© 2014 Nature America, Inc. All rights reserved. oiid rti–pcfc niois a b ue i high- in used be can throughput RNAi screens antibodies protein–specific Modified protein modified the contain that structures subcellular or protein modified the contain not do or do that types cell or cells vidual tissues, the modified protein–specificcomplex antibodies in can identify indi example, For resolution. temporal and spatial high at protein modified the of analysis immunocytochemical allow mass spectrometry as such approaches, other by performed easily not are which of of range wide a in acids) amino surrounding plus residue (modified protein modified the recognize can that specifically be generated antibodies identified, in which it is embedded sequence acid amino surrounding the and residue modified the of identification the through proteins, modified translationally Mass has revolutionized spectrometry our ability to identify post- signaling) Wnt (e.g., modulation its and cascade signaling of the execution for mechanism molecular the of part often is and signaling kinase (MAP) protein (ERK)/mitogen-activated kinase– Protein of signaling pathways (e.g., Ras–extracellular signal-related kinase phosphorylation. is mediated phosphorylation of substrates is the output of a number modification for studied known, widely most are the acetylation, and methylation modifications histone example, protein numerous Although with transcriptional with or transcriptional translational control mechanisms compared proteome the of activity can the alter rapidly more much modification protein post-translational in environment, or the development during conditions changing to response In partners binding and/or affecting localization stability, activity, potentially their thus and proteins individual of the post- structure changing a proteome, the is alters proteins that in mechanism acids ­translation amino of modification Covalent I purification and specificity testing takes 6 d of time.discontinuous affinity purification is not sufficient to obtain a modified protein-specific antibody preparation. preparation specificity. sequence complexity epitopes containing the modified amino acid. Dot blot and western blot assays are used to assess antibody subtraction and affinity using purification, stringent washes to remove antibodies that recognize the unmodified protein and low to purify polyclonal antibodies that specifically detect the modified protein of interest. complexes. However, antibodies directed at modified sites on individual proteins are often nonspecific. Here we describe a protocol immunocytochemistry and immunoprecipitation of the modified protein to purify protein-protein and protein-nucleic acid partners. P Published online 23 January 2014; St. Louis, Missouri, USA. Correspondence should be addressed to S.A. ( 1 Swathi Arur modified proteins antibodies for detecting post-translationally Generation and purification of highly specific Department of Genetics, of The Department University Texasof M.D. Anderson Cancer Center, Houston, Texas, USA. NTRO ost-translational modifications ost-translational alter modifications protein structure, affecting activity, stability, localization and/or binding 27,2 D 27,29–3 UCT 8 A . Such modified protein–specific antibodies can be used ntibodies ntibodies that specifically recognize post-translationally modified proteins have a number of uses including I ON 3 1 , and, at the single-cell level, individual organelles organelles individual level, single-cell the at ,and, & Tim Schedl 27,2 in vivo in 8 . The modified antibodies protein–specific 16,23–2 T 4 he he approach is designed to overcome the common occurrence that a single round of subtraction and studies of the modified protein, some some protein, modified the of studies 1 or to test hypotheses related to control d o 6 i . Once a site modification has been : 1 2 0 . 1 0 3 8 / n p r o t . 2 0 1 4 in vivo . 0 1 7 [email protected] 4,14,19–2 32–4 13,1 1–1 2 5 8 0 2 1 ) - . . . .

tein pro phosphorylated the of utilization or pathway signaling a of elegans on of ERK substrates MAP kinase that in function study our of out grew protocol and strategy the of Development the Development protocolof immunoprecipitation. and blotting multiple distinct assays including immunocytochemistry, western proteins, modified which can then be post-translationally used in detect that antibodies polyclonal specific highly of purification modifications very yield that methods high quality,refine specific antibodies and for detection of post-translational develop to need a is there generate or to purify specific to antibodies a modified protein. Thus, laboratories individual by attempts failed of number the are were found form to the with unmodified cross-react case of regularly used antibodies to H3pS10 (ref. the in example, for proteins; nonhistone to reactivity showed or ble were antibodies either not to specific the histone modification (ChIP) analysis, found that nearly 50 of immunoprecipitation the 200 commonly availa chromatin using genome-wide modi fications histone of distribution the identify to designed project, modENCODE The residue. modified the contain that proteins other identifying or protein target the of forms unmodified and used antibodies can be nonspecific, recognizing both the modified modification- However, specific antibodies are not straightforward to generate, and widely investigated. being protein modified chromatin in histone modified the contain that regions genomic of analysis used widely a for protein; histones to modified specific antibodies is of use the example modified the contain that complexes or protein-RNA protein-DNA protein-protein, to purify be used These approaches require that the antibody be specific to the the to specific be antibody the that require approaches These 42–4 ) ) or T.S. ( germ cell biology. We identified candidate substrates by by substrates candidate We identified biology. cell germ 5 . Importantly, modified protein–specific antibodies can can antibodies protein–specific modified .Importantly, 4,14,20,24,46,4 2 [email protected] Department of Genetics, of Department Washington University in St. Louis, in vivo in T . Here we present a protocol that allows the the allows that a protocol . Here present we he he approach uses iterative rounds of 7 . natureprotocols O ). ne ne full round of antibody

| VOL.9 NO.2VOL.9 4 protocol 6 Caenorhabditis Caenorhabditis ), preparations 4 8 . Unknown | 2014 |

375 - - -

© 2014 Nature America, Inc. All rights reserved. NOS-3 NOS-3 protein) and no staining in extracts from from extracts in staining no but type wild in band single a showed it as specific, was preparation antibody anti-pNOS-3 the that demonstrate to proteins. We used western blotting of of blotting Wewestern proteins. used phosphorylated NOS-3 (pNOS-3) and for nonphosphorylated NOS-3 (non-pNOS-3) specific antibodies purified and generated of regulation ERK–mediated MPK-1 of translational of relief to the of repression leading FBF-1/-2, cofactors related Pumilio- to binding disrupts phosphorylation where substrate MPK-1/ERK an as identified was protein, RNA-binding related in NOS-3 with illustrated is function protein modified of in studies antibodies, non-phospho-specific of mutant null a in occur to failed it as activity, ERK on dependent vivo in phosphorylated are products gene these that show to antibodies (DDX19) and glycogen synthase kinase 3 19 helicase RNA DEAD-box substrates candidate to phosphoprotein-specific antibodies purified We substrate. the to unique were that acids amino surrounding and the T or S contained that phosphorylated peptide a to antibodies generated then We an in ERK2, for phospho-acceptor the as substrates. nine (T) residue N-terminal to proline (P), which is used typically candidate the to We to mutagenesis used site-directed map the (S) serine or threo antibodies phoprotein-specific vivo in phosphorylated were products gene the that demonstrated to be remained it substrates, high-likelihood identified approach this (ref. EKR2 activated by proteins candidate the of quantitative (iii) and Ras; in mutation gain-of-function weak a or ERK in loss-of- mutation weak function a either of enhancement for screen RNAi an (ii) conserved proteins evolutionarily that contain ERK docking sites; of identification bioinformatic (i) approach: three-part a using from ref the germline. TZ, transition zone. Scale bar, 20 equivalent to total NOS-3 staining MPK-1 (red, dpMPK-1, in found in a mutually exclusive population of germ cells that lack activated in in proximal germ cells that also contain activated MPK-1/ERK (red, dpMPK-1, lines shows that purified anti-pNOS-3 antibody staining (green in ( nos-3(0) of the kinase ( phospho-residues, identifies a single band in wild type and in the absence non-pNOS-3 antibody, raised against the peptide in which encodes the kinase that generates the modified sites. Purified anti- single protein species in wild type that is absent in pNOS-3 antibody, raised against the peptide shown in ( western blots of total lysates from adult wild-type (WT), pNOS-3 antibody preparations is shown in methods. ( modifications that would be difficult or impossible to obtain by other unique reagents that allow the analysis of the spatial distribution of protein germ-line tissue. Phospho- and non-phospho-specific antibodies provide Figure 1 376 c nos-3(0) protocol , c The utility of phosphoprotein-specific antibodies, as well as as well as antibodies, phosphoprotein-specific of utility The d mpk-1 ), whereas purified anti-non-pNOS-3 antibody staining (green in ) Immunocytochemical staining of wild-type adult hermaphrodite germ

| VOL.9 NO.2VOL.9 . by ERK. Therefore, we turned to the generation of phos of generation the to turned we Therefore, ERK. by at the identified sites and that the phosphorylation was was phosphorylation the that and sites identified the at

. Tubulin western blot sample loading control, bottom ( 2 | ) and Spatial control of NOS-3 phosphorylation in the 7 a , the , the . , b ) The specificity of purified anti-pNOS-3 and purified anti-non- mpk-1(0) mpk-1 C. elegans C. | 2014 nos-3 fem-3 /ERK–null mutant ( ), but it fails to identify a protein species in d | ). The sum of the pNOS-3 and non-pNOS-3 signal is

–null mutant worms (specificity for the the for (specificity worms mutant –null natureprotocols mRNA ERK ortholog ERK 2 7 . Asterisks (*) indicate the distal end of in vitro in 2 7 . To understand spatial control control spatial To. understand mpk-1(0) a and Figure Figure tests of phosphorylation phosphorylation of tests C. elegans C. 2 8 µ . b m. Adapted with permission β , respectively, which are ) worms. Purified anti- (GSK3 fem-3 nos-3(0) T in vitro in able 1 T able . NOS-3, .a Nanos- NOS-3, nos-3 mpk-1 lysates ( lysates translation, we we translation, 2 C. elegans β and in without the 28). Although Although 28). 2 ) ) and used the kinase assay. kinase , identifies a –null mutant a ERK–null , c b mpk-1(0) ) is found ). Fig. 1 Fig. d

) is

a , ) - -

The protocol describes the generation of polyclonal polyclonal of or S proline-directed generation by phosphorylated sites with the experience significant have we where describes antibodies, phosphoprotein-specific protocol The the workflowof Overview below). (see bodies by staining immunocytochemical with the anti phospho-specific change in subcellular localization of the protein, which is detected a causes CGH-1 of Interestingly, testing. phosphorylation cificity spe and protocol purification antibody the of in description the points of number a illustrate to DDX6, human and Dhh1p yeast to orthologous is which CGH-1, helicase RNA the to antibodies relieving fully thus 1/-2, FBF- to co-regulators binds longer no that form phosphorylated the to NOS-3 of conversion complete essentially is there 1/ERK, protein NOS-3 for total observed to staining the is identical two patterns and the that the sum of tissue in the germ-line patterns mulation are and present in non-phospho-NOS-3 mutually exclusive accu ( MPK-1/ERK activated lacks that tissue germ-line the of region the in only present is phospho-NOS-3 (ref. MPK-1 on ( NOS-3 phosphorylated have MPK-1/ERK activated containing cells only that we found antibodies, NOS-3 phospho-specific the with tissue the of germ-line staining immunocytochemical using mutants (specificity to the nonphosphorylated form in of NOS-3).observed was By band single a whereas NOS-3), for no staining in extracts from ( antibody anti-non-pNOS-3 the that demonstrated we Similarly, kinase). the for site modified the of (specificity worms mutant a Fig. 1 Fig. 1 Fig.

d c pNOS-3 In this protocol, we describe the generation of phospho-specific Tubulin * * * *

b Mitotic c 2

) ) was specific, as it produced a single band in wild but type Mitotic ) and that phosphorylation was genetically dependent dependent genetically was phosphorylation that and ) 7 . These results indicate that, in cells with activated MPK- Mitotic Mitotic WT

TZ 27). Antibody staining demonstrates that non- that demonstrates staining Antibody 27).

