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Generation and Purification of Highly Specific Antibodies for Detecting PROTOCOL Generation and purification of highly specific antibodies for detecting post-translationally modified proteins in vivo Swathi Arur1 & Tim Schedl2 1Department of Genetics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA. 2Department of Genetics, Washington University in St. Louis, St. Louis, Missouri, USA. Correspondence should be addressed to S.A. ([email protected]) or T.S. ([email protected]). Published online 23 January 2014; doi:10.1038/nprot.2014.017 Post-translational modifications alter protein structure, affecting activity, stability, localization and/or binding partners. Antibodies that specifically recognize post-translationally modified proteins have a number of uses including immunocytochemistry and immunoprecipitation of the modified protein to purify protein-protein and protein-nucleic acid complexes. However, antibodies directed at modified sites on individual proteins are often nonspecific. Here we describe a protocol to purify polyclonal antibodies that specifically detect the modified protein of interest. The approach uses iterative rounds of subtraction and affinity purification, using stringent washes to remove antibodies that recognize the unmodified protein and low sequence complexity epitopes containing the modified amino acid. Dot blot and western blot assays are used to assess antibody preparation specificity. The approach is designed to overcome the common occurrence that a single round of subtraction and affinity purification is not sufficient to obtain a modified protein-specific antibody preparation. One full round of antibody purification and specificity testing takes 6 d of discontinuous time. INTRODUCTION Covalent modification of amino acids in proteins is a post- of a signaling pathway or utilization of the phosphorylated pro- ­translation mechanism that alters the proteome, changing the tein42–45. Importantly, modified protein–specific antibodies can structure of individual proteins and thus potentially affecting be used to purify protein-protein, protein-DNA or protein-RNA their activity, stability, localization and/or binding partners1–18. complexes that contain the modified protein; a widely used In response to changing conditions during development or in example is the use of antibodies specific to modified histones for the environment, post-translational protein modification can analysis of genomic regions that contain the modified histone in much more rapidly alter the activity of the proteome compared chromatin4,14,20,24,46,47. with transcriptional or translational control mechanisms4,14,19–21. These approaches require that the antibody be specific to the Nature America, Inc. All rights reserved. Inc. Nature America, 4 Although numerous protein modifications are known, for modified protein being investigated. However, modification- example, histone methylation and acetylation, the most widely specific antibodies are not straightforward to generate, and widely used antibodies can be nonspecific, recognizing both the modified © 201 studied modification is phosphorylation. Protein kinase– mediated phosphorylation of substrates is the output of a number and unmodified forms of the target protein or identifying other of signaling pathways (e.g., Ras–extracellular signal-related kinase proteins that contain the modified residue. The modENCODE (ERK)/mitogen-activated protein (MAP) kinase signaling13,15) project, designed to identify the distribution of histone modi- and is often part of the molecular mechanism for execution of fications genome-wide using chromatin immunoprecipitation the signaling cascade and its modulation (e.g., Wnt signaling)22. (ChIP) analysis, found that nearly 50 of the 200 commonly availa- Mass spectrometry has revolutionized our ability to identify post- ble antibodies were either not specific to the histone modification translationally modified proteins, through the identification of or showed reactivity to nonhistone proteins; for example, in the the modified residue and the surrounding amino acid sequence case of regularly used antibodies to H3pS10 (ref. 46), preparations in which it is embedded16,23–26. Once a modification site has been were found to cross-react with the unmodified form48. Unknown identified, antibodies can be generated that specifically recognize are the number of failed attempts by individual laboratories to the modified protein (modified residue plus surrounding amino generate or purify antibodies specific to a modified protein. Thus, acids)27,28. Such modified protein–specific antibodies can be used there is a need to develop and refine methods that yield very in a wide range of in vivo studies of the modified protein, some high quality, specific antibodies for detection