General Principles of Immunoprecipitation相关交流-蚂蚁淘在线

Lysisbuffer.

Thechoiceoflysisbufferdependsonwhatthecellswerelabeledwithandwhetheryouwanttoobtainanactivekinase.ThelowestbackgroundisobtainedwithRIPAbufferwithoutEDTA.EDTAincreasestheprecipitationofbothactinandmyosingreatly.Sinceactinisnotphosphorylated,thisdoesn"tmattermuchfor32P-labeledsamples.RIPAishowevermoredenaturingthanNP40bufferandshouldnotbeusedforfragileenzymes.

32Pi-labeledcellsandisolationofproteinkinases.

RIPAbuffercontainingphosphatebufferandsupplementedwith100to200µMsodiumvanadate,50mMsodiumfluorideand2mMEDTAshouldbeusedwithcellswhichhavebeenlabeledwith32Pi.Itisalsothechoiceforcellsfromwhichyouwanttoobtainproteinswithproteinkinaseactivity,providedthekinaseisstable.TheEDTAshouldchelatealltheMg++inthecellsandpreventphosphorylation.Phosphorylationcanoccurfollowingcelllysis,withthecellularpoolofATPservingasthephosphatedonor.Thevanadateshouldinhibitmosttyrosineproteinphosphatasesandthefluorideshouldinhibitsomeserine/threoninespecificproteinphosphatases.Whethertheadditivesneedtobepresentinallthewashesisn"tknown.Theymaybecompletelydispensableafterthefirstspin.Tamarahasfoundthatthevanadateworksmuchmoreeffectivelyifitisaddedtothelysisbufferfreshly!Phosphate-bufferedRIPAisthebestchoice.PhosphateisagoodbufferatpH7.2andalsofunctionsasaninhibitorofphosphatases.RIPAis:1%NP-40orTritonX-1001%sodiumdeoxycholate0.1%SDS0.15molarNaCl0.01molarsodiumphosphate,pH7.21%Trasylol,a1:100dilutionofwhatcomesfromMobayIfTrisissubstitutedforphosphate,use50mmolarTris-HCl,pH7.2.Addsodiumfluoride,EDTAand/orvanadateasnecessary.Addthevanadatefreshlyeachtimeyouuseit.

NP40bufferisournameforRIPAlackingDOCandSDS.ThislysisbufferpreserveskinaseactivitybetterthanRIPA,butgivesahigherbackgroundintheimmunoprecipitate.NP40buffercanbemadewitheitherNP40orTritonX-100,andcanbebufferedwitheitherphosphateorTris,dependingonyourneeds.NP40bufferis:1%NP-40orTritonX-1000.15molarNaCl0.01molarsodiumphosphate,pH7.21%Trasylol,a1:100dilutionofwhatcomesfromMobayIfTrisissubstitutedforphosphate,use50mmolarTris-HCl,pH7.2.Addsodiumfluoride,EDTAand/orvanadateasnecessary.Addthevanadatefreshlyeachtimeyouuseit.

PBS,inthecontextofimmunoprecipitation,is:0.15molarNaCl0.01molarsodiumphosphate,pH7.2ThePBSusedintissuecultureisamorecomplexphysiologicalphosphate-bufferedsalinewhichcontainscalcium,magnesium,andpotassium

TNis:0.15molarNaCl0.05molarTris-HCl,pH7.2

CD4buffer.ChrisRuddusedavariantofNP-40bufferforhisearlyCD4/lckexperiments.Itcontains3%NP-40andpH8.4Tris.Itisnotclearthatthisvariantreallyoffersanyadvantages.

Bewareoffragilekinases.ThekinaseactivitiesofSyk,thep60srcproteinsoftsRSVs,ofp105fpsofPRCIIvirusandapparentlyofp70fgrofGR-FeSVarelABIle.MostoftheiractivityislostinRIPA.Fortheseproteins,itisimportanttouseNP-40buffer,containingEDTAandphosphataseinhibitors,forboththelysisofcellsandthewashingofIPs.Itisalsoessentialtoworkonlyinthecoldroomwiththeseproteins!

BoilinginSDS.Forsamplesthataretobetotallydenatured,lysethecellsinasmallvolume--0.1mlper35mmdish--of0.5%SDS,1mmolarfreshDTT,andsomebuffer,maybe0.05molarTris-HCl,pH8.0.Scrapethemup,boilfor30to60secandthendilutethemwith4volumesofRIPAwithoutSDSbutcontaining1mmolarDTT.ThepurposeoftheDTTistopreventaggregationduetodisulfideformationbetweennewlyexposedcysteines.FreshDTTshouldbepresentthroughtheinitialstepsoftheimmunoprecipitation.Whetheritneedstobepresentafterthatisn"tknown.ThereasontousefreshDTTisthatitispossIBLethattheDTTwillloseitsreducingpowerifstoredatadiluteconcentration.It"ssimplestthereforetomakeupafreshbatchofRIPAeachtimeyouneeditfromRIPAwithoutDTTanda1molarstockofDTT.

