免疫生物学系/组织移植与免疫研究中心
来自 : 蚂蚁淘
ThesearebasicallyTim'sproceduresandhavebeenusedsuccessfullybynumerousmembersofourlabandbyothersaroundus.
A.CheckingPeptideThiolGroups
Dothisbeforethiolcoupling,eithertoKLHortoresin.Peptidethiolgroupshaveatendencytogetlostaftersynthesis.
- Makeup5mMEllman'sreagent(dithio-bis-2-nitrobenzoicacid)in0.1MNaPipH7.2.
- Weighoutabout1mgofpeptideintoataredtube.
- Add0.5mlreagent.Itshouldgobrightyellow.
- Dilutethemixture1/50inbuffer.ReadA412againstreagentatthesameconcentration.
- Calculatetheapparentmolecularweightofthepeptidebasedonthiolgroups,usingamolarextinctioncoefficientof14,000.Comparethistotheexpectedmolecularweightofthepeptide.Theyshouldagreewithinafactorofthree,withtheapparentmolecularweightusuallyhigher.Ifthethiolconcentrationisanomalouslylow,i.e.,theapparentmolecularweightisveryhigh,theremaybesomethingwrongwiththepeptide-anywayitprobablywillnotcouplewell.YoumayneedtoregeneratethethiolgroupsbyreducingthepeptidewithexcessDTTandrunningaP2column.
B.CouplingofPeptidetoKLH
Thisrecipeisfortwobunniesforaboutfiveinjectionsperbunny
- Weighout100mgofkeyholelimpethemocyanin(KLH).Dissolvein2mlwater.Itgenerallytakesabout4hourstodissolve-youwillneedtosonicateandvortex.Bepatientandputonarotatorat4degC.Dialyzeagainst2lof0.1MNaPhoshatepH7.8overnight.Thisistoremoveanycontaminatingthiolsoraminocompounds.
- Spin10minutesatfullspeedinmicrofugetoremoveaggregates(don'tbesurprisedtoseeasubstantialpellet).
- SplittheKLHinto2aliqoutsfor-SHand-NH2coupling.
- For-NH2coupling,add5mgpeptidetoonealiquot,followedbyglutaraldehydeto0.1final.Addthepeptideasasolidifitissoluble,otherwisefroma100mg/mlstockinDMSO.Precipitationdoesnotmatterandoftenhappens.Afteraddingtheglut,checkthepHwithpHpaper,andadjustto7.8ifnecessaryusingNaOH.Incubate8-12hrsat4degrees,rotatinggently.
- AddatinypinchofNaBH4tokillremainingglut.Makesurethesampleisinalargetubesinceittendstofizzup.Incubate8-12hrsat4degrees.Thisistheglutconjugate.
- Forthe-SHcoupling,warmtheotheraliquotofKLHtoroomtemp.Add1/9thvolumeofIodoaceticacidN-hydroxysuccinimideesterat100mg/mlinDMSO.MaketheDMSOstockfresh,andprotecttheiodoacetamidereagentfromlight.WemakeourownIAA-NHSester,butitcanbepurchasedfromSigma.
- After10minutesatroomtemptheKLHwillstarttogetalittlecloudy.LoaditontoaP-10columnequilibratedwith0.1MNaPhosphatepH7.8.Makesurethecolumnisatleast10timesthevolumeofthesample.PooltheKLHcontainingfractionsbycolor(itwillbesortofgreyishgreen).Add5mgofpeptidetothem,asinstep5above.Incubateatleast8hrsat4degrees,rotatinggently.
- Poolthecoupledpeptidefromthetwoproceedures.Diluteto5mlwith0.15MNaCl.Ifthereisaprecipitate,sonicatevigorouslytobreakitup.Splittheimmunogeninto1mlaliquots(eachaliquotwillimmunizetworabbits)andfreezeit.
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