of post-translational of which are not easily performed by other approaches, such as modifications in vivo. Here we present a protocol that allows the mass spectrometry27,28. The modified protein–specific antibodies purification of highly specific polyclonal antibodies that detect allow immunocytochemical analysis of the modified protein at post-translationally modified proteins, which can then be used in high spatial and temporal resolution. For example, in complex multiple distinct assays including immunocytochemistry, western tissues, the modified protein–specific antibodies can identify indi- blotting and immunoprecipitation. vidual cells or cell types that do or do not contain the modified protein27,29–33, and, at the single-cell level, individual organelles Development of the protocol or subcellular structures that contain the modified protein32–40. Development of the strategy and protocol grew out of our study Modified protein–specific antibodies can be used in high- on substrates of ERK MAP kinase that function in Caenorhabditis throughput RNAi screens41 or to test hypotheses related to control elegans germ cell biology. We identified candidate substrates by NATURE PROTOCOLS | VOL.9 NO.2 | 2014 | 375 PROTOCOL Figure 1 | Spatial control of NOS-3 phosphorylation in the C. elegans a b germ-line tissue. Phospho- and non-phospho-specific antibodies provide unique reagents that allow the analysis of the spatial distribution of protein WT nos-3(0) mpk-1(0) WT nos-3(0) mpk-1(0) modifications that would be difficult or impossible to obtain by other Non-pNOS-3 pNOS-3 methods. (a,b) The specificity of purified anti-pNOS-3 and purified anti-non- pNOS-3 antibody preparations is shown in a and b, respectively, which are western blots of total lysates from adult wild-type (WT), nos-3–null mutant Tubulin Tubulin (nos-3(0)) and mpk-1/ERK–null mutant (mpk-1(0)) worms. Purified anti- pNOS-3 antibody, raised against the peptide shown in Table 2, identifies a c DAPI single protein species in wild type that is absent in nos-3(0) and in mpk-1(0), dpMPK-1 which encodes the kinase that generates the modified sites. Purified anti- pNOS-3 non-pNOS-3 antibody, raised against the peptide in Table 2 without the Pachytene Oocytes phospho-residues, identifies a single band in wild type and in the absence TZ of the kinase (mpk-1(0)), but it fails to identify a protein species in Mitotic nos-3(0). Tubulin western blot sample loading control, bottom (a,b). Pachytene (c,d) Immunocytochemical staining of wild-type adult hermaphrodite germ * Oocytes lines shows that purified anti-pNOS-3 antibody staining (green in c) is found TZ in proximal germ cells that also contain activated MPK-1/ERK (red, dpMPK-1, Mitotic in c), whereas purified anti-non-pNOS-3 antibody staining (green in d) is * DAPI found in a mutually exclusive population of germ cells that lack activated d dpMPK-1 MPK-1 (red, dpMPK-1, in d). The sum of the pNOS-3 and non-pNOS-3 signal is Non-pNOS-3 * Mitotic equivalent to total NOS-3 staining27. Asterisks (*) indicate the distal end of TZ Oocytes the germline. TZ, transition zone. Scale bar, 20 µm. Adapted with permission Pachytene Mitotic Loop from ref. 27. * TZ Oocytes Pachytene using a three-part approach: (i) bioinformatic identification of evolutionarily conserved proteins that contain ERK docking sites; (ii) an RNAi screen for enhancement of either a weak loss-of- function mutation in ERK or a weak gain-of-function mutation in Ras; and (iii) quantitative in vitro tests of phosphorylation mutant worms (specificity of the modified site for the kinase). of the candidate proteins by activated EKR2 (ref. 28). Although Similarly, we demonstrated that the anti-non-pNOS-3 antibody this approach identified high-likelihood substrates, it remained (Fig. 1b) was specific, as it produced a single band in wild type but to be demonstrated that the gene products were phosphorylated no staining in extracts from nos-3–null mutant worms (specificity in vivo by ERK. Therefore, we turned to the generation of phos- for NOS-3), whereas a single band was observed in mpk-1–null phoprotein-specific antibodies to the candidate substrates. mutants (specificity to the nonphosphorylated form of NOS-3). By We used site-directed mutagenesis to map the serine (S) or threo- using immunocytochemical staining of the germ-line tissue with nine (T) residue N-terminal to proline (P), which is typically used the phospho-specific NOS-3 antibodies, we found that only cells Nature America, Inc. All rights reserved. Inc. Nature America, 4 as the phospho-acceptor for ERK2, in an in vitro kinase assay. containing activated MPK-1/ERK have phosphorylated NOS-3 We then generated antibodies to a peptide that contained the (Fig. 1c) and that phosphorylation was genetically dependent phosphorylated
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