Antisera.Soasnottowastelysate,it"ssmarttoimmunoprecipitateinantibodyexcess.Antibody:antigenequivalencecannotbepredicted.1µlofTamara"santi-p56antibodywillbindallthep56lckin5x105LSTRAcells.SinceLSTRAcellscontain40foldmorep56thandootherTcells,1µlshouldbeenoughfor107regularTcells.Theamountofantibody327necessarytoprecipitateallofthep60c-srcincellshasnotbeencarefullymeasured.

Bugs:Thedogmaisthat2µlofstockbugswillprecipitatealloftheIgin1µlofrabbitantiserum.Goatseradonotbindtobugsaswellasrabbitsera.Thereforeusemorebugswhenyouareusingagoatantibody.Inmostcases,theprecipitationofratormouseseraisonlypossibleifyouaddsomegoatanti-ratoranti-mouseserum,usuallythesamevolumeasthevolumeofprimaryserumthatyouareusing,15minpriortotheadditionofthebugs.Pelletswhichcontainlessthan15µlofbugsarehardtohandleandeasytolose.Therefore,irrespectiveoftheamountofantiserumused,itisagoodideatouseatleast15µlofbugs.Surprisingly,excessbugsdonotseemtoincreasethebackground.Thebugsoughttobeinthesamebufferasthecelllysate.Toaccomplishthis,andtogetridofanythingwhichhasleachedoutofthebugswhiletheywerebeingstored,dilutethebugswiththebufferofinterest,spinthebugsdownandresUSPendtheminthebufferyouwant.

Secondaryantibodies:

MostrabbitserabindwelltoS.Aureus/proteinA.Sotoodomostsubclassesofmousemonoclonalantibodies.Goatserabind,butnotaswellasrabbitsera.RatantibodiesbindpoorlytoProteinA.IfyouareusinganantibodythatbindspoorlytoproteinA,youshoulduseasecondantibody,usuallyfromarabbitoragoat,tofacilitatebindingoftheantigen/antibodycomplexestothebugs.

Celllysis.

I"mafirmbelieverinlysingthecellsinthecoldroom!20minisatrADItionalintervalforcelllysis.Whether20minisreallynecessarytosolubilizetheproteinthatyou"reinterestedinisn"tknown.Surprisingly,backgroundsseemtobesomewhatlowerifthecelllysateinkeptconcentrated,0.25to0.30mlper35mmdish.Pelletedsuspensioncellsshouldberesuspendedgentlyinthelysisbuffer.Adherentcellsshouldbescrapedoffthedishbutthenallowedtodissolvedfor20minonthedish.Scrapethemtothesidetoremovethelysate.Thelysateshouldbeclarifiedbycentrifugation.60minat17Kina1.5mlEppendorftubeintheoldadaptorsintheSM24rotorisgenerallymorethanenough.Forsamplestobeassayedforkinaseactivity,30minisprobablylongenough,sincebackgroundislowerintheseexperiments.Forboiledlysates,aspinof90minisessentialunlessyouaddbugsasdescribedbelow.OtherwisetheDNAgooeseverythingup.Evenifyou"renotusing0.5%SDS,nuclearlysiscanbeaseriousproblem,especiallywithmammaliancells.Begentle.Duringlysis,allIdotothecellsisscrapethemoffthedishwitharubberpoliceman,letthemsit,scrapethemagainandtransfertothecentrifugetube.InclusionofEDTAinthelysisbufferhelps.SodoestheuseofNP40buffer.

Pre-Clearing.

Whenthebackgroundinanimmunoprecipitationishigh,somepeopleliketopre-clearthelysatebyaddingeithernormalserumandbugs,orbugsalone,tothelysateandthenspinningthemout.ThetheoryisthatthiswilldragdownanythingthatcouldgettrappedinanIP.Thiscaninfacthelpreducethebackgroundinsomecircumstances.Theoretically,itshouldalwayshelp.Inmyexperiencehowever,itoftenmakesnodifferenceatall.PackingdowntheDNAgoo.Manypeopleliketoadd30µlofwashedStaph.A.bugstothelysatepriortocentrifugationtohelppackdownanyreleased,gooeyDNA.Thiscancuttheclarificationspintimeinhalfandhasnoobviousdeleteriouseffect.

Ifthenuclei/DNAinthepelletinsistongettingintoyoureppendorftipafterthespin,thebestsolution,besidesre-spinningthesample,istoremovethegoowiththepipetandthrowitaway.GenerallyitsallinonegooeystrandandtheremainingliquidwillyieldlowbackgroundIPs.

Itiseasiertoaddthelysatetotheantibodythanviceversa.ThereforeIusuallysetupallthetubesfortheexperimentwithantibodyinthembeforetheclarifyingspinisfinished.Withlargevolumesoflysate,thelysatecanjustbeshotin.Forsmallvolumesitisnecessarytoputthelysatedirectlyintothedropofserum.Obviouslythisnecessitatesthechangingofeppendorftips.Immunoprecipitateswhichareallowedtoformovernightseemtohaveahigherbackgroundthanonesworkeduprightaway.Thismaybeduetotimedependentaggregationordenaturationofcellularproteins.45minofincubationofantibodyandlysateand20minwithbugsseemslongenough.

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General&nbsp;Principles&nbsp;of&nbsp;Immunoprecipitation

32Pi-labeledcellsandisolationofproteinkinases.

Secondaryantibodies:

Celllysis.

Pre-Clearing.

General Principles of Immunoprecipitation