER suite: protein structure and function prediction. Nat Methods 12:7–8 12. Blythe MJ, Flower DR (2005) Benchmarking B cell epitope prediction: underperformance of existing methods. Protein Sci 14:246–248 13. Gao J, Faraggi E, Zhou Y et al (2012) BEST: improved prediction of B-Cell epitopes from antigen sequences. PLoS One. doi:10.1371/ journal.pone.0040104 14. Gao J, Kurgan L (2014) Computational prediction of B cell epitopes from antigen sequences. Methods Mol Biol 1184:197–215 15. Sanne MM, Hensen SMM, Derksen M et al (2014) Multiplex peptide-based B cell epitope mapping. Methods Mol Biol 1184:295–308 16. Huang J, He B, Zhou P (2014) Mimotopebased prediction of B-cell epitopes. Methods Mol Biol 1184:237–243

47

17. Sun P, Ju H, Zhang B et al (2015) Conformational B-cell epitope prediction method based on antigen preprocessing and mimotopes analysis. Biomed Res Int. doi:10.1155/2015/257030 18. Tsigelny IF (ed) (2002) Protein structure prediction: bioinformatic approach. International University Line, La Jolla, CA 19. Benoiton NL (ed) (2006) Chemistry of peptide synthesis. Taylor & Francis, New York 20. Howl J (ed) (2005) Peptide synthesis and applications. Humana, Totowa, NJ 21. Kates SA, Albericio F (2000) Solid phase synthesis: a practical guide. Marcel Dekker, New York 22. Fields GB (1997) Solid-phase peptide synthesis. Academic Press, New York 23. Hermanson GT (2008) Bioconjugate techniques. Academic Press, San Diego, CA 24. Sommer J, Garbers C, Wolf J et al (2014) Alternative intronic polyadenylation generates the Interleukin-6 trans-signaling inhibitor sgp130-E10. J Biol Chem 289:22140–22150 25. Lateef S, Gupta S, Jayathilaka G et al (2007) An improved protocol of coupling synthetic peptides to KLH for antibody production using MBS as bifunctional linker. J Biomol Tech 18:173–176 26. Posnett D, McGrath H, Tam JP (1988) A novel method for producing anti-peptide antibodies. J Biol Chem 263:285–288 27. Tam JP (1988) Synthetic peptide vaccine design: synthesis and properties of a highdensity multiple antigenic peptide system. Proc Natl Acad Sci U S A 85:5409–5413 28. Westermeier R (2001) Electrophoresis in practice. WILEY-VCH, Weinheim 29. Hames BD (1998) Electrophoresis of protein. Oxford University Press, Oxford

Chapter 3 Production and Purification of Polyclonal Antibodies Masami Nakazawa, Mari Mukumoto, and Kazutaka Miyatake Abstract Polyclonal antibodies consist of a mixture of antibodies produced by multiple B-cell clones that have differentiated into antibody-producing plasma cells in response to an immunogen. Polyclonal antibodies raised against an antigen recognize multiple epitopes on a target molecule, which results in a signal amplification in indirect immunoassays including immune-electron microscopy. In this chapter, we present a basic procedure to generate polyclonal antibodies in rabbits. Key words Polyclonal antibody, Rabbit, Ammonium sulfate precipitation, Cardiac puncture, Subcutaneous injection

1

Introduction The original meaning of the term “polyclonal antibody” is the total population of antibodies present in an animal’s serum. Polyclonal antibodies are derived from multiple B-cell clones that have differentiated into antibody-producing plasma cells in response to an immunogen. In contrast to a monoclonal antibody which recognizes a single epitope, a polyclonal antibody against a single molecular species of antigen usually recognizes multiple epitopes on the target molecule. This allows multiple antibodies to bind through the antigen amplifying the signal in indirect immunoassays. Polyclonal antibodies with low nonspecific-binding are a powerful tool for immune-electron microscopy as they produce a stronger signal than monoclonal antibodies. All animals that have an immune response can produce polyclonal antibodies. The choice of host animal is based on several factors; the most important one is the intended amount of polyclonal antibody. For most work (required antibody amount less than 100 mL), the rabbit is the most common species for polyclonal antibody production. In this chapter, we present a basic procedure to generate polyclonal antibody in rabbits.

Steven D. Schwartzbach et al. (eds.), High-Resolution Imaging of Cellular Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 1474, DOI 10.1007/978-1-4939-6352-2_3, © Springer Science+Business Media New York 2016

49

50

2

Masami Nakazawa et al.

Materials

2.1 Immunization of Rabbits

1. Institutional Animal Care and Use Committee approval. 2. Sterile phosphate buffered saline (PBS): A 10× PBS stock solution is made by mixing 87.7 g NaCl, 30 g Na2HPO4·12H2O, 2.9 g NaH2PO4·2H2O, and distilled water for a final volume of 1 L. Dilute the 10× PBS tenfold with distilled water to prepare PBS as needed. Sterilize by autoclaving at 121 °C for 20 min. 3. Antigen solution: resuspend antigen in sterile PBS for a final concentration of 1 mg/mL. A minimum of 5 mg antigen is required to immunize two rabbits. 4. New Zealand White female rabbits, 2-kg weight, specific pathogen free. 5. Freund’s complete adjuvant (Sigma-Aldrich, St. Louis, MO; see Note 1). 6. Freund’s incomplete adjuvant (Sigma-Aldrich, St. Louis, MO). 7. 5-mL Luer-Lock, all-plastic type syringes for preparing emulsion (see Note 2). 8. 22-gauge 1 1/2″ syringe needles. 9. 18-gauge double hub micro-emulsifying needle with reinforcing bar. 10. Alcohol swabs. 11. A rabbit restrainer. 12. Sterile 1.5-mL plastic sample tubes. 13. Sterile 1-mL plastic syringes.

2.2 Indirect Enzyme-Linked Immunosorbent Assay (Indirect ELISA, See Note 3)

1. PBS: A 10× PBS stock solution is made by mixing 87.7 g NaCl, 30 g Na2HPO4·12H2O, 2.9 g NaH2PO4·2H2O, and distilled water for a final volume of 1 L. Dilute the 10× PBS tenfold with distilled water to prepare PBS as needed. 2. Antigen solution: dilute antigen with PBS for a final concentration of 5 μg/mL. 3. PBS-T: PBS containing 0.05 % Tween 20. 4. 1 % BlockAce (DS Pharma Biomedical, Osaka, Japan) in water. 5. Microtiter plate washer. 6. Microtiter plate reader with 490 nm filter. 7. Test antisera. 8. Secondary antibody: horseradish peroxidase-conjugated goat anti-rabbit IgG-(H + L). 9. 10 mg o-phenylenediamine dihydrochloride tablets. 10. 30 % hydrogen peroxide.

