首页 > BPS Bioscience > Bpsbioscience/GAL4 Reporter Kit (Glucocorticoid Receptor Pathway)/60522/500 reactions

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Bpsbioscience/GAL4 Reporter Kit (Glucocorticoid Receptor Pathway)/60522/500 reactions

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Background:Theglucocorticoidsignalingpathwayplaysanimportantroleindevelopment,fluidhomeostasis,cognition,immuneresponseandmetabolism.Glucocorticoidsareaclassofsteroidhormonesthatbindtotheglucocorticoidreceptor,causingittotranslocatetothenucleus.Upontranslocation,thereceptorcanregulatethetranscriptionofalargenumberofgenes,includingthosethatregulateglucosemetabolismandinflammatoryresponses.Description:TheGlucocorticoidReceptorPathwayReporterKitisdesignedformonitoringtheactivityoftheglucocorticoidsignalingpathwayinculturedcells.Thekitcontainsatransfection-readyexpressionvectorfortheglucocorticoidreceptorligandbindingdomainthatisfusedtotheDNAbindingdomain(DBD)ofGAL4(GAL4DBD-GR).ThisfusionconstructactivatesfireflyluciferaseexpressionunderthecontrolofamultimerizedGAL4upstreamactivationsequence(UAS).Thisallowsforspecificdetectionofglucocorticoid-inducedactivationoftheglucocorticoidreceptorwithouttheneedforindividualtranscriptionaltargetsandwithlowcrossreactivityforothernuclearreceptorpathways.TheGAL4/UASreporterispremixedwithconstitutivelyexpressingRenilla(seapansy)luciferasevector,whichservesasaninternalpositivecontrolfortransfectionefficiency.Thekitalsoincludesanon-inducIBLefireflyluciferasevectorpremixedwithconstitutivelyexpressingRenillaluciferasevectorasanegativecontrol.Thenon-inducibleluciferasevectorcontainsafireflyluciferasegeneunderthecontrolofaminimalpromoter,butwithoutanyadditionalresponseelements.Thenegativecontroliscriticalfordeterminingpathway-specificeffectsandbackgroundluciferaseactivity.Synonym(s):GlucocorticoidReceptor,GR,Nuclearreceptorsubfamily3groupCmember1,NR3C1,GAL4,GAL4/UASFormat:COMPONENTS:Instructionsforuse:Seeassaykitdatasheetfordetailedprotocol.Storage/StABIlity:>1yearfromdateofreceiptwhenstoredat-20°CApplication(s):•Monitorglucocorticoidsignalingpathwayactivity.•Screenactivatorsorinhibitorsoftheglucocorticoidsignalingpathway.Reference(s):PaguioA,etal.(2010)ImprovedDual-LuciferaseReporterAssaysforNuclearReceptors.CurrChemGenomics.4:43-49.Notes:MaterialsRequiredbutNotSupplied•Mammaliancelllineandappropriatecellculturemedium•96-welltissuecultureplateor96-welltissueculturetreatedwhiteclear-bottomassayplate(Corning#3610)•Transfectionreagentformammaliancellline.[WeuseLipofectamine™2000(LifeTechnologies#11668027),however,othertransfectionreagentsshouldworkequallywell.]•Opti-MEMIReducedSerumMedium(LifeTechnologies#31985-062)•Dualluciferaseassaysystem:Dual-Glo®LuciferaseAssaySystem(Promega#E2920):Thissystemassayscellsdirectlyingrowthmedium.Itcanbeusedwithanyluminometer.Automatedinjectorsarenotrequired.ORDual-Luciferase®ReporterAssaySystem(Promega#E1910):Thissystemrequiresacelllysisstep.Itisidealforluminometerswithautomatedinjectors.•LuminometerThesevectorsarereadyfortransienttransfection.TheyareNOTSUITABLEfortransformationandamplificationinbacteria.Warning(s):Avoidfreeze/thawcycles.ScientificCategory:Cell-BasedAssayKitProductType:AssayKitDatashownislot-specific.Contactusforspecificinformationonotherlots.

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