Immunoprecipitation and Immune Complex kinase assayaccording to Tamara Hurley相关交流-蚂蚁淘在线

Immunoprecipitation and Immune Complex kinase assayaccording to Tamara Hurley

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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.)

Youhaveachoicetoeitherpreconcentrate/prebindtheantibodytothebugsandthenaddlysatetothisortomixlysatewithantibodyandthenaddthebugs.Bartprefersthelatter.

6,7)PreconcentratingAbtobugs:

Mixbugs(20microliters/IP)withantibody-incubate20min,4°C.Addlysate-incubate45min,4°C.

Skiptostep8.

OR

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).

KINASEASSAY

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.

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Immunoprecipitation&nbsp;and&nbsp;Immune&nbsp;Complex&nbsp;kinase&nbsp;assayaccording&nbsp;to&nbsp;Tamara&nbsp;Hurley

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KINASEASSAY

Immunoprecipitation and Immune Complex kinase assayaccording to Tamara Hurley