Production and Purification of PAbs

51

11. 0.5 M H2SO4. 12. Multichannel pipet and disposable pipet tips. 13. 96-well ELISA plate. 14. Citrate phosphate buffer: A 10× citrate phosphate buffer stock solution is made by mixing 36.8 g Na2HPO4·12H2O, 10.2 g citric acid, and distilled water for a final volume of 100 mL. Store stock solution at 4 °C. Dilute 10× citrate phosphate buffer stock solution tenfold as needed. 2.3 Preparation of Blood Sample by Cardiac Puncture and Preparation of Serum from Blood

1. Sterile 50-mL plastic syringes. 2. Sterile 2.5-mL plastic syringes. 3. 26-gauge 1/2″ syringe needles. 4. 18-gauge 1 1/2″ syringe needles. 5. A rabbit restrainer. 6. Weight scales. 7. Sterile 50-mL plastic centrifuge tubes with conical bottom. 8. Sterile PBS: A 10× PBS stock solution is made by mixing 87.7 g NaCl, 30 g Na2HPO4·12H2O, 2.9 g NaH2PO4·2H2O, and distilled water for a final volume of 1 L. Dilute the 10× PBS tenfold with distilled water to prepare PBS as needed. Sterilize by autoclaving at 121 °C for 20 min. 9. 5 % (w/v) sodium pentobarbital solution: dissolve sodium pentobarbital in sterile PBS (see Note 4). 10. Alcohol swabs. 11. A rabbit restraint board.

2.4 Precipitation of Immunoglobulin G with Ammonium Sulfate

1. Saturated ammonium sulfate solution: add 800 g ammonium sulfate to 1 L of distilled water. Heat until the salt dissolves completely, and let cool to room temperature. Adjust the pH to 7.4 with ammonium hydroxide. Allow undissolved ammonium sulfate to settle to the bottom of the container and be careful not to remove the ammonium sulfate crystals when using the solution. 2. Antisera. 3. Magnetic stirrer. 4. Cold room or cold chamber (4 °C). 5. PBS: A 10× PBS stock solution is made by mixing 87.7 g NaCl, 30 g Na2HPO4·12H2O, 2.9 g NaH2PO4·2H2O, and distilled water for a final volume of 1 L. Dilute the 10× PBS tenfold with distilled water to prepare PBS as needed. 6. Dialysis tubing, MWCO 14,000. 7. 200- or 300-mL glass beaker.

52

Masami Nakazawa et al.

2.5 SDS Gel Electrophoresis and Western Blotting

1. 29.2 % acrylamide/0.8 % bisacrylamide stock solution: mix 29.2 g acrylamide and 0.8 g bisacrylamide with distilled water for a final volume of 100 mL. Store at 4 °C in the dark for up to 3 weeks. Unpolymerized acrylamide is a neurotoxin and care should be taken to avoid inhalation and skin contact. 2. Separating gel solution: 1.875 M Tris–HCl, pH 8.8. 3. Stacking gel solution: 0.6 M Tris–HCl, pH 6.8. 4. 10 % ammonium persulfate in sterile water. Prepare immediately before use. 5. N,N,N′,N′-tetramethylethylenediamine (TEMED). 6. 10 % (w/v) SDS: in distilled water. 7. SDS-PAGE running buffer: 25 mM Tris, 192 mM glycine, 0.1 % SDS. 8. 3× sample loading buffer: 180 mM Tris–HCl, pH 6.8, 30 % glycerol, 6 % SDS, 0.003 % bromophenol blue, 15 % β-mercaptoethanol. Store in aliquots at −20 °C. 9. Electrophoresis apparatus: Dual slab PAGE system AE-6500 (mini-slab) (ATTO Corporation, Tokyo, Japan). 10. Hand gel casting apparatus: glass plates with 1 mm spacers, 1 mm Teflon combs with 14 teeth. 11. Blotting apparatus: Trans-Blot SD Semi-Dry Electrophoretic transfer cell (Bio-Rad Laboratories, Hercules, CA). 12. Blotting buffer: A 10× blotting buffer stock solution is made by mixing 58.14 g Tris and 29.27 g glycine and distilled water for a final volume of 1 L. Dilute the 10× blotting buffer tenfold with distilled water to prepare working concentration of blotting buffer as needed. Store diluted blotting buffer at 4 °C. 13. 0.45 μm Immobilon-P membrane (Merck Millipore, Billerica, MA): cut into 6 × 9 cm pieces, must be wetted in 100 % methanol for 1 min then soaked in blotting buffer with gentle shaking before use. 14. Filter paper 3MM sheets cut into 6 × 9 cm pieces. 15. PBS: A 10× PBS stock solution is made by mixing 87.7 g NaCl, 30 g Na2HPO4·12H2O, 2.9 g NaH2PO4·2H2O, and distilled water for a final volume of 1 L. Dilute the 10× PBS tenfold with distilled water to prepare PBS as needed. 16. Blocking buffer: 5 % powdered skim milk in PBS. 17. Washing buffer: PBS. 18. Impulse heat sealer. 19. Plastic boxes. 20. Heat sealable plastic bags. 21. Primary antibody diluted 1000 times in 0.1 % bovine serum albumin in PBS (see Note 5).

Production and Purification of PAbs

53

22. Secondary antibody: Goat anti-rabbit horseradish peroxidase conjugated antibody diluted 10,000 times in 0.1 % bovine serum albumin in PBS. 23. Chemiluminescent substrate: SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific, Rockford, IL). 24. Luminescent image analyzer LAS-4000 (Fujifilm, Tokyo, Japan). 25. Saran wrap.

