GST-fusionproteininjectionintorabbits

TheGST-fusionproteinelutedfromtheGSH-sepharoseisdilutedandneedtobeconcentrated.Combineelutedfractions(El1+El2)in50mlfalcontube,freezeat-80oC,closewithpunctureplasticcap.Lyophilizeover-night.ResUSPendin0.5-1mlofDDW,spin,andtransfertoasmalltube.Storeat-20oC.

RabbitScreening

Itisimportanttoscreenpre-immuneserafromseveral(2-6)rabbitsonwesternblotscontainingplantproteinextract(soluble,andmembranepellet).Oftenpre-immuneseracross(at1:500dilution)reactsstronglywithplantproteins.

Preparationofproteinforantibodyproduction

Dependingontheantiginicityofyourfusionprotein,arangeof20-100mgoffusionproteinisrequiredforfirstinjection.

NOTE:WhenusingGST-fusedtosmallpeptide(10-20aa)use~250-500mgofprotein/injection.

Boost:Forsecondinjection(amonthlater5-20mgoffusionproteinisrequired/boostinjection).

Tworabbitsarebeingusedtogenerateantibodytothesameantigen.OnerabbitisinjectedwiththenativeformofGST-fusionprotein,andthesecondrabbitisinjectedwithSDS-reduceddenaturedformofGST-fusionprotein.

1.ForthenativeformtheGST-fusionproteinsample(normally100mgin0.5to1ml)ismixedwithequalvolumeadjuvant(seebelow).

2.ForthedenaturedformtheGST-fusionproteinsample(normally100mgin0.5to1ml)ismixedto1%SDS,5mMDTT,andincubatedfor15minat65oC,cooledandthenmixedwithadjutant(seebelow).Alternatively,thefusionproteincanbeseparatedonreducingSDS-PAGE,theproteiniselutedfromthegel,mixedwithadjuvantandinjectedtorabbits.

Atthedayofinjection

To0.5mlofproteinsample(nativeordenatureformed)add1mlofTiterMax,vortex.Addadditional0.5mlofproteinsolution.Withaallplastic(3ml)syringeequippedwith20gaugeneedlePipetteupanddownthecontentuntilitbecomeveryviscous.

Injectionintorabbit

Upto2mlofTiterMax:proteinsampleisinjected/rabbit.

Treatmentofpre-immuneandimmunesera

1.Beforeinjectiontotheselectedrabbit(itispreferredtoinjectsamplesinto2rabbits)collectbloodforpre-immune.

2.28daysfrominitialinjection,collectfirstbleed(~30ml),coolat4oCforseveralh.

Atthesamedayofcollectingbloodinjectaboostofyourprotein

3.Centrifugeat2,000gfor15minat4oC.Transfertheclearsupernatantintonewtube.

4.AddNa-azidefroma20%stocktofinal0.02%.Dividetheclearblood(5-15ml)into1mlaliquots,andfreeze-20oC.Keeponetubeat4oC(uptoseveralmonths)foranalysis.

5.28daysfromsecondinjection,injectanothersample,andcollectatthesamedaythesecondbleed(~30ml).Cool,spin,aliquotasinsteps3,4.

6.Ifthetiterislow,athird,andfourthinjectioncanbedone.Bloodiscollectedandseraisanalyzedseebelow.

7.Collecttheentirebloodfromtherabbit(~150-200ml=4thandfinalbleed).

Analyzeeachpre-immuneseraandvariousbleedsfrom1st,2nd,3rd,4that1:500to1:2000dilutioninTBST-5%milkonwesternblotscontaining50mgeachoftotal,solubleandmembranefractionfromplants(ArABIdopsis,Tobacco,Maize,Rice,etc.);and1mgofGST,andGST-fusionproteinsproducedinE.coli.

Antibodycanbefurtherpurifiedifneeded(SeeBar-PeledandRaikhel(1996),Anal.Biochem.)

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