James Hardwick's angiotensin assay protocol相关交流-蚂蚁淘在线

ThisspecificprocedurewasdevelopedtoassaytheactivityoftheLckkinaseexpressedfromaretroviralvectorinratfibroblasts.Youcanobviouslyusefewerormorecells,dependingonyourneeds.Don"tskimptoomuchontheamountofS.aureusbugsthatyouuseforprecipitationoryouwillrisklosses.

1.Immunoprecipitatetheproteinfrom3x106cellsusing45microlitersS.aureusbugs.ResUSPendtheimmunoprecipitatesin12microliterskinasebuffer(40mMPIPES,pH7.0,10mMMnCl2)at4°C.2.Thekinasereactioniscarriedoutatroomtemperatureinatotalvolumeof20microlitersthatcontains:

15microCigamma-32P-ATP5microlitersimmunoprecipitate2mMolar[Val5]-angiotensinII

3.Remove5microliteraliquotsofthereactionmixtureafter1,3,and5minutesofreactiontime.TheincorporationofATPislinearduringthisperiod.

The5microliteraliquotsthatareremovedateachtimepointaremixedimmediatelywith15microlitersof5%trichloroaceticacid(TCA).Letthesamplessitoniceforatleast15mintoprecipitateproteins.4.Spinthesamplesinamicrocentrifugefor5minat4°C.5.Spot10microlitersofeachsupernatantontoa2x2cmphosphocellulosepaper(Whatmanp81).Washthepaperin0.425%phosphoricacid(add5ml85%phosphoricacidto1LH2Otomake0.425%phosphoricacid.)for3min(15milliliters/pieceofpaper).Repeatthewashtwice.UnincorporatedATPwillbewashedoffthepaper.6.Addthepiecesofpaperto5mlscintillationfluidtoandcount.

Val5-angiotensinIIisobtainedfromSigmaandstoredasa50mMstockat-70°C.Note:Ifyougetmorethan100,000cpmincorporated,youmaybepeggingtheassay.Inthiscase,repeattheexperimentanduselessoftheprecipitateintheassay.ConsumptionofmostoftheATPcanoccurifyouarestudyingproteinsover-expressedbytransienttransfection.

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