3

Methods

3.1 Immunization of Rabbits

1. Collect a pre-immune blood sample from the rabbit’s marginal ear vein with a sterile 1-mL plastic syringe using a 22-gauge needle (see Note 6). Transfer the blood to a sterile 1.5-mL plastic sample tube. Keep at 37 °C for 1 h to allow clots formation. Transfer the tube to 4 °C and leave overnight. Centrifuge at 1000 × g for 20 min at 4 °C. Collect the supernatant as a pre-immune serum. Aliquot and store at −80 °C. 2. Prepare an emulsion (1.2 mL/rabbit) using 0.6 mL sterile PBS containing antigen (1 mg/mL) and 0.6 mL Freund’s complete adjuvant. Use a 22-gauge needle to load one all plastic syringe with antigen solution and the other with adjuvant. Link the two syringes with a 18-gauge double hub micro-emulsifying needle with reinforcing bar. Press the syringe barrels back and forth until a stable emulsion is formed (for 5–10 min till thickened). Let a drop of the emulsion fall into water to check the endpoint of emulsification. A stable emulsion will not disperse. 3. Immobilize a rabbit with a rabbit restrainer or by help from an assistant and wipe the back of the rabbit with alcohol swabs to sterilize. Inject the rabbit subcutaneously with 1 mL of the antigen emulsion delivered to four to ten injection sites using a 22-gauge needle (see Note 7). At least two rabbits should be immunized for each antigen. 4. Boost rabbits 2 weeks later by subcutaneously injecting an emulsion (1 mL/rabbit) of equal volumes of sterile PBS containing 0.5 mg antigen/rabbit and Freund’s incomplete adjuvant. The emulsion is prepared and injected as in steps 2 and 3. 5. Collect blood 1 week after the booster injection to determine the antibody titer. Place rabbits in a rabbit restrainer and collect the blood from the marginal ear vein using a sterile 1-mL plastic syringe with a 22-gauge needle. Transfer the blood to a sterile 1.5-mL plastic sample tube. Keep the blood at 37 °C for 1 h and centrifuge at 1000 × g for 20 min to separate the serum sample (supernatant fraction). 6. Determine the antibody titer in the serum by indirect ELISA (Subheading 3.2, see Note 3).

54

Masami Nakazawa et al.

7. Boost rabbits as in step 4 every 2 weeks until a sufficient titer (e.g., a significant ELISA signal at >1/10,000 dilution) is achieved. 8. When the antibody titer is sufficient, boost rabbits by injecting 0.5 mg antigen in sterile PBS without adjuvant into a marginal ear vein (500 μL) using a sterile 1-mL plastic syringe with a 22-gauge needle. 9. Three days after the final immunization (step 8), collect all of the blood from the rabbits by cardiac puncture (see Subheading 3.3). 3.2 Indirect Enzyme-Linked Immunosorbent Assay (Indirect ELISA)

1. Coat plate(s) with antigen by dispensing 100 μL of antigen solution (5 μg/mL in PBS) into each well of an ELISA plate using a multichannel pipet and tips. Close the lid and incubate at 37 °C for 1 h or 4 °C overnight to allow the antigen to coat the wells. 2. Rinse the plate once with PBS-T using a microplate washer apparatus. 3. Add 200 μL of 1 % BlockAce solution to each well and incubate at 37 °C for 1 h or 4 °C overnight. 4. Rinse the plate once with PBS-T using a microplate washer apparatus. 5. Add 100 μL of serum diluted in PBS to each of the coated wells, close the lid and incubate at 37 °C for 1 h (see Note 8). 6. Wash the plate five times with PBS-T using a microplate washer and remove residual liquid completely by vigorously patting on a paper towel. 7. Add 100 μL of diluted secondary antibody diluted 1:10,000 in PBS-T or as specified by the supplier to each well, close the lid and incubate at 37 °C for 1 h. 8. Wash the plate five times with PBS-T using a microplate washer and remove residual liquid completely by vigorously patting on a paper towel. 9. Prepare the substrate solution by dissolving one tablet of o-phenylenediamine dichloride in 25 mL of citrate phosphate buffer in the dark and then add 5 μL of 30 % H2O2. 10. Add 200 μL of substrate solution to each well and incubate at 37 °C for 30 min in the dark. 11. Add 50 μL of 0.5 M H2SO4 to each well to stop the peroxidase reaction. 12. Read absorbance at 490 nm with a microtiter plate reader. Generally, a signal which shows both OD > 0.7 in 30 nm are unstable even after protein conjugation and therefore should not be stored for long periods. In addition, large-size gold particles are dissociated relatively easily from sections during the IEM procedure [10]. 4. Standard electrodes may be damaged by colloidal gold. Therefore, litmus paper is used for measuring the pH of colloidal gold solutions. Colloidal gold of smaller particle size requires more protein to stabilize it than those preparations containing larger colloids. 5. Colloidal gold is a negatively charged suspension and its stability is maintained by static repelling forces. Therefore, the suspension is unstable and precipitates immediately in the presence of electrolytes. However, when macromolecules such as proteins are absorbed onto the surface of colloidal gold particles by

70

Sadaki Yokota

electrostatic van der Waals forces, a stable complex is formed that is no longer precipitated in solutions containing electrolytes. Proteins absorbed by gold particles do not lose their enzymatic activity or ligand affinity making them useful for detecting enzyme substrate reactions and receptor ligand interactions. 6. For example, if the minimum amount of protein A/G/L needed is 2 μg/0.5 mL colloidal gold, 80 μg of protein A/G/L is needed to saturate 20 mL of colloidal gold and with an additional 10 % this makes up a total of 88 μg protein. 7. The color of the colloidal gold conjugate prepared using thiocyanate is dark yellow. 8. When the protein A/G/L-gold probes are contaminated with even small amounts of free protein A/G/L, the binding of protein A/G/L-gold probes to IgG is greatly decreased due to the binding of free protein A/G/L to IgG. The most effective way to remove free protein A/G/L is the discontinuous glycerol gradient centrifugation method. Repeating the differential centrifugation twice can also be used to eliminate free protein A/G/L. 9. Protein A/G/L-gold conjugates retain biological activity for 5 years under these storage conditions. 10. Using a fixed angle rotor, centrifuge the gold–IgG or gold– F(ab′)2 conjugate for 30 min at 15,000 × g, 35,000 × g, and 100,000 × g for 15 nm and larger gold conjugate, 8 nm gold conjugate and 2–3 gold conjugate, respectively. Aggregated IgG and F(ab′)2 gold conjugates form a tight aggregate on the wall of the tube while the non-aggregated conjugates form a loose pellet at the bottom of the tube. 11. After centrifugation, excess medium attached to the tube wall is wiped off with tissue paper. This almost completely avoids contamination with free IgG or F(ab′)2. 12. Under these storage conditions, the conjugates retain binding activity for 1 year. During storage, the conjugates slowly precipitate and should be centrifuged immediately before use to remove aggregated conjugates. References 1. Roth J (1982) The protein A-gold (pAg) technique—a qualitative and quantitative approach for antigen localization on thin sections. In: Bullock GR, Petrusz P (eds) Techniques in immunocytochemistry, vol 1. Academic, London, pp 108–133 2. Tokuyasu KT (1980) Immunochemistry on ultrathin frozen sections. Histochem J 12:381–403

