You"llneedtocouplethesyntheticpeptidehaptentoaproteincarrier,withabifunctionalreagent. Forantibodydevelopment,wedon"tbothertousehighlypurifiedpeptide.Crude(unpurified)peptideorpartiallypurifiedpeptidedoesjustfine;afterall,theimpuritiesinunpurifiedpeptidesareprincipallyincomplete,partialversionsofthedesiredpeptide.Sinceyouneedtousequiteabitofpeptidetogetgoodcouplingandantibodyproduction(10sofmg),it"sprobablymosteconomicaltouseunpurifiedsyntheticpeptides. Themostusefulcouplingprotocolsarein:RFDoolittle.OfUrfsandOrfs.APrimeronHowtoAnalyzeDerviedAminoAcidSequences.UniversityScienceBooks,1987. Goodcarriersincludebovineserumalbumin,orkeyholelimpethemocyanin(ourfavorite,fromCalbiochem#374817). Goodcouplingmethodsareglutaraldehyde(aminotoamino),MBS(=m-Maleimidobenzoicacid-N-hydroxysuccinimide;aminotosulfhydryl),BDB(bisdiazobenzidine;couplestyrtotyr),EDAC(carbodiimide;couplesaminotocarboxyl). Weoftenaddanextra("adventitious")tyrtotheN-orC-terminusofthesyntheticpeptide,forBDBcoupling.We"vedonethismoreoftenthannot.Trytoputtheextratyrattheleast"interesting"endofthepeptide,sinceitwillbeattached(buried)tothecarier,andhenceyou"lltendtogetantibodytothemostinterestingendofthepeptide(awayfromthecarrier). It"susefultohaveaverylowspecificactivity(say,10,000cpm/mgpeptide)14Clabelinaglyoralaresidueinthepeptide.Youcanthenfollowcouplingefficiencyofthepeptideintothecarrier. WetypicallyimmunizerabbitsbytheVaitukaitisprotocol,withmultipleintradermalimmunizationsatonce.Weshavetheupperbackoftherabbitpriortoimmunization.Wetendtouse1mgofpeptideperimmunization(3-5intradermalsitesateachimmunization),andre-immunizeat2-6weekintervals.Intradermal(intracutaneous)means:raiseawheal(injectintotheskin,justammorsobeneeaththesurface).Aftertheinitialimmunizationand2-3boosts,webleed3-4weekslater,andtesttheantibodies,bywesternblot(immunoblot)orimmunoprecipitationof[125-I]-labeledantigen(eitherthepeptide,orthewholemoleculefromwhichthepeptidederives). Fortheinitialimmunization,wemixtheimmunogen(hapten-carrierconjugate)withanequalvolumeofcompleteFreund"sadjuvant.Forboosts,weuseincompleteFreund"sadjuvant.Mixingtheoilyadjuvantwiththeaqueousconjugateisbestdonewithamixingneedleandtwoattachedsyringes.MixingresultsinasUSPension. Bythiskindofprotocol,weALWAYSobtainsyntheticpeptideantibodieswhichrecognizetheantigen(intactfulllengthmolecule,fromwhichthepeptidewasderived),especiallyonimmunoblot.Sometimestheepitopeisweak,andtheantobodiesareoflowtiter(e.g.,1:100forimmunoblot).Butwealwaysgetausableantibody. Citationswherewe"veusedtheseprocedures: BarbosaJA,GillBM,TakiyyuddinMA,O"ConnorDT:ChromograninA:posttranslationalmodificationsinsecretorygranules.Endocrinology128:174-190,1991.Here,weusedantibodiestochromograninAsyntheticpeptidestoexaminerouteofproteolysis(post-translationalprocessing)ofthemolecule. GillBM,BarbosaJA,DinhTQ,GarrodS,O"ConnorDT:ChromograninB:isolationfrompheochromocytoma,N-terminalsequence,tissuedistributionandsecretoryvesicleprocessing.RegulPeptides33:223-35,1991.Here,weusedantibodiestochromograninBsyntheticpeptidestoexaminerouteofproteolysisofthemolecule. GillBM,BarbosaJA,Hogue-AngelettiR,VarkiN,O"ConnorDT:ChromograninAepitopes:cluesfromsyntheticpeptidesandpeptidemapping.Neuropeptides21:105-18,1992.Here,weusedantibodiestochromograninAsyntheticpeptidestoimmunoprecipitatethemolecule. TakiyyuddinMA,DeNicolaL,GabbaiFB,DinhTQ,KennedyB,ZieglerMG,SabbanEL,ParmerRJ,O"ConnorDT:Catecholaminesecretoryvesicles.Augmentedchromograninsandaminesinsecondaryhypertension.Hypertension21:674-9,1993.HerewedevelopedanRIAforchromograninA,basedonanN-terminalsyntheticpeptide.