TZ

TZ TZ nos-3(0)

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Loop Tubulin Fig. 1 Fig.

Oocytes

Oocytes Oocytes WT d

Oocytes ), that phospho- phospho- that ), nos-3(0) pNOS-3 dpMPK-1 DAPI Non-pNOS-3 dpMPK-1 DAPI mpk-1

mpk-1(0) –null –null - - -

© 2014 Nature America, Inc. All rights reserved. traction traction and/or affinity purification are necessary. Finally, western sub of rounds additional whether determine to tests specificity as conducted are analyses blot western and dot-blot round, each After phospho-epitopes. complexity sequence low to bind that to remove antibodies washes high-stringency uses approach ond and affinity to purification remove undesired antibodies. The sec ( way the along purification of extent the assess to tests specificity and antibodies to undesired remove approaches methodological ref. (see phospho-epitopes complexity sequence low with react that antibodies remove to insufficient cases, most in affinity is, of purification round single a non-phosphopeptide, the with react that antibodies to remove sufficient be can subtraction of round methods the with complex epitope,react phosphopeptide similar to described previously that antibodies for enrich to procedure fication puri affinity an and protein, nonphosphorylated the with react low uses a procedure with subtraction to remove that undesired antibodies react that ( phospho-epitopes complexity antibodies sequence and non- the protein with react that ­phosphorylated antibodies as well as phosphopro interest, the of to tein specifically react that that antibodies mix complex includes a contain will serum polyclonal this site(s); phosphorylation the contains that peptide a against antibodies polyclonal raise to is procedure the of phase first The proteins. fusion standard as well as modifications, specific protein other many to antibodies protocol polyclonal The purifying to kinases. applied MAP be also as can such site) (p(S/T)P kinases T Characters in T able able Fig. Fig. Total protein antibody specificity Desired antibody specificity Non-phosphopeptide Peptide immunogen A Non-phospho-specific antibody epitope Antibody specificity to low complexity ntibody reactivity 2 8 2 ). Therefore, the workflow of the protocol two incorporates 1 1 ). The first approach uses iterative rounds of subtraction subtraction of rounds iterative uses approach first The ). | bold 48,4

Assortment of antibody reactivity after immunization with phosphopeptide. denote phosphorylated residues; 9 . however,In practice, a single we although that find underlined characters denote C-terminal cysteine added for coupling peptide toKLHand BSA. X X p[ p[ P-X X X X X 2 4 1 2 1 1 Table -X - -X -X -X -X S S p[ 7 / / 3 2 3 2 2 -X T T -X -X -X -X -X S A ] ] 8 / -P-X -P 4 3 4 3 3 mino acidsequence -X T 1 -[S/T]-P-X -X - -X -X ] p[ ). The protocol protocol The ). 9 -P 4 4 4 X 7 -[S/T]-P-X - S 10 p[ / T S ] / -P-X T ] 7 -P-X -X 7 -X 7 8 -X 8 7 -X - - - - 8

-X 8 -X 9 X 9 such as phospho-tyrosine were first described in 1981 by Ross Ross al. by et 1981 in described first were modifications phospho-tyrosine as protein such recognized specifically that Antibodies other methods with Comparison interest. of phosphoprotein to the preparation the antibody of the specificity to used validate tein mutants gene or product–null RNAi are samples knockdown phosphopro of staining immunohistochemical and/or blotting gle round of subtraction against the nonphosphorylated peptide peptide nonphosphorylated the against subtraction of round gle by a sin protein nonphosphorylated to the react that antibodies study; under protein the the of removal first antisera, through of resulting (ii) purification of region phosphorylated the to ing correspond phosphopeptide a with immunization (i) follows: antibodies phosphoprotein-specific phosphoprotein-specific antibodies. for methods of purification ylation ies were then used to G follow phosphor substrate the of kinetics site by using a non-phosphopeptide immunogen; the two antibod also generated antibodies to the corresponding unphosphorylated and immunogen, phosphopeptide a using by substrate G protein Nairn sarcoma virus (RSV)-transformed that mammalian cells. Subsequently, demonstrate Rous in present to are proteins tyrosine-phosphorylated numerous used pan- were antibodies purified ­phospho-tyrosine These Sepharose. phospho-tyrosine–modified purification with affinity by followed hemocyanin (KLH) limpet keyhole phospho-tyrosine-modified with immunization by ated 10 X 10 - Salient features of current methods for purification of pol C - C 5 0 et et al. . Polyclonal anti-phospho-tyrosine antibodies were gener were antibodies .Polyclonalanti-phospho-tyrosine in in vitro with with other phosphoproteins complexity recognition epitope will likely cross-react or a few adjacent amino acids, where this low sequence Antibodies that recognize the phospho-site, but only one phospho-epitope Total protein antibody specificity, not including the purification workflow outlined in immunogen in the first row of this table and the the modified protein and is generated from the peptide sufficient sequence complexity to specifically recognize Desired antibody specificity, which is typically of the subtraction The corresponding non-phosphopeptide that is used in purification phosphoprotein-specific antibodies and used for affinity Conceptual phosphopeptide immunogen used to generate non-phosphopeptide Antibodies that specifically recognize the 5 1 generated antibodies to a phosphorylation site on the . These initial studies provided the basis of current current . of provided basis the studies initial These natureprotocols Description 48,49,51–6 Figure

| VOL.9 NO.2VOL.9 3 are summarized as summarized are 2 protocol

| 2014

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© 2014 Nature America, Inc. All rights reserved. and remove antibodies that bind to the low sequence complexity complexity to bind low the sequence that remove antibodies and for enrich epitopes sequence to the unique/complex recognize that antibodies washes 4.0) (pH high-stringency and if necessary) subtraction, (and purification affinity of cycles iterative uses protocol this Instead, immunizations. in additional be generated to likely also are phospho-epitopes complexity sequence lower purification and affinity subtraction of round single a perform then and rabbits of cohort new a with starting process immunization the repeat to is proteins cellular other with cross-reacting antibodies purified single-round of problem the to solution the that proposed have protocols Previous interest. of protein the to specific not is that in a resulting preparation phospho-sites, their of as part epitopes complexity sequence low the contain that cellu phosphoproteins other lar with cross-react and to bind then which epitopes, complexity sequence low recognize that antibodies and epitope sequence complex or unique the recognize that antibodies both for enrich will purification affinity of round single A proteome. organismal an in proteins phosphorylated other of a number on ( acids amino rounding sur few a or one only and phospho-residue the recognize that proteome. However, the complex mixture also contains antibodies to uniquely identify,complexity in most sequence cases, a specific sufficient protein in of an are organismal epitopes the that such ( acids amino surrounding of number a plus (p(S/T)) that recognize the mixture antibodies including phospho-residue a contains complex the with phosphopeptide after immunization ( proteins ismal preparation shows cross-reactivity toward other cellular or organ the as interest, of protein phosphorylated the to specific are that is to purification usually insufficient obtain polyclonal antibodies conditions. mental the experi various of under testing phosphopeptide the to and bound elution antibodies step, final the in (iii) Sepharose; to of linked round phosphopeptide the to single preparation antibody a the binding by protein phosphorylated the to react that linked to Sepharose, followed by affinity enrichment for antibodies 378 above). described (as epitopes purification Affinity Subtraction protocol We and other groups have found that a single round of affinity We affinity of round a have that single found groups and other

| VOL.9 NO.2VOL.9 Step 94 Steps 83–92 Steps 70–73 Steps 65–69 Steps 58–63 Steps 55–57 Steps 47–54 f e d c b a h g with sequentialhighandlowpHbuffer Stringent washofphosphopeptidecolumn Sera generatedagainstphosphopeptid Collect theflow-throughfromnon- Bind theflow-throughtophospho- Bind tonon-phosphopeptidecolum | Fig. Fig. Test forphosphopeptidespecificity Test forphosphoproteinspecificity 2014 phosphopeptide column Elute boundantibody 3 4 9 ) | peptide column . However, antibodies that recognize the the recognize that antibodies However, .

28,4 natureprotocols Table 9 . The polyclonal antiserum generated generated antiserum polyclonal .The 1 ), where these epitopes are found found are epitopes these ), where n s e of interest specific totheprotein fractions arenot If theantibody Step 95 epitopes arepresent sequence complexity protein andtolow If antibodiestototal Step 96 non-phosphopeptide fractions reacttothe If theantibody Step 93 Table 1 ) - - - -

return from present, then both the subtraction and affinity purification are repeated; If antibodies to total protein and to low sequence complexity epitopes are remains then the preparation is returned from (Step 93; purple arrow), whereas if reactivity to other phospho-proteins phosphopeptide remains, then the preparation is returned from undergoes an additional round of purification; if reactivity to the non- retains reactivity to undesired epitopes ( specificity tests indicate that the anti-phosphopeptide antibody preparation specific monoclonal antibody is that the single complex epitope epitope complex single the that is antibody monoclonal specific ERK H3(Ser10) activated to antibody clonal mono the are examples used proteins widely two modified generated; of been have number a to specific are that antibodies Monoclonal phosphoprotein. that for antibodies polyclonal of to purification alternative excellent an are interest of protein the generation ( for purification of phosphoprotein-specific antibodies, which includes sera Figure 2 Genome Browser. from databases such as the University of California, Santa Cruz (UCSC) number of isoforms from ESTs/cDNA sequence and RNA-seq data obtained product, then size may be deduced from the protein amino acid length and protein under study; if there are no existing publications on the gene and/or C terminal to the phosphorylation site) will be specific to the (e.g., if there are multiple isoforms from alternative splicing N terminal Expectations for the size of the phosphoprotein and the number of bands one ( then probed with 1:500 dilution of the phospho-antibody preparation after or 100 to pCGH-1 is indicated by *). ( antibody preparation that is specific to phospho-CGH-1 (band corresponding purification, with stringent washes, were necessary to generate a polyclonal by western blots of adult preparation through multiple rounds of affinity purification as assessed Figure 3 to screen purifica and it is time-consuming antibodies, and polyclonal of tion generation the than expensive more fold several however, is, antibodies monoclonal phosphoprotein-specific of generation The laboratories. between results to reproducible ing lead uniform highly are preparations and defined is specificity and specificity tests ( a Monoclonal antibodies specific for a phosphorylation site on on site phosphorylation a for specific antibodies Monoclonal a ), ), two ( µ 1st round g g (right) per lane, were resolved on a 10% (wt/vol) SDS gel and

| | Workflow for phospho-antibody purification. ( Removal of nonspecific antibodies from the phospho-CGH-1 h a 64–6 to ), subtraction steps ( b ), ), three ( b 6 * (Step 96; blue arrow). Dsic avnae o a phosphoprotein- a of advantages Distinct . g , h c ). If the dot blot ( b ) ) and four ( C. C. elegans 2nd round a – d b ) ) Whole-worm lysates, 200 lysates. Multiple rounds of affinity , c d ), affinity purification steps ( ) ) rounds of affinity purification. * T able d c Fig. 2 8 and to phosphohistone phosphohistone to and h 3rd round 1 to 5 ), then the purified sample ) and western blot ( d (Step 95; red arrow). a * – h ) Workflow µ g g g (left) 4th roun to d b – Fig.