3. Frens G (1973) Controlled nucleation for the regulation of particle size in monodisperse gold suspensions. Nature Phys Sci 241:20–22 4. Baschong W, Lucocq JM, Roth J (1985) “Thiocyanate gold”: small (2–3 nm) colloidal gold for affinity cytochemical labeling in electron microscopy. Histochemistry 83:409–411 5. Tschopp J, Podack ER, Müller-Eberhard HJ (1982) Ultrastructure of the membrane attack

Gold Particles Preparation and Conjugation complex of complement: detection of the tetramolecular C9-polymerizing complex C5b-8. Proc Natl Acad Sci U S A 79:7474–7478 6. De Roe C, Courtoy PJ, Baudhuin P (1987) A model of protein-colloidal gold interaction. J Histochem Cytochem 35:1191–1198 7. De Mey J, Moeremans M, Geuens G, Nuydens R, De Brabander M (1981) High resolution light and electron microscopic localization of tubulin with the IGS (Immuno Gold Staining) method. Cell Biol Int Rep 5:889–899 8. Ackerman GA, Wolken KW, Gelder FB (1980) Surface distribution of monosialoganglioside Gm1 on human blood cells and the effect of

71

exogenous GM1 and neuraminidase on cholera toxin surface labeling. A quantitative immunocytochemical study. J Histochem Cytochem 28:1100–1112 9. Bonnard C, Papermaster DS, Kraehenbuhl J-P (1984) The streptavidin-biotin bridge technique: application in light and electron microscope immunocytochemistry. In: Polak JM, Varndell IM (eds) Immunolabeling for electron microscopy. Elsevier, Amsterdam, pp 95–111 10. Yokota S (1988) Effect of particle size on labeling density for catalase in protein A-gold immunocytochemistry. J Histochem Cytochem 36:107–109

Chapter 5 Helper-Dependent Adenoviral Vectors and Their Use for Neuroscience Applications Mónica S. Montesinos, Rachel Satterfield, and Samuel M. Young Jr. Abstract Neuroscience research has been revolutionized by the use of recombinant viral vector technology from the basic, preclinical and clinical levels. Currently, multiple recombinant viral vector types are employed with each having its strengths and weaknesses depending on the proposed application. Helper-dependent adenoviral vectors (HdAd) are emerging as ideal viral vectors that solve a major need in the neuroscience field: (1) expression of transgenes that are too large to be packaged by other viral vectors and (2) rapid onset of transgene expression in the absence of cytotoxicity. Here, we describe the methods for large-scale production of HdAd viral vectors for in vivo use with neurospecific transgene expression. Key words Adevnovirus, Recombinant viral vectors, Neuroscience, Cell-type specific, Reporters, Transgene expression

1

Introduction The ability to manipulate the cellular and molecular mechanisms of specific neuronal populations is critical for understanding the regulation of neural circuit function. Recombinant viral vector technology has emerged as an indispensable tool in neuroscience to manipulate neuronal function, in vivo and in vitro at both, the cellular and molecular level and in the creation of animal models of neurological disorders [1–3]. Viral vectors are suitable for a wide range of applications: expression of molecular reporters and effectors, gene silencing in combination with transgenic animals, expression of RNAi technology [4–6] or the recent development of clustered regularly interspaced short palindromic repeats (CRISPRs) and associated (Cas) systems (CRISPR/CAS9) [7–9] or when fast onset and high-level neurospecific transgene expression in vivo are needed [10, 11]. Viral vectors can be used to achieve cell type specific labeling of neuronal populations through cell-type specific promoters or through in utero electroporation [12]. However, despite the importance of viral vectors in neuroscience research, a major challenge with this

Steven D. Schwartzbach et al. (eds.), High-Resolution Imaging of Cellular Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 1474, DOI 10.1007/978-1-4939-6352-2_5, © Springer Science+Business Media New York 2016

73

74

Mónica S. Montesinos et al.

technique is the limited viral vector packaging capacity, which restricts the size of the transgene of interest, thereby confining the types of transgenes that could be expressed. Research from the gene therapy field has led to the development of four different viral vectors, which are extremely useful in neuroscience research [13]. Based on the type of virus and packaging capability, they can be classified as: (1) recombinant adenoassociated virus (rAAV), (2) recombinant lentiviral vectors (LVV), (3) recombinant adenoviral vectors (rAd), and (4) herpes simplex viral vectors (rHSV) (Table 1). Despite of the wide use of rAAV and LVV for the transduction of neurons in vivo, neither has the packaging capacity to incorporate large promoters and still coexpress a gene of interest [13]. In addition, since LVV are single stranded RNA viruses, they cannot be used with promoters that contain introns in the 5′ UTR, which regulate sequence specificity [14]. Both rAd and rHSV vectors are suitable alternatives to rAAV and LVV. These vectors contain double stranded DNA (dsDNA) genomes and are capable of packaging large amounts of foreign Table 1 Common viral vectors for neuroscience applications

Vector

Virus type

Vector capacity

rAAV

ssDNA

LVV

Pros

Cons

~5 kb ~2.5 kb scAAV

– Infects neurons and astrocytes. – Long term expression. (years) – No cytotoxicity. – Scalable production.

– Packaging capacity extremely limited.

RNA

~9 kb

– Infects neurons and astrocytes. – Long term expression. – No cytotoxicity.

– Packaging capacity limited. – Difficulty with making high titer vectors. – Cannot use promoters with intron in 5′ UTR.

rAd: – First generation – Second generation - Gutted Vector (HdAd)

dsDNA

~8.2 kb ~14 kb ~37 kb

– Possible cytotoxicity with doses – Infects neurons and over 1 × 109 particles/µL astrocytes. – Ability of package large amount of foreign DNA. – Long term expression (years). – No cytotoxicity. – Scalable production.

rHSV

dsDNA

~150 kb

– Infects neurons. – Ability of package large amount of foreign DNA.

