ClaireWalczakJanuary1996

ThesearebasicallyTim"sproceduresandhavebeenusedsuccessfullybynumerousmembersofourlabandbyothersaroundus.

A.CheckingPeptideThiolGroups

Dothisbeforethiolcoupling,eithertoKLHortoresin.Peptidethiolgroupshaveatendencytogetlostaftersynthesis.

1. Makeup5mMEllman"sreagent(dithio-bis-2-nitrobenzoicacid)in0.1MNaPipH7.2.
2. Weighoutabout1mgofpeptideintoataredtube.
3. Add0.5mlreagent.Itshouldgobrightyellow.
4. Dilutethemixture1/50inbuffer.ReadA412againstreagentatthesameconcentration.
5. Calculatetheapparentmolecularweightofthepeptidebasedonthiolgroups,usingamolarextinctioncoefficientof14,000.Comparethistotheexpectedmolecularweightofthepeptide.Theyshouldagreewithinafactorofthree,withtheapparentmolecularweightusuallyhigher.Ifthethiolconcentrationisanomalouslylow,i.e.,theapparentmolecularweightisveryhigh,theremaybesomethingwrongwiththepeptide-anywayitprobablywillnotcouplewell.YoumayneedtoregeneratethethiolgroupsbyreducingthepeptidewithexcessDTTandrunningaP2column.

B.CouplingofPeptidetoKLH

Thisrecipeisfortwobunniesforaboutfiveinjectionsperbunny

1. Weighout100mgofkeyholelimpethemocyanin(KLH).Dissolvein2mlwater.Itgenerallytakesabout4hourstodissolve-youwillneedtosonicateandvortex.Bepatientandputonarotatorat4degC.Dialyzeagainst2lof0.1MNaPhoshatepH7.8overnight.Thisistoremoveanycontaminatingthiolsoraminocompounds.
2. Spin10minutesatfullspeedinmicrofugetoremoveaggregates(don"tbesurprisedtoseeasubstantialpellet).
3. SplittheKLHinto2aliqoutsfor-SHand-NH2coupling.
4. For-NH2coupling,add5mgpeptidetoonealiquot,followedbyglutaraldehydeto0.1%final.Addthepeptideasasolidifitissoluble,otherwisefroma100mg/mlstockinDMSO.Precipitationdoesnotmatterandoftenhappens.Afteraddingtheglut,checkthepHwithpHpaper,andadjustto7.8ifnecessaryusingNaOH.Incubate8-12hrsat4degrees,rotatinggently.
5. AddatinypinchofNaBH4tokillremainingglut.Makesurethesampleisinalargetubesinceittendstofizzup.Incubate8-12hrsat4degrees.Thisistheglutconjugate.
6. Forthe-SHcoupling,warmtheotheraliquotofKLHtoroomtemp.Add1/9thvolumeofIodoaceticacidN-hydroxysuccinimideesterat100mg/mlinDMSO.MaketheDMSOstockfresh,andprotecttheiodoacetamidereagentfromlight.WemakeourownIAA-NHSester,butitcanbepurchasedfromSigma.
7. After10minutesatroomtemptheKLHwillstarttogetalittlecloudy.LoaditontoaP-10columnequilibratedwith0.1MNaPhosphatepH7.8.Makesurethecolumnisatleast10timesthevolumeofthesample.PooltheKLHcontainingfractionsbycolor(itwillbesortofgreyishgreen).Add5mgofpeptidetothem,asinstep5above.Incubateatleast8hrsat4degrees,rotatinggently.
8. Poolthecoupledpeptidefromthetwoproceedures.Diluteto5mlwith0.15MNaCl.Ifthereisaprecipitate,sonicatevigorouslytobreakitup.Splittheimmunogeninto1mlaliquots(eachaliquotwillimmunizetworabbits)andfreezeit.

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