Description:
Transcriptionfactors(TFs)areagroupofcellularproteinsthatplayessentialrolesinregulatinggeneexpression.Theyactasasensortomonitorcellularchangeandconvertthesignalsintogeneexpression.AspecificTFisoftenregulatedbymultipleupstreamproteins.MonitoringtheactivationofaTFiscriticaltouncoveringthemechanismofsignalpathwaysandcellularregulations.Signosisdeveloped48differentfilterassaysformonitoringtheactivitiesofTFs.Unlikegelshiftassays,96samplescanbeanalyzedinoneexperiment.Principle:
InSignosis"TFfilterplateassays,aspecificbiotin-labeledTFDNAbindingsequenceismixedwithnuclearextractstoallowformationofTF-DNAcomplexes.AfilterplateisusedtoretainboundDNAprobeandremovefreeprobe.Theboundpre-labeledDNAprobeisthenelutedfromthefilterandcollectedforquantitativeanalysisthroughDNAplatehybridization.ThecapturedDNAprobeisfurtherdetectedwithstreptavidin-HRP.Luminescenceisreportedasrelativelightunits(RLUs)onamicroplateluminometer.