– Difficulty in scalability of production for high titer vectors. – Potential cytotoxicity. – Instability in transgene expression.

HdAd and Their Use in Neuroscience

75

DNA. Although rHSV vectors have been used with some success in neurons, they are less than ideal due to long term instability of transgene expression, possible toxicity due to helper virus (HV) contamination, and difficulties with scalable production [13]. However, the latest generation of rAd vectors—HdAd—is ideal for cell-type promoter-specific long-term expression in neurons [15]. HdAd vectors are completely devoid of any viral genes, which increases their packaging capacity up to 37 kb of foreign DNA and eliminates possible residual viral gene expression that could cause chronic toxicity [16–19]. This high packaging capability allows the delivery of elements to enhance, prolong, and regulate long-term transgene expression [20]. HdAd are able to infect a wide variety of mammalian tissues succeeding in specific cell-type expression by using different promoters and additional 3′ UTR elements. Additionally, since HdAd vector genome exists in a non-integrating form in transduced cells, the risks of germ-line transmission and insertional mutagenesis are negligible [3]. Furthermore, unlike rHSV, HdAd production is easily scalable giving large amounts of HdAd with extremely high titers [20, 21]. Because HdAd are dsDNA viruses, the onset of their expression is extremely rapid when compared to other viral vectors [16]. To exploit this, our laboratory has generated an extremely high level expression cassette, pUNISHER, which in combination with either second generation rAd or HdAd, can be effectively used to express dominant negative proteins in order to understand cellular and molecular mechanisms of neuronal function [10, 11]. To determine the infectious units and purity of HdAd when used in vivo, quantitative polymerase chain reaction (qPCR) is used [22]. Since HdAd is replication defective, these viral vectors are not able to be titrated in assays that rely on vector replication, as plaque assay or endpoint dilution assay, commonly used for first generation or second generation rAd vectors. Thus, HdAd vectors can be considered ideal vectors for central nervous system applications where large capacities are needed for labeling cell-type specific populations for functional analysis and molecular perturbation studies.

2

Materials All solutions are prepared using ultrapure water (18 mΩ cm at 25 °C). All reagents used are of molecular biology grade and stored at room temperature (RT), unless otherwise specified.

2.1 Virus Amplification

1. Biological safety cabinet. 2. Inverted microscope. 3. Pipette aid. 4. 1, 5, 10, 25 mL serological pipettes. 5. Sterile 1.5, 15, and 50 mL centrifuge tubes.

76

Mónica S. Montesinos et al.

6. 100 % glycerol, sterilized by autoclaving. 7. Tabletop centrifuge. 8. Water bath. 9. 10 % bleach. 10. 100 mM Tris–HCl: dissolve 6.057 g Tris base in 500 mL ultrapure water and adjust the pH to 8.0 with 1 N HCl. Autoclave to sterilize. 11. Wet ice. 12. −80 °C ultralow freezer. 13. Vortex. 14. 37 °C CO2 incubator. 15. 6 and 24-well tissue culture dishes. 16. 60 mm, 10 cm, and 15 cm tissue culture dishes. 17. Human embryonic kidney cells (HEK 293), American Type Culture Collection (ATCC). 18. Hemocytometer. 19. Helper Virus pNG163R-2 (HV): a first generation rAd serotype 5 (Ad5) (see Note 1). Store at −80 °C. 20. 10 % FBS DMEM: DMEM supplemented with 2 mM lglutamine, 4.5 g/L glucose and sodium pyruvate, 10 % fetal bovine serum (FBS), and 100 U/mL penicillin–streptomycin (Pen/Strep) (see Note 2). 21. 5 % FBS DMEM: DMEM supplemented with 2 mM lglutamine, 4.5 g/L glucose and sodium pyruvate, 5 % FBS, 100 U/mL Pen/Strep. 22. Low melting point agarose. 23. Microwave. 24. Black sharpie. 25. Sterile cotton-plugged Pasteur pipettes. 26. Dry ice. 27. Ethanol, 200 proof. 28. Cell scraper. 29. 1× phosphate buffered saline (PBS), pH 7.4 (Corning, Manassas, VA, USA) store at 4 °C. 30. Plasmid pΔ28E4 (see Note 1). 31. Restriction enzyme PmeI. Store at −20 °C. 32. Agarose gel electrophoresis system. 33. Gel imaging system. 34. 116 producer cells (see Notes 1 and 3). 35. 10 % FBS MEM: MEM supplemented with 2 mM l-glutamine, 10 % FBS, 100 U/mL Pen/Strep, and 100 μg/mL hygromycin.

HdAd and Their Use in Neuroscience

77

36. 5 % FBS MEM: MEM supplemented with 2 mM l-glutamine, 5 % FBS, 100 U/mL Pen/Strep. 37. ProFection Mammalian Transfection System (Promega, Madison, WI, USA). 2.2

Virus Purification

1. Items 1–13 in Subheading 2.1. 2. Amplified HV or HdAd cellular pellet. 3. 5 % sodium deoxycholate, filter sterilized. 4. 2 M magnesium chloride (MgCl2), filter sterilized. 5. Benzonase (384 U/μL) (Sigma, St. Louis, MO, USA). Store at −20 °C. 6. 1.2, 0.8, and 0.45 μm sterile syringe filters. 7. 5 and 20 mL syringes. 8. 10 mM Tris–HCl pH 8.0: dissolve 0.61 g Tris base in 500 mL ultrapure water and adjust pH to 8.0 with 1 N HCl. Autoclave to sterilize. 9. 1.25 g/mL cesium chloride (CsCl) solution: mix 54 g CsCl and 146 g 10 mM Tris–HCl, pH 8.0, filter sterilized (see Note 4). 10. 1.35 g/mL CsCl solution: mix 70.4 g CsCl and 129.6 g 10 mM Tris–HCl, pH 8.0, filter sterilized (see Note 4). 11. Ultra-Clear centrifuge tubes for SW41Ti rotor (Beckman Coulter, Brea, CA, USA). 12. Ultra-Clear centrifuge tubes for SW55Ti rotor (Beckman Coulter, Brea, CA, USA). 13. Optima L-90K ultracentrifuge with SW41Ti and SW55Ti rotors (Beckman Coulter, Brea, CA, USA). 14. Ring stand. 15. Three-prong extension clamp, 25 mm max grip size. 16. Forceps. 17. Kimwipes. 18. 22 gauge needles. 19. Dialysis buffer 1: 10 mM HEPES–NaOH pH 7.4, 1 mM MgCl2, for HV dialysis. Store at 4 °C. 20. Dialysis buffer 2: Dialysis buffer 1 containing 250 mM sucrose for HdAd dialysis. 21. 1 and 4 L beakers. 22. pH meter. 23. Slide-A-Lyzer dialysis cassette G2: 10,000 molecular weight cutoff (MWCO), 3 mL capacity (Thermo Scientific, Waltham, MA, USA). 24. Tape.