f ) d

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© 2014 Nature America, Inc. All rights reserved. these experiments are challenging and difficult to owing conduct and difficult are challenging experiments these phorylated phos being indeed is protein desired the that ensure to controls specificity extensive devise to family. important is gene it a situations, such to In belong may protein the when occurs usually this and unique, not is phospho-epitope a of context sequence for Sometimes, time. real in protein methods modified a no tracking specifically currently are There analysis. real- cytological from time obtained be can that behaviors molecular dynamic of observation allow not may samples fixed However, tion. such prepara antibody protein–specific modified the with used and resolu immun for obtained be usually can temporal samples tion High antiserum. low-titer from preparation vendor.antibody It is to very difficult generate a antibody specific by immune response, assessed usually the ELISA through custom a strong elicit must protein) (or the fusion peptide The modified Limitations contain antibodies. to specific not thought was that antiserum frozen stored from from respectively, gens, immuno protein fusion or peptide and modified-peptide using by proteins, total to and poly proteins modified specific to antibodies obtain clonal to useful be will protocol the Therefore, investigation. of lines discontinued resurrecting antibodies, cific spe generate may washes, stringent with purification affinity of rounds multiple after antiserum, such that possible It is freezers. in unused stored or discarded were and antibodies specific tain con to not assumed were they thus and purification, affinity of round one undergone having after specificity to show failed that numerous research preparations groups have antibody that polyclonal generated likely is it Similarly, to antibodies. necessary specific were obtain purification affinity of rounds more or two (ref. FEM-3 total detect to proteins, fusion to antibodies polyclonal purify to steps purification affinity the used also Wehave tide. pep synthesized a chemically into incorporated be can that acid amino modified any to applicable be should it thus and bodies, anti and acetylation-specific H3 as methylation– as well histone antibodies phosphoprotein-specific of a number to generate used successfully been has protocol The Applications antibody. monoclonal corresponding the of generation the in expense and time the supporting produced, be can interest of phosphoprotein the identifies specifically that an epitope that recognize that antibodies principle of proof vides pro it Furthermore, antibody. monoclonal a of generation the way,for impetus interesting providing in a biologically regulated spatially and/or temporal is and kinase(s) predicted the through occurs phosphorylation the that onstrate phase of characterization a project where the is preparation antibody used to dem initial the in useful most is phosphoprotein a to specific antibodies polyclonal of purification and generation The outcome. successful a of guarantee no lower-sequence-complexity with phospho-sites, related other not with interest, of cross-reacting phosphoprotein the to specific is that one the identify to clones antibody–secreting monoclonal many among to the inherent redundancy in biology. in redundancy inherent the to 2 7 ) and GFP (S.A., unpublished data). In all these cases, cases, these all In data). unpublished (S.A., GFP and ) in vivo in and not another family member; often times, times, often member; family another not and de novo de immunizations and potentially potentially and immunizations 27,2 8 (S.A., unpublished data, (S.A., unpublished in vivo in o , that it occurs occurs it that , cytochemistry cytochemistry ------

teins. The binding pocket of an antibody can interact with its its with interact can antibody an of pocket binding The teins. pro phosphorylated other or form unphosphorylated the nize recog not but high interest of protein with phosphorylated the bind to affinity specifically must antibodies ­phospho-specific design. Phosphopeptide design Experimental phosphopeptide antibodies. T injecting the antigen and generation of the serum should be one one be should serum the of generation and antigen the injecting for choose to animal the Ideally, choice. of extract cell or tissue against 1:5,000 and 1:1,000 at sera the with analysis blot western sera from the animals to be preimmune injected from of the vendor,ml 1 and Obtain perform interest. of type cell or tissue the of extracts whole-cell against sera the of generation for the injected to be the animals of prescreening we recommend immunization: antisera. polyclonal of Generation sites modified both recognize ously simultane that generated be could complexity sequence high of ylated site-directed mutagenesis suggested that two sites were phosphor MPK- GSK3 For substrates 1/ERK coupling. in used disulfide cysteine form terminal can the with that bonds residues cysteine or members protein family related with cross-reaction avoid to used were lengths shorter cases some in and protein, total the to antibodies of tion a flanking given side acids of the site p(S/T)P to avoid amino excessive genera six than greater extensions C-terminal or N- used not have we design, immunogen peptide In antibodies. specific to raise we successfully have that used peptides (BSA). (KLH) purification and antibody cross-linking the peptide to the carrier proteins for immunization ( site that to terminal C four and terminal N acids amino four about with site p(S/T)P located have a centrally have we used that gens organism(s) relevant the of proteome the in site this of uniqueness the determine to formed analysis BLAST location. this by constrained is immunogen an as used be to is that sequence peptide study,the under protein the in site(s) specific a at occurs phosphorylation organism an of proteome the in protein specific a identify uniquely cases, most in should, they that complexity amino acids ten to six to up of range the in (epitope) determinant antigenic a Phosphorylated residue. able able GSK-3 DDX-19 N CDC-48.2 PAR-5 CGH-1 NOS-3 ame Table in in vivo 2 2 |

Phosphopeptide immunogens used to generate 1 67–6 ). A cysteine (C) is placed at the N or C terminus for for C N terminus or the at placed is (C) ). A cysteine 27,2 9 8 . Six to ten amino acids are of sequence sufficient and were in proximity sufficient that antibodies natureprotocols β and NOS-3, NOS-3, and To be a useful experimental tool, tool, experimental useful a be To . Typical phosphopeptide immuno phosphopeptide Typical DPK A LRK EILN ERP STENSE IIEY mino acidsequence . Screening of animals before before animals of Screening a a a a pSPIQEKK-C pTPLSAKK-C a pSPLYS-C and IVIG pTPTSRP pTPEHKK-C in vitro in a Table pSPSRSS

| VOL.9 NO.2VOL.9 a kinase assays and and assays kinase 2 pTPQA-C

phosphoprotein- 7 lists examples of lists examples protocol 0 a should be per be should pTPKHRKK-C 67–6 | 9 2014 . Because Because . a pTPGI-C |

379 ------­ © 2014 Nature America, Inc. All rights reserved. tion of the peptide immunogen early after immunization. after early immunogen peptide the of tion form of the protein, which presumably arise from dephosphoryla (iv) that antibodies react with the specifically nonphosphorylated and pro affinity; lower with the immunogen the bind in should and teome present proteins phosphorylated other of number a with cross-react will problematically that epitopes complexity a amino acids and few thus surrounding represent low-sequence- and residue the that only react with p(S/T) one or antibodies (iii) forms; protein, and nonphosphorylated both the phosphorylated total to the have specificity may thus and residue, p(S/T) the not residues, but only flanking with that react (ii) antibodies affinity; tifies the phosphorylated protein under study and binds with high epitopes, the including residue, p(S/T) such that it uniquely iden sequence complex sufficiently with react that specificity desired schematically in shown is which generated, be will reactivities of array diverse a have that antibodies polyclonal phosphopeptide, erated. gen are that species antibody various the and Immunization information. more for section TROUBLESHOOTING the to Refer purified. be can thus antibodies and generated, been has response a that indication an are Usually, with. to proceed values values these good are considered preimmune at 1:100,000) to 2.873 (for postimmune, at 1:100,000) or at OD dilution, in of 280 levels terms nm, (for 1:100,000 0.088 the at injected, antigen the to specifically response of sign good a considered is 800) versus 20,000 (e.g., sera preimmune versus sera postimmune of titer A high 1:100,000. and 1:10,000 1:1,000, of dilutions at serum, postimmune the and serum preimmune the between compared are titers ELISA Typically, generated. antibod ies various the of concentration the in differences in ing result response immune the in differences to owing animal to animal from differ can titers antibody antigen, peptide given a by the performed vendor.bleeds, blot usually before terminal For dot or ELISA via assayed be can antibody phosphopeptide to the response The purify. to difficult be often will phosphopeptide the to titer low a with Antisera bleed. production the by lowed fol injection booster one and injections biweekly three and tion injec priming one of consists which antisera, the of generation the against extract. cell or response tissue pre-existing of amount least the has that permission from ref slightly slower than the non-phospho-BSA complex. Adapted with unconjugated BSA (left lane), with the phosphopeptide-BSA running weight/retarded mobility (middle and right lanes) compared with the the conjugated peptide-BSA complex should run at a higher molecular Brilliant Blue staining. If the conjugation has gone to completion, and phosphopeptide-conjugated BSA (right lane), followed by Coomassie BSA material (left lane), non-phosphopeptide-conjugated BSA (middle lane) assessed by electrophoresis (10% (wt/vol) SDS-PAGE) of the unconjugated efficiently conjugate the phosphopeptide to the BSA. Conjugation can be phosphopeptide ( If the affinity purification fails to recover antibodies that recognize the Figure 4 specifically bind to the complex sequence phospho-epitope 380 phospho-epitope sequence complex the to bind 1–46). ­specifically (Steps preparation column Peptide protocol Typically, we follow a 10-week immunization schedule for the the for schedule immunization 10-week a Typically, follow we Table

| VOL.9 NO.2VOL.9

After immunization with the 2 mg of KLH-conjugated KLH-conjugated of mg 2 the with immunization After | Testing the efficiency of peptide conjugation to activated BSA. 1 . These include the following: (i) antibodies with the the with antibodies (i) following: the include These . Fig. | . 2014

2 8 2 . and | natureprotocols T able 1 ), this may occur owing to a failure to Antibodies that that Antibodies

------resin (Steps 45,46). (Steps resin Sepharose the on uncoupled remained have may that sites tive any reac to block buffer) (blocking buffer a high-salt Tris-based antibody, its activity. Finally,blocking the column is with washed purified the to bind then and step elution later the would in eluted be which interactions, ionic with through associated CNBr-Sepharose the loosely is that complex peptide-BSA any nate elimi to 42–44) (Steps buffer acidic then and buffer alkaline of cycles repeated with washed stringently is column the coupling, After CNBr-Sepharose. the inactivate will that EDTA) (Tris, ers buff amine-based any remove to and reaction the for required peptide- 23–25) (Steps solution pH the basic a into dialyzed is complex coupling, BSA Before (CNBr-Sepharose). Sepharose chemistry amine-based with matrix column the to coupled is complex peptide-BSA the ( 18–22) PAGE(Steps by SDS- assessed is BSA to peptide the coupling of and efficiency the 7–17), (Steps column desalting a using by purified then is BSA, forming stable thioether linkages. The peptide-BSA complex to maleimide-activated or non-phosphopeptide phosphopeptide the of cysteine C-terminal or N- added the cross-link to used is chemistry maleimide–based stage (Steps 1–6), homobifunctional first the In stages. two in columns Sepharose peptide the of tion produc the describes protocol The beads. the to antibodies the the column, and the higher local maypeptide density help anchor of capacity the increases which bead, Sepharose per bound tides in increase the pep an of number ninefold obtain approximately and then coupling the complex peptide-BSA BSA to Sepharose, to we can peptide the coupling first By residues. cysteine through conjugated peptides nine to up have can molecule BSA single a protein) (or peptide to a single linked typically is bead A Sepharose purification. for affinity column phos phopeptide a and protein total recognize that antibodies subtract to column non-phosphopeptide a columns: peptide two uses tocol the in present antibodies immune serum, and thus the efficient recovery is important. of The pro proportion small a ­represent 10 16 22 36 50 64 98 (kDa) Fig. Fig. 7 4 , by using cyanogen bromide–activated bromide–activated ,cyanogen by using 4 ). In the second stage (Steps 26–46), 26–46), (Steps stage second the In ). Unconjugated BSA

