Description:
Transcriptionfactors(TFs)areagroupofcellularproteinsthatplayessentialrolesinregulatinggeneexpression.Theyactassensorstomonitorcellularchangeandconvertthesignalsintogeneexpression.Often,TFsregulategeneexpressionnotonlythroughdirectlybindingtotheirDNAbindingsiteonthetargetpromoterregion,butalsoby interactingwithothertranscriptionfactorsorregulators.Sincechangesintranscriptionalinteractionpatternscanhavedramaticeffectsongeneexpressionandcellularresponsestostimuli,monitoringmultipleTF/TFandTF/co-regulatorinteractionssimultaneouslycouldyieldagreaterunderstandingofregulatorymechanisms.Signosis’TFProteinInteractionPlateArrayIcanmonitorTF/TForTF/co-regulatorinteractionsamong48differentTFssimultaneouslyinrelationtooneTForco-regulatorofinterest.ApplicableGrid:
ListofApplicableTFs:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| A | AP1 | CDP | GATA | NF-1 | Pit | Stat3 | AP1 | CDP | GATA | NF-1 | Pit | Stat3 |
| B | AP2 | CREB | GR/PR | NFAT | PPAR | Stat4 | AP2 | CREB | GR/PR | NFAT | PPAR | Stat4 |
| C | AR | E2F-1 | HIF | NF-E2 | PXR | Stat5 | AR | E2F-1 | HIF | NF-E2 | PXR | Stat5 |
| D | ATF2 | EGR | HNF4 | NFkB | SMAD | Stat6 | ATF2 | EGR | HNF4 | NFkB | SMAD | Stat6 |
| E | Brn-3 | ER | IRF | OCT4 | Sp1 | TCF/LEF | Brn-3 | ER | IRF | OCT4 | Sp1 | TCF/LEF |
| F | CEBP | Ets | MEF2 | p53 | SRF | TR | CEBP | Ets | MEF2 | p53 | SRF | TR |
| G | CAR | FAST-1 | Myb | Pax-5 | SATB1 | YY1 | CAR | FAST-1 | Myb | Pax-5 | SATB1 | YY1 |
| H | CBF | GAS/ISRE | Myc-Max | Pbx1 | Stat1 | TFIID | CBF | GAS/ISRE | Myc-Max | Pbx1 | Stat1 | TEIID |
Principle:
Signosis’TF-TFInteractionPlateArraycansimultaneouslyprofilethetranscriptionalinteractionofmultipleTFswithaTForco-regulatorofinterest.Inthisassay,aseriesofuniquebiotin-labeledprobesareprovidedthatcorrespondwiththeconsensussequencesofindividualTFDNA-bindingsites.Therefore,eachproberepresentsanindividualTF.Whentheprobemixisincubatedwithnuclearextract,individualprobesbindtheircorrespondingTF.TheTForco-regulatorofinterestisthenimmunoprecipitated,alongwithtranscriptionallyinteractingTFs,usingacorrespondingantibodyandproteinGorAagarosebeadsinacolumn.Unboundprobesandproteinsarewashedaway.Theboundprobesarethendetachedfromthecomplexandaresubsequentlydenatured.Thebiotin-labeledDNAstrandsarehybridizedonawhiteplateanddetectedwithstreptavidin-HRPandsubstrate.ThedetectedsignalsreflecttheinteractingTFswiththeparticularTForco-regulator.Luminescenceisreportedasrelativelightunits(RLUs)onamicroplateluminometer.
Data:
AnalysisofNFkBinteractionpatternsinHeLaandTNFalpha-treatedHeLa.ThecellsweretreatedwithandwithoutTNFalphafor1hour.Thenuclearextractswerepreparedandincubatedwithcis-elementprobemixandantibodyagainstNFkBp65.TheNFkBinteractingTFswerepulleddownandtheboundprobeswereelutedandhybridizedtotheplate.