78

Mónica S. Montesinos et al.

25. Stir plate. 26. 0.22 μm low protein binding syringe filter. 27. 0.5 mL Protein LoBind tubes (Eppendorf, Hauppauge, NY, USA). 28. Optical Density (OD) buffer 1: 10 mM HEPES–NaOH pH 7.4, 1 mM MgCl2, 0.1 % sodium dodecyl sulfate (SDS), 10 % glycerol, used for HV. 29. OD buffer 2: 10 mM HEPES–NaOH pH 7.4, 1 mM MgCl2, 0.1 % SDS, 250 mM sucrose, used for HdAd. 30. Biophotometer Plus Spectrophotometer (Eppendorf, Hauppauge, NY, USA). 31. UVette cuvettes, 220–1600 nm (Eppendorf, Hauppauge, NY, USA). 2.3 Determination of Viral Particles (vp) of HV Necessary to Cause Cytopathic Effect (CPE) in 48 h

1. Purified HV. 2. Biological safety cabinet. 3. Inverted microscope. 4. Pipette aid. 5. Sterile 1.5 mL centrifuge tubes. 6. 24-well tissue culture dishes. 7. 37 °C CO2 incubator. 8. Human Embryonic Kidney cells (HEK 293), American Type Culture Collection (ATCC). 9. 5 and 10 % FBS DMEM, as prepared in Subheading 2.1.

3

Methods All cell culture is carried out in a biological safety cabinet. Cells are maintained in a 37 °C incubator with 5 % CO2 (see Note 5). Please note that before HdAd amplification, HV has to first be amplified and the viral particles (vp) necessary to cause CPE in 48 h determined as explained in Subheading 3.3.

3.1 Virus Amplification 3.1.1 Amplification of the Helper Virus

1. Split 1 confluent 15 cm tissue culture dish of HEK 293 cells 1:10 using 10 % FBS DMEM (ten plates total). Change medium as needed. These dishes will become confluent in about a week and will be used later in the amplification process. 2. Seed a 6-well dish with HEK 293 cells at 1.5 × 106 cells/well in 3 mL 10 % FBS DMEM. The next day, aspirate medium from the dish and add 300 μL fresh 5 % FBS DMEM. 3. Dilute the HV serially from 10−1 to 10−7 in 5 % FBS DMEM (see Notes 6 and 7). Add 100 μL of each dilution into each well (see Note 8).

HdAd and Their Use in Neuroscience

79

4. Place the dish in the incubator for 30 min for virus adsorption then overlay the infected cells with melted and cooled 1.25 % low melt agarose prepared with 5 % FBS DMEM. Allow to solidify and then return to the incubator. 5. Check daily, up to 10 days, for plaque formation (see Note 9). While viewing the cells under an inverted microscope, mark the location of each plaque on the bottom side of the dish with a black sharpie. 6. Using a cotton-plugged Pasteur pipette and a pipette aid, pick individual plaques by sucking up the agarose directly above the marked location. Scrape on the bottom of the dish to help loosen the plaque. Transfer each plaque to a 15 mL tube containing 1 mL 5 % FBS DMEM. Repeat for each plaque and store tubes at −80 °C until needed. 7. Seed a 24-well dish with HEK 293 cells at a density of 1.25 × 105 cells/well in 0.5 mL 10 % FBS DMEM which will reach about 90 % confluence in 24 h (see Note 10). 8. The next day, perform three freeze–thaw cycles at −80 °C in a dry ice–100 % ethanol bath, and 37 °C water bath, in order to lyse the cells and release the virus from each plaque previously stored at −80 °C. Centrifuge each plaque at 3000 × g, 4 °C, 5 min then transfer the supernatant containing the virus to a new 15 mL tube. Discard the pellet into a biohazard waste container. 9. Remove the medium from the 24-well dish and add 0.2 mL of each virus plaque pick to individual wells. The remaining supernatant can be stored at −80 °C for later use. 10. For virus absorption, place the dish in the incubator for 1 h then add 1 mL 5 % FBS DMEM to each well and return to the incubator until CPE is reached (see Note 11). 11. After 40–48 h, select the wells that have reached CPE and detach the cells from the bottom of the dish using a cell scraper. Transfer the content of each well to a separate 15 mL tube and discard wells without CPE. Wash each well of the dish with 1 mL 1× PBS and transfer to the matching 15 mL tube. 12. Centrifuge each tube at 3000 × g, 4 °C, 5 min and discard supernatant to a 10 % bleach waste container. The virus is in the cellular pellet. Resuspend each pellet in 1 mL 100 mM Tris– HCl, pH 8.0. Add 0.1 mL 100 % glycerol and store at −80 °C. 13. Three days before infection, subculture the ten confluent 15 cm dishes of HEK 293 cells (step 1) 1:3, resulting in thirty 15 cm dishes. The thirty 15 cm dishes will be about 90 % confluent in 3 days. 14. Seed a 10 cm dish with HEK 293 cells at a density of 1.5 × 107 cells/dish in 10 mL 10 % FBS DMEM to reach about 90 % confluence in 24 h.

80

Mónica S. Montesinos et al.

