Signosis/PromoterBinding TF Profiling Array I/FA2001/1 Ea

Description:

Tocharacterizetranscriptionfactors(TFs)thatbindstoaspecificpromoterorthatregulatetheexpressionofaspecificgeneviaitsupstreampromoter,twocommonapproachesareapplied.FirstistoemploygelshiftassaywithDNAbindingsitesofTFsthataresilico-identifiedwithinthepromoter.Secondistoremovalorknockoutthebindingsite(s)ofaspecificTFinordertomeasurewhethertheexpressionofapromoter-linkedreporterisincreasedordecreased.Often,aseriesofreporterconstructswiththepromoterdeletionsormutationsneedtomakebecausemanybindingsitesofoneorafewTFsarepresentwithinapromoter.SignosishasdevelopedafastmethodtofacilitatethecharacterizationofpromotersthrougharevisedTFactivationarray.Thisassaywillhelptotestwhetherselected48or96TFsbindtothepromoterornot.

Principle:

Promoter-bindingTFprofilingplatearraysareacompetitionassayofSignosis’TFactivationplatearrays.IntheTFactivationplatearrays,ifallof48or96targetedtranscriptionfactorsexistintheassayedsamples,theywillform48or96typesofcomplexes,eachTFwithitscorrespondingbiotin-labeledoligo(justlikethecomplexinthegelshiftassay).Afterasimplespinseparationofthecomplexesfromunboundfreebiotin-labeledoligoswithamembrane-basedcolumn,TF-boundoligoselutedfromthecolumnandusedforplatehybridizationinwhichacomplementaryDNAofbiotin-labeledoligo.Thecapturedoligoisthendetectedwithstreptavidin-HRPandachemiluminescentsubstrate.IfanyTFisnotpresent,itwillnotformacomplex,leADIngtonodetectionofTFinthefollowingplateassayofbiotin-labeledoligo.Inpromoter-bindingTFprofilingarrays,PCRfragmentcontainingthepromoterofyourinterestismixedwithasetof48or96biotin-labeledoligoscorrespondingto48or96TFsalongwithanassayedsample.IfunlabeledpromoterDNAfragmentcontainsaTFbindingsequence,itwillcompetewiththebiotin-labeledoligotobindtotheTFinthesample,leadingtonoorlessbiotinlabeledTF/DNAcomplexformationandnoorlowerdetection.ThroughcomparisoninthepresenceandabsenceofthecompetitorpromoterDNAfragment,promoter-boundTFscanbeidentified.

Data:

Promoter-BindingTFProfilingAssay:HeLacellsweretreatedwithorwithoutPMA.PMAwasusedtoacticveTFsincludingAP1andNFkB.NuclearextractswerepreparedandincubatedwithTFbindingoligoprobemix:controlHeLacellswithoutPMAtreatmentwiththeprobemix(blue);PMA-treatedHeLacellswiththeprobemixalone(red)andtheprobemixplusIL6promoterDNAfragment(yellow).