PIC Crosslinking and Immune Precipitation

ReferencesFishburnet.al.,2005,MolecularCell,vol.18#3:ExperimentalProcedurespg.376ImmobilizedTemplateassay(Hahnlabwebsite)NotesDTTmustbeomittedfromallbuffers1MKOAcseemstobetheupperlimitforsolubilityofNP40.Ifaprecipitateforms,lowertheKOAcconcentrationslightly.AnoUVcontrolisnotrequiredforthisprocedure(seestep7).Procedure1.Preparetemplatesperstandardprotocol2.Dialyzeextractsusing0.02microndiscsonbufferC+75mMammoniumsulfate+proteaseinhibitorsfor1hrat4degtoremoveDTT(100-150microliterextractper10mlbufferC)3.AddPEASlabeledactivatortotemplatesandbind10atRT4.Makereactionmixandform2XPICs(200microliter)perstandardprotocol5.WashPICs3x400microlitertxnwashbuffer6.ResuspendPICsin200microlitertxnwashbuffer7.Donotsplitreactionsinto100microliteraliquotsasisstandardforcross-linkingreactions8.Cross-linksamplesintheUVoven:energy=1250unitswith365nmbulbs9.Concentratebeads,discardsupernatant10.Resuspendin20microlitertxnwashbuffer+2microliter1MDTT11.Incubate10atRT,mixoccasionally12.Concentratebeads,discardsupernatant13.Resuspendbeadsin100microliterdisruptionbuffer(txnwashbuffer+1MKOAcfinalconcentration)14.Incubate30atRTwithgentlemixing(Dynalmixer)todisruptPICs15.Concentratebeads,transfersupernatanttonewtubecontaining10microliterM2-±FlagSepharose16.IncubateIPreactions1hratRTwithmixingonDynalmixer17.CollectSepharoseresinbycentrifugation,discardsupernatant18.Washresin1x100microliterdisruptionbuffer19.Washresin1x100microlitertxnwashbuffertoreducesaltubiquitousadsf20.Eluteboundproteinswith15microliter1XSDSsamplebuffer+1.5microliter1MDTT:incubateat70degfor10withmixinginThermomixer21.Transfereluatestonewtubes,storeat-20degAnalysisAnalyzebyPhosphorimagerandWestern1.Runsampleson4-12%Bis-TrisorotherNuPAGE2.TransfertoImmobilonFLmembrane3.Airdrymembrane4.ExposetoPhosphorimagerscreenfor2-7days5.Rewetmembraneinblockingbuffer-PBS6.PerformWesternfor±Flagand/orotherproteinsofinterestperstandardprotocol