15. Perform three freeze–thaw cycles as described above to the pellets previously stored at −80 °C (step 12). Centrifuge at 3000 × g, 4 °C, 5 min then transfer the supernatant containing the virus to a new 15 mL tube. Discard the pellet into a biohazard waste container. 16. Remove the medium from the 10 cm dish and add 3 mL of fresh 5 % FBS DMEM plus 1 mL of viral supernatant. Incubate for 1 h for virus absorption. Add 3 mL of 5 % FBS DMEM to the dish and return to the incubator until CPE is reached (about 2 days). 17. Detach the cells from the bottom of the dish using a cell scraper. Transfer the contents to a 50 mL tube. Wash the dish with 4 mL 1× PBS and transfer to the tube. Centrifuge 3000 × g, 4 °C, 5 min and discard all but about 1 mL of supernatant to a 10 % bleach waste container. Resuspend the pellet in the residual 1 mL. Store at −80 °C or proceed directly to the next step (see Note 12). 18. Carry out three freeze–thaw cycles as above. Add 30 mL fresh 5 % FBS DMEM to the 50 mL tube and centrifuge at 3000 × g, 4 °C, 5 min, then transfer the supernatant to a new 50 mL tube and discard pellet into a biohazard waste container (see Note 13). 19. Remove the media from the 30 confluent 15 cm dishes (step 13), add 7 mL fresh 5 % FBS DMEM and 1 mL viral supernatant (step 18) to each dish. Incubate for 1 h for virus absorption. After, add 7 mL of 5 % FBS DMEM to the dish and return to the incubator until CPE is reached (about 2 days). 20. Detach the cells from the bottom of the dish using a cell scraper and transfer the contents to 50 mL tubes. Wash five 15 cm dishes with the same 5 mL 1× PBS and transfer to 50 mL tubes. Repeat for remaining dishes (see Note 14). Centrifuge 3000 × g, 4 °C, 5 min and discard supernatant to a 10 % bleach waste container. Resuspend all pellets in one 50 mL tube in a final volume of 13.5 mL of 100 mM Tris–HCl, pH 8.0 and store at −80 °C or proceed to virus purification Subheading 3.2. 3.1.2 Amplification of the HdAd

1. Digest 10 μg of pHdAd overnight in a total volume of 50–100 μL with the appropriate restriction enzyme. For expression cassettes using p∆28E4, use PmeI. The following day, heat inactivate the enzyme at 65 °C for 20 min (see Note 15). 2. To confirm complete digestion of the plasmid, run 1 μL (200 ng) of the digested DNA on a 0.8 % agarose gel (see Note 16). 3. The day before the transfection, the process by which the linearized pHdAd is introduced into the 116 producer cells, seed a 60 mm dish with 2.91 × 106 116 producer cells/dish in 3 mL 10 % FBS MEM to reach about 70 % confluence in 24 h (see

HdAd and Their Use in Neuroscience

81

Note 17). 3 h before transfection, replace the medium with 5 mL of fresh 10 % FBS MEM. 4. Transfect the 60 mm dish with the digested pHdAd using the ProFection® Mammalian Transfection System according to the manufacturer’s instructions. Mix well to distribute the precipitate evenly over the cells and avoid localized acidification. Incubate the dish for 16–24 h in incubator. 5. Passage 0 (P0): 16–24 h after transfection, remove the medium from the transfected 116 producer cells, wash twice with 1 mL 5 % FBS MEM and then add 1 mL of additional medium to the dish. Infect the cells with diluted HV to cause CPE in 48 h as determined in Subheading 3.3 (see Note 18) (Fig. 1). Adsorb the virus for 1 h in the incubator, then add 1.5 mL additional 5 % FBS MEM to the dish and return to incubator. 6. The next day, add 1 mL of 5 % FBS MEM to the P0 60 mm dish.

Fig. 1 HdAd Production using Cre/loxP. The HdAd is constructed as a bacterial plasmid (pHdAd) containing the expression cassette, two inverted terminal repeats (ITRs) needed for viral DNA replication, and a packaging signal (Ψ). The pHdAd is digested with PmeI to excise the plasmid coding region from the desired expression cassette and ITRs. The pHdAd is optimally constructed to be >28 to 12 h. Carefully examine each stacked glass insert for any dust or imperfections between the layers of glass. (c) Kwik-Sil plugs. Kwik-Sil plugs are made in a manner similar to Dombeck et al. [4], using a custom mold. The mold yields plugs with a 5 mm diameter, 1.4 mm thick piece of Kwik-Sil (see Note 12) attached to an 8 mm diameter coverslip. The mold consists of a 1.9 mm thick piece of aluminum, with a 5 mm diameter through hole and a counterbore with a diameter of 8.2 mm and depth of 0.5 mm (Fig. 1e). Lubricate surface of counterbore with thin layer of petroleum jelly and insert a clean 8 mm cover glass (see Note 9). The glass will adhere slightly to the petroleum jelly. Flip the mold over, revealing the 5 mm through hole. Fill mold with Kwik-Sil (World Precision Instruments, Inc., Sarasota, FL), level by running a razor blade along the top of the mold, and allow to cure (5–20 min). A challenge in forming thick Kwik-Sil plugs is the formation of bubbles during the curing process. Best results are obtained by discarding the initial portion of a Kwik-Sil tube and mixing slowly. This approach can yield plugs free of bubbles 50–80 % of the time. After curing, remove plugs from mold and discard plugs with bubbles. (d) Agarose in ACSF (2–4 %), prepared as above (see Note 13). 2.3 Two-Photon Microscope

3

1. Two-photon microscope (see Note 14). 2. Image acquisition and microscope control software (see Note 15).

Methods

3.1 Virally Mediated Expression of Fluorescent Reporters

1. Obtain IACUC approval for all procedures. 2. Anesthetize animal. Anesthetic regime depends on age and species. For ferrets, we induce anesthesia with ketamine (50 mg/kg), and maintain with isoflurane (1–2 % in O2) (see Notes 16–18). 3. Aseptically clean and shave top of head (see Note 19).

