Description:

Tocharacterizetranscriptionfactors(TFs)thatbindstoaspecificpromoterorthatregulatetheexpressionofaspecificgeneviaitsupstreampromoter,twocommonapproachesareapplied.FirstistoemploygelshiftassaywithDNAbindingsitesofTFsthataresilico-identifiedwithinthepromoter.Secondistoremovalorknockoutthebindingsite(s)ofaspecificTFinordertomeasurewhethertheexpressionofapromoter-linkedreporterisincreasedordecreased.Often,aseriesofreporterconstructswiththepromoterdeletionsormutationsneedtomakebecausemanybindingsitesofoneorafewTFsarepresentwithinapromoter.SignosishasdevelopedafastmethodtofacilitatethecharacterizationofpromotersthrougharevisedTFactivationarray.Thisassaywillhelptotestwhetheraselected96TFsbindtothepromoterornot.

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).

Signosis 是一家专业的生物分析供应商，致力于基于微孔板分析技术的生命科学研究产品的开发、生产和销售。Signosis 成立于2007年，坐落于世界研发中心--美国硅谷的中心地带。公司专注于细胞因子、转录因子、miRNA及疾病标志物等重要调控因子和标志物的研究，基于自有专利技术，开发完成400多种先进而独特的用于发现、筛选和分析的生命科学研究产品。Signosis 产品销售遍布北美、欧洲、亚洲及中东等主要国家和地区，客户涵盖美国国立卫生研究院、哈佛大学、耶鲁大学等著名研究机构以及众多制药公司