npDDX-19/BSA

pDDX-19/BSA 71–7 3 . By contrast, . By contrast, ------© 2014 Nature America, Inc. All rights reserved. antibody is tested for reaction to the phosphopeptide and to to and phosphopeptide eluted the the to assay, reaction for dot-blot tested the is using antibody By OD pooled. the in be can fractions fractions the peak the and in visualized, reading automatically be protein can the then 280-nm UV machine, a to readout connected is that if used is Alternatively, collector 74–82). fraction a (Steps elution activity IgG which contain determine to fractions we procedure First, dot-blot purification. simple a affinity undergone use and has subtraction that of preparation round one the on performed then are 74–96). (Steps purification affinity and su of rounds iterative testing, specificity Workflow: neutralized. immediately and 2.2, pH at buffer high are affinity then eluted with strong acidic conditions, glycine at bind that epitopes high-complexity recognize that antibodies ies or low with The recognition. low affinity complexity sequence repeated three or four times in an to effort remove more antibod column the from elution their in resulting inter phosphopeptide the with antibody actions low-affinity the destabilize will wash 4.0) (pH phosphopeptide the and antibody the between are interaction the buffers stabilizes buffer 4.0) alkaline the (pH performed; acidic and 9.5) (pH alkaline with washes than those that bind to the complex sequence epitopes. Sequential affinity lower with bind to expected are which epitopes, plexity com sequence low the bind that antibodies remove to used are residues other than the p(S/T) residue ( few a or one only containing epitopes, complexity sequence low ( study under phosphoprotein complex sequence epitopes, at high affinity, that are to specific to bind the that antibodies phosphopeptide: the with associate that populations distinct two contains column non-phosphopeptide is and from used The fraction for the purification. flow-through affinity phosphopeptide the contains column second The tion. phopeptide will not bind and be present in the flow-through frac ( protein total the with react that antibodies away subtract and bind will that non-phosphopeptide the contains column first The above). described as (prepared columns two over passed sequentially is in shown is purification the for workflow The study. under protein phosphorylated the ( sites phosphorylation complexity sequence low with react will that antibodies remove ( form phorylated nonphos the including protein, total with antibodies cross-react remove will that to step subtraction a involves protocol the 58–73). (Steps purification affinity and 47–57) (Steps subtraction Workflow: round of purification. that the activity toward the non-phosphopeptide is lost after the second retains residual activity toward the non-phosphopeptide. Row 2 shows Row 1 shows that after the first round of antibody purification, the antibody dilution of the anti-phospho CGH-1 peptide column eluate (from non-phosphopeptide was spotted on nitrocellulose and probed with 1:1,000 and non-phosphopeptides. An amount of 10 ng each for the phospho- and Figure 5 the non-phosphopeptide (Steps 83–93; 83–93; (Steps non-phosphopeptide the

| Dot blot to test specificity toward the CGH-1 phosphopeptides Table 47,77–7 To obtain phosphoprotein-specific antibodies, antibodies, phosphoprotein-specific obtain To 1 ), whereas antibodies that react with the phos the with react that antibodies whereas ), 9 . The stringent alkaline and acidic washes are are washes acidic and alkaline stringent The . Table Table 1 Figure Figure ), and an affinity purification step to to step purification affinity an and ), 1 ), and thus they are not specific to specific not are they ), thus and Table 2 , where the crude antiserum antiserum crude the where , 1 ) and antibodies that bind bind that antibodies and ) Table 75,7 Fig. Fig. Two specificity tests tests specificity Two 6 , whereas the acidic acidic the whereas , 1 ). washes Stringent 5 ). If the purified purified the If ). Fig. b traction traction 2

). ­ - - - - -

phosphopeptide, then antibodies that recognize the total pro ( total tein the recognize that antibodies then phosphopeptide, to the in addition the non-phosphopeptide, with reacts antibody isms that are undergoing signaling that generates the phospho- the generates organ that or signaling undergoing cells are that isms wild-type from lysates with assay blot western the over non again passed subtraction, of round second a undergo must preparation the protein, total the recognize that antibodies bands in a western blot or signal in immunohistochemistry, then in immunohistochemistry, in or blot a signal bands western inappropriate If show study. knockdown RNAi or under product–null gene the protein the to specific is antibody purified the staining immunocytological ( blots western in used is (CGH-1) site rylation RNAi-mediated depletion of the protein containing the phospho ( assay genetic-based is the determining for 97). (Step tests specificity Genetic-based is that preparation interest. of phosphoprotein the to antibody specific an obtaining to key is sub tractions, and washes stringent with purification, affinity of rounds ref. of 3c Fig. antibodies Supplementary in anti-pDDX-19 of purification affinity for and here pCGH-1 for (shown washes stringent with purification affinity ( blot western the on bands nonspecific by assessed as epitopes, complexity sequence lower ( two subtraction of or rounds one with accomplished usually is non-phosphoprotein others by com ( sequence phospho-epitopes plexity lower the with react that antibodies are ration major impediment to obtaining a phosphoprotein-specific prepa ( repeated are fication complexity puri sequence and affinity the subtraction are both then present, low epitopes to and protein total to antibodies If ( phospho-epitopes complexity sequence lower the with react that antibodies remove further to used is washes gent ( a or one plus few that acids amino are to site similar the complex phospho-epitope p(S/T) the contain which proteins, other to reacting is preparation antibody the then the product, gene to relevant addition in bands blot western shows antibody purified 94–96; (Steps study under phosphoprotein the to specificity for tested is preparation antibody the modification, Table - phosphopeptide column ( column phosphopeptide Table 1 ). Thus, a second round of affinity purification with strin 4 9 . Although removal of antibodies that react with the the with react that antibodies of removal .Although 1 ) remain in the preparation. Thus, to remove the the remove to Thus, preparation. the in remain ) Phospho-peptide in vivo in Fig. Fig. Fig. Fig. natureprotocols specificity of the antibody preparation preparation antibody the of specificity 2 Figs. 1a 1a Figs. Fig. Fig. Fig. Fig. 5 , 2 ), removal of antibodies against the the against antibodies of ), removal h 7 to assess whether the signal from from signal the whether assess to → 3 Fig. Fig. 3 and b ), often requires four rounds of of rounds four requires ), often 2 ). In practice, we find that the the that find we practice, In ). 8 and 2 ). Thus, the use of multiple multiple of use the Thus, ). , Table g

Non-phosphopeptide 6 → ). First, a null mutant or ). mutant a First, null

| Round 2,anti-pAb Round 1,anti-pAb b VOL.9 NO.2VOL.9 1 ). Second, by using a using by Second, ). The gold standard standard gold The ) 2 8 , as also observed , as observed also Fig. Fig. protocol Fig. Fig. Fig. Fig. 6 ) and/or in in and/or ) | 2014 2 3 , ). If the the If ). h → |

381 d ). ­ ------

© 2014 Nature America, Inc. All rights reserved. total protein from the phosphopeptide phosphopeptide the from protein total with react will that antibodies However, CGH-1; total unmodi (e.g., and forms) fied modified the (both tein pro total for specific are that antibodies cycles. third as purification, did affinity the second and by followed subtraction of consisted cycle first the wherein cycles, complete three in purification affinity by followed fraction from via subtraction purified the phospho was antibody non-phospho-NOS-3 the example, For reiteratively. purified be to needs also sometimes antibody non-phospho the fraction, antibody phospho-specific goal desired the being specificity per above, non-phosphoprotein with are as described formed tests Specificity eluted. then and epitopes, complexity column, washed to stringently remove to antibodies low sequence non-phosphopeptide the over passed then is flow-through The ec wt te hsh-ptps n ttl rti ( protein total and that phospho-epitopes antibodies the with removing react column, phosphopeptide the over the in shown from workflow reversed is purification of order the situation, NOS-3; this In (e.g., non-phosphoprotein unmodified the for specific are that antibodies polyclonal generate to useful 117–142). (Steps antibodies total and 98–103) (Steps non-phosphoprotein–specific of Purification ( protein total of amount the affect not should they but (pCGH-1) preparation antibody protein–specific from the signal modified eliminate or reduce should MPK-1-ERK) (e.g., enzyme modifying the of mutant null a or knockdown RNAi then known, is modification to specificity. assess Second, used that produces the the if enzyme protein be to need may experiments genetics-based additional null mutants or knockdowns should be modified accordingly, and and wild-type of immunocytochemistry and blots western from results for expectations the then analysis, BLAST from as assessed sequences, surrounding and site phosphorylation the in iden tity sequence with homolog(s) or paralog(s) a is there if that ( purification round affinity of additional an undergo should it and proteins modified or products gene other to cross-reaction shows preparation the unphosphorylated CGH-1 as it is present in the lysate and comigrates with the single band recognized by the anti-pCGH-1 antibody preparation (data not shown), whereas the bottom bandof CGH-1.(#) isThe probablyslower migrating band (*) in the wild type recognized by anti-total-CGH-1 is probably phospho-CGH-1, as it is absent from thethat the antibody is specific to MPK-1 ERK–mediated phosphorylation In indicating that the anti-pCGH-1 antibody preparation is specific for CGH-1. reveals a single band (*) in wild-type worms, which is absent in in which both bands are absent in closely spaced doublet (*, upper band, #, lower band) in wild-type worms anti-tubulin antibody (bottom). Probing with anti-total CGH-1 reveals a antibody preparation after four rounds of affinity purification (middle) and (gift of D. Greenstein, University of Minnesota) (top), the anti-pCGH-1 cgh-1(0) antibody preparation. Western blot of wild type (WT), Figure 6 382 protocol In many cases, it is useful to generate generate to useful is it cases, many In mpk-1(0)

| VOL.9 NO.2VOL.9

adult whole-worm lysates probed with anti-CGH-1 total antibody | Genetic tests for specificity of the purified anti-pCGH-1 polyclonal , the anti-pCGH-1 preparation fails to detect a band, indicating Figs. 1 1 Figs. | 2014 Figure Figure and |

2 natureprotocols 7 Fig. Fig. (

6 Fig. 1 Fig. ). 2 . The crude antiserum is first passed passed first is antiserum .crude The cgh-1(0) 2 , h b → ). As with purification of the the of purification with As ). Fig. Fig. d . Probing with anti-pCGH-1 ; repeat Steps 58–73). Note Note 58–73). Steps repeat ; 6 mpk-1(0) In some situations, it is is it situations, some In ). - - mpk-1(0) T able able 3–6 2–4 2 1 (ml) S lysate and absent after staining with the anti-pCGH-1 antibody preparation. erum i. 1b Fig. 3 3

and cgh-1(0) | P

Table Scaling up phospho-antibody purification. eptide (mg)

6 4 2 2 ,

d , ) 1

2

). 7 - - .