176

Gordon B. Smith and David Fitzpatrick

4. Mount animal in stereotaxic apparatus or other device to stably hold head during surgery. 5. Make incision overlying target region and retract skin to expose skull overlying target area. If needed, carefully cut and retract muscle. For injections in primary visual cortex of ferrets, it is typically necessary to cut the muscle medial to the injection site and retract the muscle laterally. Injections in ferrets targeting primary visual cortex are usually performed 7–8 mm lateral and 1–2 mm anterior of lambda. 6. Drill burr hole (~1 mm diameter) in skull overlying target. Remove any fragments of skull from the hole. 7. Using a 30 G needle, carefully perform a small durotomy. Using a needle with a slightly bent tip facilitates approaching at a shallow angle and puncturing the dura without damaging the underlying brain (see Note 20). 8. Load virus into pipette. Pipettes are first backfilled with mineral oil, and inserted into injector according to injector instructions. Loading is best achieved by placing a droplet of viral solution (~5 μL) onto a small piece of Parafilm and then lowering the pipette into the droplet and drawing the virus into the pipette until ~2 μL is drawn into pipette. 9. Advance pipette into brain until desired depth is reached (see Note 21). For imaging superficial layers of cortex, injection depths of 200–500 μm below the cortical surface are typical. We often perform two injections of 580 nL each, at−400 and −200 μm below the surface. When doing multiple injections, best results are obtained performing the deepest injection first and then partly withdrawing the pipette to reach shallower depths. 10. Inject virus. Using a Nanoject-II, we perform a pulsed injection. Injection parameters for bulk labeling are typically 32.2 nL pulses at 46 nL/s, delivered every 15 s for a total injection volume of 580 nL. If Fast Green was used (see Note 4), there should be a slow spread of green dye into the tissue. If significant leakage is observed, withdrawing the pipette, and repeating the injection with a new and sharper pipette may help. 11. After each injection, leave the pipette in place for 3–5 min to ensure diffusion of virus into tissue. 12. After completing all injections, close craniotomy with agarose. Apply a single drop of warm agarose (40–41 °C) on top of the craniotomy and smooth flat to fill only the craniotomy. In some cases, we apply an additional thin layer of bone wax to cover the craniotomy and overlapping slightly (~1 mm) onto the adjacent skull. 13. Replace the muscle overlying the skull and, if possible, suture muscle with an absorbable suture (e.g., 6-0 vicryl). Suturing muscle may not be possible without tearing in

Injection & Implant for in Vivo Imaging

177

very young animals. Close skin with suture (e.g., 4-0 nylon) or tissue acrylic (e.g., Vetbond). Apply antibiotic ointment to wound margins. 14. Remove animal from anesthesia and provide necessary postoperative care. 15. Onset of detectable viral expression of fluorescent proteins is highly dependent on the promoter and age of the animal. For injections performed in young (P26) ferrets, 6 days of expression is sufficient for strong fluorescence signal using hSyn.GCaMP6s.WPRE.SV40, whereas 2–3 weeks is optimal for injections in P40 animals. 3.2 Cranial Window Implantation

1. Obtain IACUC approval for all procedures. 2. Anesthetize animal and mount in stereotaxic apparatus (see Notes 16–18). 3. Perform incision over injection site and resect skin and muscle, exposing skull. It is important to expose a sufficient area to allow strong adhesion of the headplate to the skull. An area extending 3–5 mm from the headplate edges is usually sufficient, although a larger area provides stronger adhesion. 4. Prepare skull for headplate by scraping with a scalpel blade. The goal is to remove all soft tissue from the skull to maximize adhesion (see Note 22). 5. Dry skull with gauze and/or compressed air. 6. Quickly apply a thin layer of tissue acrylic to skull, avoiding craniotomy from viral injection if still present, and allow to dry. 7. Position headplate over injection site and cement into place with C&B Metabond. Allow 20 min to harden. Ensure that there are no gaps between the headplate and skull, and fill any gaps with cement. It may be necessary to apply cement to both the interior and exterior of the headplate to create a complete seal. Also ensure that no cement is on inner edges of headplate that will prevent forming a seal with the coverslip (see Note 23). 8. Remove animal from stereotaxic apparatus and re-mount using headplate. Using the headplate described in Note 8, this is accomplished by clamping the protruding tab. 9. Perform craniotomy. Drill around interior edges of headplate until skull flap is completely loose. Take care not to apply too much pressure and break through suddenly and damage the underlying brain. Best results are typically obtained by drilling around the entire craniotomy as a unit, as opposed to completely drilling different sections sequentially. Frequent use of compressed air to clear debris is helpful. 10. Once skull flap is loose, apply ACSF to craniotomy and let sit for 3–5 min. Doing so greatly reduces the tendency of the dura

178

Gordon B. Smith and David Fitzpatrick

to adhere to the skull when lifting the skull free, thereby reducing the risk of rupturing blood vessels. 11. Remove skull flap. 12. Perform durotomy. There are several approaches; however, we find the most consistent success by first puncturing the dura with a 30 G needle, then using 90° vitrectomy scissors to excise a circular dural flap (see Note 24). If it is necessary to cut large dural blood vessels, irrigate craniotomy to clear blood after cutting, and clamp end of vessel with forceps for 10–20 s to stop bleeding. For particularly large or persistent bleeders, repeated clamping of the vessel end may be required. Gel foam (soften first by soaking in ACSF) can also be applied to help stop bleeding. Take care not to cut or damage any blood vessels on the cortical surface (see Note 25). 13. Insert coverslip. The exact procedure will depend on the approach to filling the space between the brain and the headplate lip that will hold the coverslip (see Notes 10 and 11). We have used four different approaches: agarose (see Note 26), Kwik-Sil (see Note 27), a stacked glass insert (see Note 28), or a cannula (see Note 28). See Note 11 for discussion of the advantages and drawbacks of various approaches. 14. Secure coverslip in place. For the headplate described in Note 8, insert a snap ring into the groove in the headplate. For additional security, a bead of Kwik-Sil or cyanoacrylate glue can be applied around the edges of the snap ring. 3.3 Two-Photon Imaging

1. Position animal under two-photon microscope and focus coarsely on the cortical surface, using microscope oculars (if present), reflected light or epifluorescence. 2. Block stray light entering the system. Stray light reaching the microscope detectors can be a major source of noise and can introduce imaging artifacts. The simplest way to achieve this is to operate the microscope in a darkened room. In some cases (e.g., when presenting visual stimuli) this is not possible. In that case, see Note 29. 3. Image labeled structures using image acquisition software. See Note 30 for laser power considerations, and see Note 31 for PMT voltage considerations. For functional imaging, see Note 32 for anesthetic considerations. Optimizing power, PMT voltage, and scan parameters (including dwell time on galvoscanned systems) depends on the individual sample. High quality experiments using GCaMP6s typically yield strong label suitable for imaging within 2 weeks. In one such experiment, shown in Fig. 2, visual cortex was infected with AAV1.hSyn. GCaMP6s.WPRE.SV40 and imaged after 2 weeks. Using modest laser power (