ImmunoprecipitationandImmuneComplexkinaseassay--accordingtoTamaraHurley 1)Washcellswithcold"Tris"(fromMargie"sshelf). SpindownthecellsifyouareworkingwithsUSPensioncellsinalowspeedswingingbucketcentrifuge,resuspendinapproximately1mlTrisandspinat2Kinamicrofuge. Washonplateifadherentcellsbyaspiratingmedium,adding2to10mlTris,andaspiratingagain. 2)Lysecellsat107cells/mlor(minimally)500microliters/10cmdishforadherentcellsinlysisbuffer(e.g.NP-40,RIPA).GentlyresuspendthesuspensioncellsinthelysisbufferintheeppitubeusingaplasticpasteurpipetorPipetman.Scrapeadherentcellsoffthedishwitharubberpoliceman,butleavetheminthedish.3)Incubatelysingcellsfor20minat4°C.Scrapethelysateofadherentcellstothesideandtransfertoaneppitube.4)ClarifylysatebycentrifugationinSM-24rotor(keptincoldroom)at17Krpmfor20minat4°C(longerforRIPAlysis).Ifyouareusing32P,besurenottooverfillthetubeandbesuretouseascrewcappedtube.5)Wash"bugs"(Staph.a)--thatarestoredintubeoncoldroomshelf--inlysisbuffer.Todothis,takeoutameasuredvolume--morethanyouneed--andmixwithtwicethisvolumeoflysisbuffer.Thenspindownthebugsinmicrofuge,andresuspendinthe"originalmeasuredvolume"inlysisbuffer.) Mixbugs(20microliters/IP)withantibody-incubate20min,4°C.Addlysate-incubate45min,4°C. Skiptostep8. 6)AliquotAbintubes.Addclarifiedlysateandincubate45min,4°C.7)Add20microlitersbugs/IPandincubate20min,4°C. 8)Spindownbugstowhichareboundtheantibodyandtheantigen,aspiratesupe,andresuspendbyvortexingin500microliterslysisbuffer.9)TransfertoaneweppitubeandwashIP3xwith500microliterslysisbufferandthen1xwithTNbyspinningandresuspending.10)Ifdoingkinaseassay,doadditionalwashinTNandthenproceedwithkinaseassay(below).11)Pelletscanbestoreddryat-20°Cforshortterm(oneortwodays)orat-70°Cforlonger.12)Resuspendpelletinatleast50microliterssamplebufferwith5%freshb-ME,boil2min,microfuge4min,andloadlessthanhalf(soyoucandoitagain,ifnecessary). 1)Resuspendpelletsin10microlitersofkinasebuffer(40mMPIPES,pH7.0;10mMMnCl2).Keepsamplesoniceuntiluse.2)MakeupaATPmixsuchthatyouhave2or10microcuriesofgamma-[32P]ATPper10microlitersKB.Whenusinganexogenoussubstratesuchasenolase,addthesubstratetotheATPmix.3)Add10microlitersATPmixtoeachIP,pipettingtomix,thenincubateat30°Cfor10min.4)Stopkinaseassaybyadding1mlofTN+0.01%NP-40.Microfuge2min,andaspiratesupebyhandusingaone-pieceplasticpasteur/transferpipet.Note:Ifusingasolubleexogenoussubstratesuchasenolase,stoprxnbyadding20microliters2xSDSsamplebuffer.
Youhaveachoicetoeitherpreconcentrate/prebindtheantibodytothebugsandthenaddlysatetothisortomixlysatewithantibodyandthenaddthebugs.Bartprefersthelatter.
6,7)PreconcentratingAbtobugs:OR
KINASEASSAY