Maleimide-activated recombinant proteins to Sepharose (Steps 117–130) and affinity affinity and 117–130) (Steps Sepharose to proteins recombinant the of coupling describes protocol The resides. modification the where region the than protein the of part separate a to respond cor that immunogens or peptide modifications, eukaryotic lack from recom isolated protein either fusion used binant have we Instead, react to proteins. likely nontarget are they with thus and sequences peptide shorter to ( immunogen coupled to the Sepharose). We have reused columns up to six six to up columns reused have We Sepharose). the to coupled complex peptide-BSA the of degradation prevent (to neutral to back brought pH the and eluted be must column the to bound antibodies residual serum, crude of purification or first-round for purification of rounds additional for either columns, the of 104–110). (Steps columns of Regeneration obtained. results the on depending optimized be should dilutions However, immunofluorescence. for 1:200 and immunoprecipitation for extract protein total of 10–15 blotting), western (for 1:1,000 is as such applications western blotting, and immunoprecipitation immunofluorescence various in antibody final the for dilution at 4 °C up to can be maintained stocks 3 months. A starting good long-term storage, keep the at antibody –80 °C; however, working For glycerol). (vol/vol) 25% Tween-20 and (vol/vol) 0.01% BSA, (wt/vol) concentration—2.5% (final buffer stabilization a into is and above ready for use, it should be described concentrated to ~1 mg ml tests specificity relevant the passed has 111–116). (Steps tion prepara antibody purified the of storage and Concentration 131–142). (Steps purification BSA (mg) 6 4 2 2 Anti-pCGH-1 Table Anti-CGH-1

Tubulin bromide (g) C 1 yanogen ) are problematic, as they have specificity specificity have they as problematic, are ) Once the purified antibody preparation preparation antibody purified the Once 0.9 0.6 0.3 0.3

WT

* # * obtained (ml) B ed volume

Escherichia coli Escherichia mpk-1(0) 3 2 1 1 µ # g of antibody per mg mg per antibody of g For reuse or storage or storage For reuse

cgh-1(0) collected (ml) mpk-1(0) −1 Fractions , which will will which , and placed 30–50 20–40 20 10

- - - © 2014 Nature America, Inc. All rights reserved. • Polyclonal antisera • (andcorresponding non-phosphopeptides)Phosphopeptides • • • Reagents antibody for detection of • • • • • • Others • • Detergents • • • • • Corrosive solvents organic • • • • • • • • • Salts • • intheprotocol kitsused of Reagents aspart REAGENTS M will antibody phosphopeptide-specific the to purify used be will Scaling of the protocol and materials. purification. of efficacy decreased in result times storage Longer column. the making of storage. Storage should not be more than 3 months from the time of duration the on dependent highly be will this although times, different vendors such Biosystems, asOpen Covance and Yenzyme. We have Design). intheExperimental in-house (asdescribed We have several used KLH-conjugated phosphopeptide sera through acommercial vendor or Custom polyclonal (e.g., antisera are rabbit) the2mgof generated with unavoidable, then the terminal cysteine should be placed on the adjacent end. conjugation efficiencyto KLHandBSA. of aninternal cysteine is If as itmaywith the cysteine,terminal adisulfide form bond thus reducing the  powderthe peptide to room temperature (22°C)before opening it  the manufacturer out material asneeded at−20°Candweighed >90% purity. We have stored inpowdered thepeptides from form the non-phosphopeptide synthesized, be followed by HPLC to purification recommended thephosphopeptide and20mgof thatatleast20mgof from obtained canbe made in-houseor they commercial vendors; itis canbe Custom design) inExperimental asdescribed (designed peptides cat. no. WBKLS0500) Electrochemiluminescence (ECL)reagent for HRP(Millipore Immobilon, (Invitrogen, HRP, anti-rabbit goat cat. no. A10547) conjugated antibody Secondary to peroxidase horseradish (HRP) Nitrocellulose ScientificPierce, (Thermo membrane cat. no. PI-88013) β BSA for stabilization antibody (Sigma Aldrich, cat. no. A7888) BSA for western (Sigma blotting Aldrich, cat. no. A2153) Cyanogen bromide–activated Sepharose (Sigma-Aldrich, cat. no. C5338) Nonfat milk(Nestle Carnation) dry Glycerol (Fischer Scientific, cat. no. BP2291) sulfate (Sigma-Aldrich, lauryl Sodium cat. no. L6026) Tween-20 (Sigma-Aldrich, cat. no. P9416) DMSO (Sigma-Aldrich, cat. no. D8418) Hydrochloric (Fischer acid Scientific, cat. no. A144C-212) hydroxideSodium (Sigma-Aldrich, cat. no. S8045) acetic (FischerGlacial acid Scientific, cat. no. BP2401-212) Methanol (Fischer Scientific, cat. no. BP-1105) azideSodium (Sigma-Aldrich, cat. no. S8032) bicarbonate(NaHCOSodium BlueCoomassie (Sigma-Aldrich, Brilliant cat. no. B0149) Glycine (Sigma-Aldrich, cat. no. G8898) Tris base(Sigma-Aldrich, cat. no. T6791) Potassium phosphate (KH phosphateSodium (Na Potassium chloride (KCl; Sigma-Aldrich, cat. no. P9333) chlorideSodium (NaCl; Sigma-Aldrich, cat. no. S7653) cat. no. 77667) Purification buffer (Imject maleimide-activated BSAkit, Pierce, cat. no. 77667) Conjugation buffer (Imject maleimide-activated BSAkit, Pierce, ATER -Mercaptoethanol (Sigma Aldrich, cat. no. M6250)

CR CR I I T T I I I CAL CAL ALS Avoid, possible, internal if cysteine immunogen, inthepeptide powder peptide The ishydroscopic. Warm containing thevial 2 HPO 2 PO 4 3 ; Sigma-Aldrich, cat. no. S7907) 4 ; Sigma-Aldrich, cat. no. S7795) ; Sigma-Aldrich, cat. no. P9791) The amount of serum that

• • • • • • • • EQUIPMENT ( indicated as nent compo each up scale then used, to is be amount a larger if used; be will ( serum crude of ml 1–2 collected that assumes here procedure The fractions of number the and volume bed column resulting the and Sepharose CNBr-activated of amount BSA, the maleimide-activated of amount the peptide, starting of determine the size of the peptide column that is made, the amount the buffer atroom temperature for up to 1year. NaCl deionized water.of in800mlof Adjust 50%NaOH. thepHwith Store and 0.5MNaCl. For 1liter, acetic and29.2g acid glacial dissolve 5.25mlof columns)peptide Column washbuffer acidicwashbuffer for used (alsocalled washing when plastic. corrosion of gloves andalabcoat. pipette Pipette to out by theacid avoid usingaglass ! the volume to 1,000 ml. Store the buffer at room temperature for up to 1 year. NaCl deionized water.29.2 gof in800mlof Adjust thepHto 9.5. Make up NaOH. 50%(wt/vol) 9.5 with For 1liter, NaHCO3 dissolve 8.4gof and columns)peptide Coupling buffer alkalinewashbuffer for used (Also called washing when pipettePipette to out by theacid avoid usingaglass corrosion. plastic inafumehood; used wearit shouldbe goggles, gloves andalabcoat. ture for upto 1year. deionized water. 500mlwith volume of Store thesolution atroom tempera acetic made upto acid afinal glacial and 3.5mlof methanol in 200mlof BlueCoomassie Brilliant at room temperature for upto 1year. bubbling. Make thesolution upthevolume to 1,000ml. of Store thesolution SDSanddissolve 10%(wt/vol) itgently 50mlof add to avoid excessive deionized water.glycine in800mlof Once thesolution dissolved, hasfully Tris-glycine SDS, 5× temperature for upto 1year. not over-bubble. Make upthevolume to 1,000ml. Store thesolution atroom to facilitate dissolution. Care must taken be to ensure thatthesolution does deionized solution. sulfate in800mlof lauryl Heat thesolution to ~37°C SDS, 10%(wt/vol) 2× SDSsampleimmediately before use. thisbuffer is1week. shelf-life of The temperature for upto 1year. Add 1%(vol/vol) bromophenol blue. thevolume Bring to 10ml. Store thebuffer atroom glycerol, 0.1% (vol/vol) SDSand0.5mlof 10%(wt/vol) 4mlof SDS samplebuffer, 2× to 1 year. the of solution should be 7.4. Store the solution at room temperature for up salts are dissolved, make up the volume to 1,000 ml and test the pH. The pH Na PBS REAGENT SETUP •

(Millipore, cat. no. UFC901024) membrane Amiconwith Ultracel-10 filter centrifugal unit Ultra-15 cat. no. 66380) Slide-a-Lyzer dialysis cassettes, 10KMWCO Scientific, (Thermo ECL gel/blot developer (ODYSSEY Fc; Li-Cor Odyssey) Scientific,Spectrophotometer (Thermo cat. no. 4001-000) Econo-Pac columns (Bio-Rad Life Sciences Technologies, cat. no. 732-2032) Cold room, room at4°C set Rotator Scientific, shaker (Thermo cat. no. 2309-1CEFSQ) Balance (FineBalance; Mechatronics, Sartorium cat. no. SECURA513) choice used canbe worked with Yenzyme ourantibodies; however, for 90%of any company of Hydrion (Fischer Scientific, pHstrips 5.5–8.0plastic cat. no. 13-640-516) CAUT 2 HPO

For 1 I ON 4 and 0.24 g KH of The strong The inafumehood; used wear baseshouldbe goggles, liter of liter 1× of PBS, dissolve 8 g NaCl,of 0.2 g KCl,of 1.44 g of

Column washbuffer is0.1Msodiumacetate (pH4.0) Mix 0.1MNaHCO3 and0.5MNaCl. Adjust thepHto For SDS, 10%(wt/vol) 1liter sodium of dissolve 100 gof

!

For 1liter, Tris dissolve 15.1gof baseand94gof Table CAUT

Add 2.5ml of 0.5MTris-HClAdd 2.5mlof (pH6.8), 2mlof

Dissolve 1 g of Coomassie Brilliant Blue CoomassieDissolve powder Brilliant 1gof 2 I PO natureprotocols ON 3 ). 4 Glacial acetic isa strong Glacial acid acid, and in 800 ml deionized of water. Once all the β ! -mercaptoethanol to the

CAUT | VOL.9 NO.2VOL.9 I ON protocol The stro The

| 2014

Table ng base

|

383

3 - ). -

© 2014 Nature America, Inc. All rights reserved. 13| 12| 11| 10| 9| 8| 7| Desalting the peptide-  6| 5| 4| also clogthe desalting column(below). undissolved peptide and should bediscarded. Ifitisnot removed, thispeptide willdisruptthe conjugation efficiency and 3| mixture toincrease solubilization, and continue rotation atroom temperature for 1h. 2| affinity purification) and dissolveitin200 1| C PROCE NaCl deionizedand 29.2gof water. into 800mlof Adjust thepHto 7.5by Tris, 10mM(pH7.5)0.5MNaCl chemical hood. CAUT Freshly make thesolution for each useandkeep itatroom temperature. HCl, 10mM water. Store thesolution atroom temperature for upto 1year. NaCl, 100mM plastic. of corrosion avoid to pipette glass a using by acid the out Pipette coat. lab a and gloves goggles, wear ! plastic. of corrosion avoid to pipette glass a using by acid the out Pipette coat. lab a and gloves goggles, wear hood; fume a in used be temperature. room at month 1 is buffer blocking of life shelf The aliquot. smaller a to or 0.02%, at amount entire the to water) deionized of ml 100 in g (1 azide sodium 50 HCl. Add with 8.0 to solution the of pH water.the Adjust deionized of ml 800 in Tris Base of g 2.42 and NaCl of g 29.2 Tween-20.liter, 1 dissolve For (vol/vol) 0.05% buffer Blocking plastic. of pipettePipette to out by theacid avoid usingaglass corrosion inafumehood; used wearshould be goggles, gloves andalabcoat. 384

protocol onjugating peptidestomaleimide-activated CAUT

CR

Add the peptide-BSA mix from Step6tothe topof the columnand drain the flow-through and discard it. Labelten1.5-mlEppendorf tubesand haveitready for fraction collection. Add 500 Add 500 | I VOL.9 NO.2VOL.9 Add DMSO toafinal concentration of 1%(vol/vol),ifDMSO wasusedinStep2. Dissolve the saltsinwaterbyvortexing the mix. Add 60mlof deionized watertoone bottleof purification salts. Rotate the tubesonaninvertrotator atroom temperature for 2h. Add the dissolvedpeptide (supernatant from Step3)tothe tubefrom Step4. Add 200–500 Centrifuge the mixture at8,000 If some peptide remains undissolved (solution isturbid), add DMSO toafinal concentration of 1%(vol/vol)tothe Weigh out2mg of the peptide (either the non-phosphopeptide, for the subtraction, orthe phosphopeptide for the ON I I T ON D I Pipette the concentrated HCl with glass pipettes ina Pipette theconcentrated glass HCl with URE CAL Sodium azide is toxic, and it should be used in a fume hood; hood; fume a in used be should it and toxic, is azide Sodium

Add 86

For 1liter, NaCl dissolve deionized 5.84gof in1,000mlof STEP

µ µ Blocking buffer is 0.5 M NaCl, 20 mM Tris (pH 8.0) and and Tris8.0) mM (pH NaCl,20 M 0.5 is buffer Blocking l of purification buffereach time tothe columnand repeat Step10three times. l–1 mlof thispurification buffertoone desalting columnand letit drain bygravity. | 2014 µ Both peptide and BSA should be fully dissolved before proceeding. ! l of concentrated deionized water. HCl tol of 100mlof µ

CAUT µ l of conjugation buffertothe maleimide-BSA tube. | l of Tween-20 to the solution. Add 1% (wt/vol) (wt/vol) 1% Add solution. Tween-20the to of l natureprotocols I BSA ON

HCl is a strong acid and it should should it and acid strong a is HCl For 1liter, Tris dissolve 1.41gof base conjugated mix g at4°Cfor 2minand collectthe supernatant for use inStep5;the precipitate is µ l of conjugation buffer. ●

T BSA

IMI

N

● G

30 min–1 h T !

