Promega-Dual-Luciferase-system

PromegaCorporation·2800WoodsHollowRoad·Madison,WI53711-5399USATollFreeinUSA800-356-9526·Phone[phone]·Fax[phone]·®ReporterAssayChemistry...................................................3B.FormatoftheDual-Luciferase®ReporterAssay.............................................5C.PassiveLysisBuffer.............................................................................................62.ProductComponentsandStorageConditions............................................83.ThepGL4LuciferaseReporterVectors.........................................................9A.DescriptionofpGL4Vectors..............................................................................9B.ImportantConsiderationsforCo-TransfectionExperiments........................94.InstrumentConsiderations............................................................................10A.Single-SampleLuminometers...........................................................................10B.Multi-SampleandPlate-ReadingLuminometers..........................................10C.ScintillationCounters.........................................................................................115.PreparationofCellLysatesUsingPassiveLysisBuffer..........................12A.PassiveLysisBufferPreparation.....................................................................12B.PassiveLysisofCellsCulturedinMultiwellPlates.....................................12C.ActiveLysisofCellsbyScraping....................................................................136.Dual-Luciferase®ReporterAssayProtocol.................................................14A.PreparationofLuciferaseAssayReagentII...................................................14B.PreparationofStop&Glo®Reagent...............................................................15C.StandardProtocol...............................................................................................15D.ImportantConsiderationsforCleaningReagentInjectors..........................18E.DeterminationofAssayBackgrounds............................................................197.References.........................................................................................................218.Appendix...........................................................................................................22A.CompositionofBuffersandSolutions............................................................22B.RelatedProducts.................................................................................................22Dual-Luciferase®ReporterAssaySystemAlltechnicalliteratureisavailableontheInternetat::[e-mail]-Luciferase®ReporterAssaySystemINSTRUCTIONSFORUSEOFPRODUCTSE1910ANDE1960.PRINTEDINUSA.Revised3/09Part#TM0401.DescriptionGeneticreportersystemsarewidelyusedtostudyeukaryoticgeneexpressionandcellularphysiology.Applicationsincludethestudyofreceptoractivity,transcriptionfactors,intracellularsignaling,mRNAprocessingandproteinfolding.Dualreportersarecommonlyusedtoimproveexperimentalaccuracy.Theterm“dualreporter”referstothesimultaneousexpressionandmeasurementoftwoindividualreporterenzymeswithinasinglesystem.Typically,the“experimental”reporteriscorrelatedwiththeeffectofspecificexperimentalconditions,whiletheactivityoftheco-transfected“control”reporterprovidesaninternalcontrolthatservesasthebaselineresponse.Normalizingtheactivityoftheexperimentalreportertotheactivityoftheinternalcontrolminimizesexperimentalvariabilitycausedbydifferencesincellviabilityortransfectionefficiency.Othersourcesofvariability,suchasdifferencesinpipettingvolumes,celllysisefficiencyandassayefficiency,canbeeffectivelyeliminated.Thus,dual-reporterassaysoftenallowmorereliableinterpretationoftheexperimentaldatabyreducingextraneousinfluences.TheDual-Luciferase®Reporter(DLR™)AssaySystem(a–f)providesanefficientmeansofperformingdual-reporterassays.IntheDLR™Assay,theactivitiesoffirefly(Photinuspyralis)andRenilla(Renillareniformis,alsoknownasseapansy)luciferasesaremeasuredsequentiallyfromasinglesample.ThefireflyluciferasereporterismeasuredfirstbyaddingLuciferaseAssayReagentII(LARII)togenerateastabilizedluminescentsignal.Afterquantifyingthefireflyluminescence,thisreactionisquenched,andtheRenillaluciferasereactionissimultaneouslyinitiatedbyaddingStop&Glo®Reagenttothesametube.TheStop&Glo®ReagentalsoproducesastabilizedsignalfromtheRenillaluciferase,whichdecaysslowlyoverthecourseofthemeasurement.IntheDLR™AssaySystem,bothreportersyieldlinearassayswithsubattomolesensitivitiesandnoendogenousactivityofeitherreporterintheexperimentalhostcells.Furthermore,theintegratedform