IMI

N G 2–4h Tween-20. Freshly prepare thisbuffer each time. Western blot washbuffer slowly. ! Tween anddissolve it. Freshly prepare thisbuffer each time. 1×PBS, 0.1%(vol/vol) BSAto the100mlof with milkor 5gof nonfat dry of (we BSA.with best find results Nestle or 5g of Carnation) Add either 5g theTween-20 of all until iswelldissolved. Weigh milk nonfat dry 5gof Tween-20dissolve 1mlof 1×PBS. in1,000mlof thesolution Stir thoroughly milkfor blocking. nonfat dry For BSAinsteaduse 5%(wt/vol) 1liter, of milk. nonfat5% (wt/vol) dry For on phospho-antibodies western blots, Western blot blocking buffer (WBBB) glycine-HCl (pH2.2)elution buffer. 10,a pHof andthefree hydroxyls the0.2M of aidinneutralization will  deionized water. Store thesolution atroom temperature for upto 1year. Tris base, 1M at room temperature. Filter-sterilize (through a0.45- deionized water.glycine in800mlof HCl.Adjust thesolution with thepHof Glycine-HCl, 0.2M(pH2.2)elution buffer 10 mMTris is1year atroom temperature. using HCl. Make thesolution upthevolume to life of 1,000ml. of shelf The

CAUT

CR I T I I ON CAL Tween-20 into very thepipette anditrises tip viscous isvery Do not adjust the pH of thissolution. at be notadjust thepHof Do solution The will

For 1liter, Tris dissolve 121.2gof basein1,000mlof

This bufferThis contains 1×PBSand0.1%(vol/vol) µ m filter) thesolution for long-term storage

This bufferThis contains 1×PBS-Tand

For 1liter, dissolve 15.04gof

© 2014 Nature America, Inc. All rights reserved. 33| to use.  32| and add 90mlof 10mM HCl and allowittodrip through withgravity. 31| 30| 29| 28| 27| conical tube. 26| C 9.0. Ifthe pHislower, add more coupling bufferuntil the pHisat9.0.Store the solution at4°Cuntil use. the dialyzed peptide-BSA complex and check the pHof the solution withpHpapertoensure thatthe mixture isatapHof 25| 24| 23| Dialysis of peptide- ? peptide-BSA complex runs atahigher molecular weight ( 22| see ref. 21| 20| 19| 18| A essential toknow the protein concentration permlfor subsequent antibody purification step,Step34. for protein detection 17| the fractions withapositiveODvalue. 16| 15| from Step12. 14| pipette and save itinanEppendorf tube for processing atStep40. oupling non-phosphopeptide and oupling non-phosphopeptide phosphopeptide-

ssessing ssessing the efficiency of peptide- TROUBLES

CR Set up a Bio-Rad Econo-Pac column by breaking off the bottom and attaching a small stopcock provided with the columns. Closethe stopcock atthe bottombefore the lastof the bufferdrips through. Remove 10 Add 10mlof coupling buffer tothe columnand letitdrip through. Add the swollenCNBr-Sepharose resin (from Step28)tothe columnand keep the stopcockclosed. Openthe stopcock Add 10mlof 10mMHCl tothe columnand letitdrip through bygravity; repeat three times. Mixitonaninvertrotator atroom temperature for 60minatroom temperature. Add 7mlof 10mMHClto the CNBr-Sepharose powder. Weigh out0.3gof CNBr-Sepharose for a1-mlbed volume column( The next morning, remove the liquid from the Slide-a-Lyzer; there should beno precipitate formation. Remove 5 Placethe cassettein4-literbeaker, withstirbar, containing coupling bufferand dialyze itovernight at4°C. Add the peptide-BSA complex (maximum of 5mlvolume) tothe 10-kDaSlide-a-Lyzer cassettes. Stainthe gel for protein visualization withCoomassie Brilliant Blue(for more details, seeref. Loadthe entire amount ona10%(wt/vol)SDS-PAGE gel and runitwithaprotein marker at80V(for more details, Centrifuge the mixture at8,000 Boilthe mixture at100°Cfor 1mininaboiling waterbath. Add 10 Determine the peptide-BSA concentration either byusing the 280-nmwavelength and the Warburg-Christian method Measure the absorbance of eachof the fractions at280nmtoassessfor the presence of peptide-BSA mix.Combine all RepeatStep14nine additional times tocollectthe tenfractions. Add 500 I T I 8 CAL 2 ). µ

H STEP µ l of 2×SDSsamplebufferto 10 OOT l of the purification buffertothe center of the column(ontop)and collectthe fraction inone of the tubes This step activates the resin, and thus it should only be carried out when the peptide-BSA solution is ready I N G BSA 8 0 , the BCAprotein quantification method complex into coupling buffer for column preparation g at22°Cfor 2minand letitsitatroom temperature for 5min. BSA conjugation µ l of peptide-BSA mixfrom Step16. BSA ● Fig.

T to IMI 4 S 8 ) compared withthe unconjugated BSA. N 1 epharose orany preferred method of protein quantification. It is G 5–6 h T able ●

T IMI 3 , row 1),inadisposable15-mlpolypropylene N ● G

1 d T IMI natureprotocols N G 16 h at 4 ° µ l of the Sepharose witha 8 2 ). The conjugated

| C VOL.9 NO.2VOL.9

protocol | 2014 µ l of

|

385 © 2014 Nature America, Inc. All rights reserved. 52| 51| 50| 49| remove small particulate matter byusing a0.45- 48| 47| S small amount of buffer behind. ! up to3months at4°C. 46| 45| 44| 43| 42| ? to protein beadswillnot reveal any protein bands on the gel. The Sepharose conjugatedtothe peptide-BSA complex willreveal protein bands onthe gel, whereas Sepharose unconjugated complex tothe Sepharose beads. Stainthe gel with Coomassie Brilliant Bluefor visualization of conjugatedpeptide-BSA. 41| add 2×SDSsamplebufferand boilthe samplesfor 1minat100°Cinaboiling waterbath. 40| the flow-through for assessment of coupling efficiency between the peptide-BSA mix and the Sepharose inStep40. 39| 38| lower capacity for binding to the antibodies. column. In this case, drying out will cause less peptide-BSA to be coupled to the column, resulting in a column that has a ensure that the resin does not stick to the side of the column and dry out by adding more of the buffer used to load the top should also be sealed with a Parafilm wrap to prevent inadvertent loss of liquid. Additionally, care should be taken to ! 37| 36| 35| more than2mg of the peptide-BSA mixwith1mlof resin. 34| 386 53| protocol

ubtracting ubtracting the antibodies fromnon-phosphopeptide the crude serum

CAUT CAUT TROUBLES

Capand Parafilm-wrap the column. Closethe stopcockand add the dilutedserumtothe column,and ensure that there isnot much space ontop. Add 20mlof 100mMNaCl tothe columnand letitdrip bygravity, and discard the flow-through. Remove the non-phosphopeptide columnfrom 4°C. Dilutethe serum1:10withchilled100mMNaCl. Keep itonice. Remove large debris byspinning the seruminaclinical centrifuge ormicrocentrifuge at4°C2,000 Thaw1–2mlof crude immune serum(for a1-mlcolumn)onice. Add 0.2%(wt/vol)sodiumazide in4mlof blocking buffertothe columnfor future storage. The columncanbestored Add 10mlof blocking buffertothe column.Letitdrip through, and then discard it.Repeatthisfivetimes. RepeatSteps42and 43five more times. Add 10mlof acidic wash buffertothe column.Let itdrip through, and then discard it. Add 10mlof alkaline wash buffertothe columnfrom Step39.Letitdrip through, and then discard it. Resolvethe samplesfrom Step40onanSDS-PAGE (asinref. Take 10 Remove the stopcockatthe bottomand the capatthe topand allowthe peptide-BSA todrip through. Save100 Continue mixing of the contents of the columnat4°Covernight onarotating shaker ordrum. Gently invert-rotate the columnwiththe resin, peptide-BSA mixture and the bufferatroom temperature for 1h. Capthe topof the column. Fillupthe columnwithcoupling buffer. Add the dialyzed peptide-BSA mixture from Step25tothe column.Giventhe capacityof the CNBr-Sepharose, useno | VOL.9 NO.2VOL.9 I I ON ON For all uses of the peptide columns, at no point should the column be allowed to completely dry. Always leave a In all cases in which columns are being inverted-rotated, the closed stopcock at the bottom and the cap on the H µ OOT l eachof the flow-through from Step39and the unconjugated Sepharose beadsfrom Step33. To each, | 2014 I N G | natureprotocols µ m syringe filter. 2 8 ) toassessthe extent of conjugation of the peptide-BSA ●

T IMI N G 1 d g for 3minand µ

l of

© 2014 Nature America, Inc. All rights reserved. 75| Note thatPVDFshould not beused. 74| Identifying fractions with IgG activity ? 73| on ice. RepeatSteps71and 7214more times. 72| 71| 70| E 69| 68| 67| 66| 65| S of the binding of the antibodies tothe phosphopeptide column. 64| column; collectthe flow-through. 63| 62| phosphopeptide column.Capand Parafilm-wrap the column. 61| 60| 59| 58| A 57| 56| phosphopeptide column. represents, at least in part, the antibodies that are specific to the phosphopeptide and that will next be passed over the  55| 54| luting luting the antibodies anti-phosphopeptide tringent washing of the column phosphopeptide

ffinity-purifying phosphopeptide antibodies phosphopeptide fromffinity-purifying fraction subtracted with the non-phospho column TROUBLES

CR Letthe spots dry completelyatroom temperature. Spot1 Test 1–2 Collectthe elution withthe appropriately marked tube, and mixitbyinversion toneutralize the sample. Placethe tube Add 1mlof elution buffer(pH2.2)tothe columnfrom Step69. To 151.5-mlEppendorf tubes, add 100 RepeatSteps67and 68three additional times. Add 10mlof acidic wash buffer(pH4.0)tothe column. Letitdrip through, and then discard it. Add 10mlof alkaline wash buffer(pH9.5)tothe column.Letitdrip through, and then discard it. Repeatthisstepthree times. Add 10mlof 10mMTris (pH7.5)and 0.5MNaCl tothe columnand letitdrip through the column,and then discard it. Savethe flow-through from the phosphopeptide columnat4°C;if necessary, itcanbetestedto determine the efficacy The next morning, continue the incubations atroom temperature for 1hand then letthe flow-through drip through the Gently invert-rotate the columnonarotator/shaker ordrum overnight at4°C. Closethe stopcockand add the flow-through collectedfrom the non-phosphopeptide column(from Step57)tothe Add 20mlof 100mMNaCl tothe columnand letitdrip bygravity, and discard the flow-through. Letthe storage solution drip through the columnafteropening the stopcockand cap. Remove the phosphopeptide columnfrom 4°C. Combine the flow-throughs from Steps55and 56and placethem on ice at4°C. Add 5mlof 100mMNaCl tothe columnand collectthe flow-through. The next morning, collectthe flow-through from the column. Incubate the columnonarotator/shaker at4°Covernight, withgentle agitation. I T I CAL µ

H STEP l of neutralized eluate from eachfraction collected inStep72toastripof nitrocellulose paperinalinear array. µ OOT l of eachfraction withpH papertoverifyneutralization. The flow-through is the set of antibodies that did not bind to the non-phosphopeptide column, and it I N G ●

T µ IMI l of 1MTris base. Labelthe tubes1to15. ● N

T G IMI 2 h ● N

T G IMI 30 min–1 h N G 1 h natureprotocols

| VOL.9 NO.2VOL.9 ● protocol

T IMI | N 2014 G 1 d

|

387 © 2014 Nature America, Inc. All rights reserved. Fig. 94| T ?  (as areplacement for loading the dilutedserum). up toatotalvolume of 15mlbyadding 100mMNaCl before loading ontothe non-phosphopeptide columninStep52 (Steps 50–57; pass the pooledantibody fractions (Step82)overthe non-phosphopeptide columnfor anadditional round of subtraction 93| 92| 91| 90| 89| nitrocellulose membrane. 88| 87| 86| 85| 10 ng, 1ng and 0.1ng onto the same stripof nitrocellulose. 84| 83| T 82| ? 81| 80| rabbit anti-pCGH-1, it is an anti-rabbit IgG secondary antibody).  phosphatase (asneeded) for 30minatroom temperature. secondary antibody conjugatedtoHRPoralkaline 79| after eachwash. three times inWBBBfor 5mineach,discarding the liquid 78| platform rotator/shaker atroom temperature for 30min. 77| 76| 388 esting esting purified antibodies for phosphoprotein specificity esting esting purified antibodies for specificity phosphopeptide protocol

TROUBLES TROUBLES

CR CR

If,asexpected, the purifiedphospho-antibody preparation detects the phosphopeptide inthe dot blot(Step93; Ifthe purifiedphospho-antibody preparation has reactivity tothe non-phosphopeptide inthe dot blot( Proceed asdetailed inStep81. Wash the membrane three times inWBWBfor 5mineach. Discard the solution containing the dilutedprimary antibody. Incubate the membrane onarotator atroom temperature for 1h. Dilutethe pooled, neutralized phospho-antibody columneluate from Step721:1,000inWBBBand add 10mltothe Discard the blocking bufferand washthe membrane three times for 5mineachinWBWB. Incubate the membrane inWBBBfor 30minatroom temperature. Letthe spotsdry. Spot1 Dissolve10 Pool allthe fractions thatare positivefor IgG inStep81( Developitwiththe ECLreagent asperthe manufacturer’s instructions. Wash the membrane three times for 10mineachwithWBWB. Incubate the membrane inWBBBcontaining After30min,discard the WBBBand washthe membrane Add WBBBand placethe membrane onahorizontal Placethe membrane inaflatplastic dish. | VOL.9 NO.2VOL.9 5 I I ), then testit for specificity(i.e., effectiveness of removal of antibodies that recognize low-sequence-complexity T T I I CAL CAL µ

H H STEP STEP l eachof the non-phosphopeptide and the phosphopeptide from Step83,inserial dilutions of 1 OOT OOT Fig. 2 | 2014 µ Columns must be regenerated (Steps 104–110) before use in additional rounds of purification. The secondary antibody should be specific to species of the antibody being purified (in the case of the g I I N N µ G G , |

l natureprotocols −1 g → of the phosphopeptide and non-phosphopeptide, eachinsteriledeionized water. b ); the number of fractions pooledinStep82willdepend onthe efficiency of elution—make

● ● Fig.

positive fractions should be pooled and taken further. Positive signal for each fraction indicates that the fraction has IgG. All the with appropriate secondary antibody (anti-rabbit) and developed with ECL. column elution (Step 72) is spotted on nitrocellulose, dried and then probed anti-IgG reactivity. A volume of 1 affinity column, dot blotting is used to identify which fractions contain antibody. After elution and neutralization of the bound antibody from the Figure 7 T T IMI IMI 7 ). N N G

G | 1 d Dot blotting to identify fractions containing the antipeptide 3 h 7 6 2 1 µ l of each fraction from the phosphopeptide 4 3 9 8 5 10 Fig. µ g, 100ng, 5 ), then

© 2014 Nature America, Inc. All rights reserved. Parafilm and store itat4°C. 110| 109| 108| 107| 106| 105| 104| R and re-purify the antibody. 103| 102| (Steps 70–82). 101| 100| 99| 98| P reduced signal afterRNAi knockdown (asdescribed inrefs. The phosphoprotein-specific antibody should show no signal abovebackground inthe kinase-null mutants orastrongly is todetermine whether the signal from the purifiedantibody isspecificto the kinase that phosphorylates the site, if known. should undergo anadditional round of affinitypurification ( signal. Ifinappropriate bands are observedinawesternblotof agene product–null mutant sample, then the antibody antibody displaysno signal abovethe background ingene product–null mutants, ortoverifythatitshows astrongly reduced RNAi knockdown followed bywesternblotanalysis and immunohistochemistry toverifythatthe phosphoprotein-specific antibody isspecifictothe gene product under study. Carryouteither null mutant analysis (as described in refs. 97| Genetic tests for gene phospho-antibody product specificity and kinase specificity column inStep52(asareplacement for loading the dilutedserum);continue toStep82again. fractions from Step82,adjusting the volume upto15mlwith100mMNaCl and loading onto the non-phosphopeptide non-phosphopeptide and affinitypurification against the phosphopeptide (repeat Steps50–82; whole-cell extract westernblotfrom Step94,then re-purify the phospho-antibody, i.e., perform subtraction against the 96| column inStep61(asareplacement for loading the flow-through); continue toStep82again. pooling fractions from Step82,adjusting the volume upto15mlwith100mMNaCl and loading onto the phosphopeptide tion overthe phosphopeptide columnagainbyrepeating the stringent washes and elution (Steps58–82; 95| phosphorylation event isknown orpredicted tooccur(asdescribed inrefs. phospho-epitopes ( urifying non-phosphoprotein-specific antibodies urifying non-phosphoprotein-specific egenerating egenerating the peptide columns Prepare crude serum as described in Steps 47–49, remove the phosphopeptide column from 4 °C and perform Steps 51–56. Akey testfor specificity of aphosphoprotein antibody preparation istoassesswhether the signal from the purified Ifthe first-round antibodies detect non-phosphopeptide inthe dot blotfrom Step93and have multiple bands onthe Collectthe flow-through from the phosphopeptide columnand add ittothe non-phosphopeptide column. Ifnonspecific bands are detected inthe westernblotfrom Step94( Assess the specificity of the non-phosphopeptide antibody via dot blots(Steps83–92)and westernblotting (Step94). Eluteand neutralize the non-phosphopeptide-specific antibody and identify antibody-containing fractions as described Perform affinitypurification withstringent washes according toSteps58–69. Add 0.2%(wt/vol)sodium azide in4mlof blocking buffertothe column,closethe stopcock,capit,wrap itwith Fillthe columnwith10mMTris (pH7.5)and 0.5MNaCl. Add 10mlof 10mMTris (pH7.5)and 0.5MNaCl tothe columnand letitdrip through, then and discard it. RepeatStep106once more. Add 2×10mlof 10mM Tris (pH7.5)tothe columnand letitdrip through, and then discard it. Add 2×10mlof 10mM Tris (pH8.8)tothe columnand letitdrip through, and then discard it. Add 2×10mlof 100mM glycine (pH2.2;elution buffer)tothe columnand letitdrip through, and then discard it. Ifthe elutednon-phosphopeptide antibody preparation detects the phosphopeptide, then follow Steps98–101again T able 1 )), byusing westernblotsprepared from totalextracts of cellsororganisms inwhich the ●

T IMI N G 30 min ●

T IMI 27,2 Fig. N G 8 2 d 2 ; , Figs. 1a h → d ; repeat Steps58–82).Anadditional specificitytest Fig. and 27,2 3 ), purifythe phospho-antibody prepara

6 8 ). ; Fig. natureprotocols 3 ● ).

T IMI N Fig. G 1 d 2 , h

| → VOL.9 NO.2VOL.9 Fig. b ), bypooling protocol 2 , 27,2 h → | 2014

8 d - ) or ) by

|

389 © 2014 Nature America, Inc. All rights reserved. stopcock. 134| 133| 132| 131| P 130| 129| ! 128| 127| 126| 125| 124| 123| 122| 121| 120| 119| 118| used for coupling to1mlof Sepharose. 117| P concentrated dialyzed antibody for long-term storage. 116| 115| 114| the membrane, usually 100–400 113| 112| 111| C 390

protocol oncentrating oncentrating purified polyclonal antibody and storage urification urification of antibodies from crude antisera to the fusion protein urifying polyclonal antibodies to fusion proteins; making the columns with recombinant protein CAUT

| VOL.9 NO.2VOL.9 Add 10mlof coupling buffertothe columnand let itdrip through. The next morning, atroom temperature, letthe protein solution drip through from the column. Incubate the columnwiththe protein and Sepharose at4°Covernight onarotating shaker. Perform Steps35and 36. Add 1mlof the recombinant protein solution from Step117tothe columnfrom Step121. Add 5mlof coupling buffertothe columnand let itdrip through. Add 20mlof 10mMHCltothe columnand letitdrip through. Add the swollenresin toaBio-Rad Econo-Pac columnasdescribed inSteps29and 30. Perform Steps26–28. Dialyze 500 Add toafinal concentration of 2.5%(wt/vol)BSA, 0.01%(vol/vol) Tween-20 and 25%(vol/vol)glycerol tothe Dialyze the antibody into 1×PBSovernight at4°Cbyusing the Dialyzer cassettes. Determine the antibody concentration byusing aspectrophotometer. Withextreme care and without disturbing the membrane draw outthe reduced, concentrated antibody from the topof Spinthe tubesat4,000 Add 10mlof pooledpurifiedphospho-antibody from Step82toa10-kDaAmicon concentrator (Millipore). Add the dilutedcrude serumfrom Step132tothe column,and care should betaken toclosethe columnwiththe Remove the affinitycolumn (from Step130)from 4°Cand prewash the columnwith20ml of 100mM NaCl. Perform Steps48and 49. Thaw1–2mlof crude immune serumfrom −70°C. Perform Step46for columnstorage and future use. Add 50mlof blocking buffertothe column,and letitdrip through. Perform Steps65–69. Add 10mlof columnwashbuffertothe columnand letitdrip through. I ON Do not allow the solution to collect in the column at any point by plugging the column. Allow for a steady flow rate. | 2014 µ g ml | natureprotocols −1 of purified recombinant protein into coupling buffer. Typically, 500 g for 45mininahigh-speed centrifuge at4°C. µ l of antibody-containing solution. ●

T IMI N G 1 h concentration, 16 h dialysis ●

T IMI N G 1 d µ g mg −1 of afusion protein is ●

T IMI N G 2 d

© 2014 Nature America, Inc. All rights reserved. T Troubleshooting advice canbefound in ? at −80°C.These antibodies canbestored for several yearsat−80°C. 142| affinity purification, stringent washes and elution as describedinSteps58–73. 141| 140| ? 139| 138| 137| 136| 135| able able 22 (INTRODUCTION) clonal antisera custom poly Generation of S

TROUBLES TROUBLES tep Store the concentrated antibodies in2.5%(wt/vol)BSA, 0.01%(vol/vol)Tween-20 and 25%(vol/vol)glycerol Perform westernblotting tototalprotein (and genetic specificitytests)asinSteps94–97.If not specific, repeat Regenerate the columnasdescribed inSteps104–110. Elutebound antibodies asdescribed inSteps70–73. Perform stringent washes asinSteps65–69. Add 4×20mlof 10mMTris (pH7.5)and 0.5MNaCl tothe columnand letitdrip through, and then discard it. The next morning, openthe stopcockand the capand letthe solution drip through, and then discard it. Perform Steps53and 54. 4 4 |

Troubleshooting table. - H H OOT OOT I I go to completion with peptide did not Conjugation of BSA phospho-antigen low titer against the The sera produced phospho-antibodies a very low number of phospho-antibodies or Peptide yielded no P N N roblem G G

T able dissolve dissolve efficiently The peptide did not is used in the conjugation dissolved or if a wrong solution peptide and/or BSA is not fully peptide used is incorrect, if the can occur if the quantity of Inefficient or failed conjugation used was low complexity The region of the peptide that was used for conjugation had an internal cysteine The peptide designed P ossible reason 4 .

dialyzed samples there is no precipitate left. Pool all the with 10% (wt/vol) DMSO and repeat until re-dissolve this in fresh conjugation buffer, Spin out what has not dissolved. Then Add DMSO to the peptide (at 10% wt/vol). completely Ensure that the peptide is dissolved purification same volume of the column (Step 5 on) for Or, use five- to-tenfold more sera with the than one animal species may be useful. results in titer generation. Thus, using more species of animals sometimes give different Anecdotally, we have found that different not form internal disulfide bonds vendor in a reduced state, such that they do (11–14-mer) and obtaining them from the interest by making slightly longer peptides chances of obtaining an antibody of the phospho-epitope, there are better when there may be a cysteine right next to difficult, if not impossible to resolve. Thus, and result in antibodies that will be very tion, as that will mask the phospho-epitope conjugate the peptide to hapten for injec An internal cysteine should not be used to S olution natureprotocols

| VOL.9 NO.2VOL.9 protocol ( | 2014 continued

|

- 391 ) © 2014 Nature America, Inc. All rights reserved. Steps Steps 7–17, desalting the peptide-BSA conjugated mix: 30 min–1 h Steps 1–6, conjugating peptides to maleimide-activated BSA: 2–4 h The total time for the purification procedure from start to finish is 5 d and 7 h, owing to multiple overnight incubation steps. ● T 392 Steps 18–22, assessing the efficiency of peptide-BSA conjugation: 5–6 h able able 81, 81, 93 73, 139 22, 41 22 S 93 protocol

tep T

IMI | VOL.9 NO.2VOL.9 4 4 N | G

Troubleshooting table (continued). | 2014 non-phosphopeptide also recognizes the antibody preparation The phosphopeptide peptide column from the phospho- fractions upon elution There are no IgG is not neutral glycine-eluted antibodies The pH of the collected with Sepharose not in the right ratio go to completion/was BSA complex did not Conjugation of peptide- P roblem | natureprotocols

Refer Refer to not optimal temperature and the reagent is ECL kit used was left out at room blotting is old and does not work The secondary antibody used for column during antibody elution phosphopeptides coming off the wash buffer, resulting in some preparation with the coupling buffer and washed thoroughly and coupled during The phosphopeptide column was not binding to the phosphopeptide column phosphopeptide column was used for column, the elution from the non- from the non-phosphopeptide Instead of using the flow through Peptide design was flawed titrated to a pH of 8 The 1 M Tris used for neutralization was the column and Sepharose mix dried in The peptide-BSA complex Sepharose beads was in excess to the The peptide-BSA complex at room temperature not allowed to continue for 2 h The conjugation reaction was Conjugation buffer has precipitates used to dissolve the peptide Conjugation buffer was not P immunogen generated from the phosphopeptide for the types of antibodies that can be ossible reason Figure 1 for possible options

Use 1 M Tris that has not been titrated or pH- such that the beads do not dry out Ensure that there is enough coupling buffer 1 mg of Sepharose added in a ratio of 2 mg of the complex to Ensure that the peptide-BSA complex is room temperature for 2 h Incubate the peptide-BSA mix at too long and ensure that it has not been at 4 °C for Check the date on which the kit was bought the kit Use the conjugation buffer supplied with S phosphopeptide column flow-through that is then passed over the phosphopeptide column and collect the Pass the antibody back over the non- functioning the ECL reagent is optimally Use a positive control sample to ensure that the secondary antibody works Use a positive control sample to ensure that protocol with care taken to follow each step of the Prepare the phosphopeptide column again, column to pass over the phosphopeptide non-phosphopeptide column is the one used Ensure that the flow-through from the antisera (INTRODUCTION) Refer to generation of custom polyclonal neutralize the strong acidic glycine solution adjusted. This allows for the hydroxyl ions to olution

© 2014 Nature America, Inc. All rights reserved. contain activated MPK-1, one of a number of criteria that indicate that the rounds of affinity purification. pCGH-1 is only observed in germ cells that (pCGH-1 in green, bottom), by using the antibody preparation after four activated ERK C. elegans germ-line tissue. Immunocytochemical staining of a wild-type adult Figure 8 phosphorylated CGH-1,whilestillpresent inthe cytoplasm, trafficking, translation and stability. We find thatMPK-1/ERK granules thatare thought tofunction inregulation of mRNA as onperinuclear Pgranules unique attributesof amodification-specific antibody reagent. TotalCGH-1is present in the cytoplasm of germcells, aswell Intriguingly, phosphorylation of CGH-1altersthe subcellularlocalization of the protein ( that contain activatedMPK-1/ERK ( germ-line tissueshows thatthere iscompleteoverlapbetweenthe cellsthatstainwithpurifiedpCGH-1antibody and those event required MPK-1/ERK, asthe pCGH-1band was not detected inthe preparation wasspecifictoCGH-1,as no signal was detected from type adult hermaphrodite lysates( were required toremove antibodies thatreacted withproteins other thanpCGH-1,asassessedbywesternblotsof wild- to remove antibodies thatbound tothe unmodified peptide. Four rounds of affinitypurification withstringent washes usually necessary. As shown bydot blotting ( To generate modified protein–specific polyclonal antibodies, multiple rounds ofsubtraction and affinitypurification are ANT Steps 131–142, purifying antibodies from crude antisera to the fusion protein: 1 d Steps 117–130, purifying polyclonal antibodies to fusion proteins; making the columns with recombinant protein: 2 d antibody Steps 111–116, concentrating purified polyclonal antibody and storage: 1 h for concentration and 16 h for dialyzing the Steps 104–110, regenerating the peptide columns: 30 min Steps 98–103, purifying non-phosphoprotein-specific antibodies: 2 d Step 97, genetic tests for phospho-antibody gene product specificity and kinase specificity: 1 d Steps 94–96, testing purified antibodies for phosphoprotein specificity: 1 d Steps 83–93, testing purified antibodies for phosphopeptide specificity: 3 h Steps 74–82, identifying fractions with IgG activity: 2 h Steps 70–73, eluting the anti-phosphopeptide antibodies: 30 min to 1 h Steps 65–69, stringent washing of the phosphopeptide column: 1 h Steps 58–64, affinity-purifying phosphopeptide antibodies from the fraction subtracted with the non-phospho column: 1 d Steps 47–57, subtracting the non-phosphopeptide antibodies from the crude serum: 1 d Steps 26–46, coupling non-phosphopeptide and phosphopeptide-BSA to Sepharose: 1 d Steps 23–25, dialysis of peptide-BSA complex into coupling buffer: 16 h at 4 °C (*) (*) indicate the distal end of the germline. Scale bars, 20 affinity-purified pCGH-1 preparation is specific ( WT * I * * C Mitosis Mitosis Mitosis I

PATE | hermaphrodite germ line for DNA (DAPI in blue, top), anti- Staining for pCGH-1 and activated MPK-1 ERK overlap in the 6 7 D (dpMPK-1 in red, middle) and anti-phospho-CGH-1 RESULTS Pachytene 19,83,8 Pachytene Pachytene 4 Fig. , which are germ-line Fig. Figs. 3 3 8 ). Genetic testsusing westernblots( ), asexpected, giventhatERKisthe kinase thatphosphorylates CGH-1onSer67. Oocytes Oocytes Oocytes and Fig. µ

m. m. 6 ). ). Asterisks 5 ), tworounds of subtraction for the anti-pCGH-1 antibody were dpMPK- pCGH-1

DAPI

1 mutant. Scale bars, 40 phosphorylated Ser67 and thus not expected to be phosphorylated in the is a missense mutation in the proline (Pro68Leu) that is C terminal to antibody is specific to CGH-1 and fails to stain specific to pCGH-1 fails to stain whereas the cytoplasmic and weak nuclear staining remains. The antibody contrast, pCGH-1–specific staining ( surround nuclei, as well as cytoplasmic and weaker nuclear staining. In CGH-1–specific staining ( proximal pachytene region that contains activated MPK-1 ERK ( and purified anti-pCGH-1 antibody ( ( Figure 9 a – cgh-1(tn691ts) c Wild typ a cgh-1 d cgh-1(0) ) ) Immunocytochemical staining of anti-total CGH-1 antibody (

e | –null mutant lysates, and thatthe phosphorylation mpk-1 Phosphorylation of CGH-1 alters protein subcellular localization. ( d ) ) mutant adult germ lines, shown as an enlargements of the Fig. –null mutant. Immunocytochemistry of whole Total CGH- 6 µ ) confirmed thatthe purified antibody m. pCGH-1 a ) highlights P granules (arrowheads) that natureprotocols 1 Fig. b cgh-1(0) – d W cgh-1(0) d b ) ) to wild type ( ild type b 9 ) does not highlight P granules, ), illustrating one of the ( d ), indicating that the cgh-1(tn691ts)

| VOL.9 NO.2VOL.9 a , b ), ), cgh-1(tn691ts) protocol (

necessary Fig. Fig. | c 2014 ), which pCGH-1 pCGH-1 a 8 ) ) and ). ). Total

| (

393 c )

© 2014 Nature America, Inc. All rights reserved. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. com/reprints/index.h at online available is information permissions and Reprints interests. CO protocol. AUT comments on the manuscript. (University of Minnesota) and C. Spike (University of Minnesota) for extensive Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)), D. Greenstein of Texas M.D. Anderson Cancer Center. We thank A. Golden (NIH/National Anderson Cancer Center and the Center for Genetics and Genomics, The University a Cancer Center Supplemental Grant (CCSG) to The University of Texas M.D. was supported by NIH grant no. GM98200, an Institutional Research Grant from of Health (NIH) grant no. GM085150. Work in S.A.’s laboratory on this project by National Science Foundation grant no. 0416502 and US National Institutes A from either fusion proteins orpeptides. lished data) and totalprotein–specific antibodies for three proteins (ref protocol isrobust, aswehavepurifiedphosphoprotein-specific antibodies for14 proteins (S.A.,A.Golden and T.S.,unpub is apparently absent from Pgranules ( 394 protocol cknowle

M

H

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