荧光素酶报告基因检测试剂盒E1910说明书

TechnicalManualDual-Luciferase®ReporterAssaySystemINSTRUCTIONSFORUSEOFPRODUCTSE1910ANDE1960.PRINTEDINUSA.Revised8/06Part#TM040AF9TM0400806TM040tm040.0806.qxp8/11/20062:49PMPage1PromegaCorporation·2800WoodsHollowRoad·Madison,WI53711-5399USATollFreeinUSA800-356-9526·Phone608-274-4330·Fax608-277-2516·www.promega.comPrintedinUSA.Part#TM040Revised8/06Page1I.Description..........................................................................................................2A.Dual-Luciferase®ReporterAssayChemistry...................................................3B.FormatoftheDual-Luciferase®ReporterAssay.............................................5C.PassiveLysisBuffer.............................................................................................6II.ProductComponentsandStorageConditions............................................8III.ThepGL4LuciferaseReporterVectors.........................................................9A.DescriptionofpGL4Vectors..............................................................................9B.ImportantConsiderationsforCo-TransfectionExperiments........................9IV.InstrumentConsiderations............................................................................10A.Single-SampleLuminometers...........................................................................10B.Multi-SampleandPlate-ReadingLuminometers..........................................10C.ScintillationCounters.........................................................................................11V.PreparationofCellLysatesUsingPassiveLysisBuffer..........................12A.PassiveLysisBufferPreparation.....................................................................12B.PassiveLysisofCellsCulturedinMultiwellPlates.....................................12C.ActiveLysisofCellsbyScraping....................................................................13VI.Dual-Luciferase®ReporterAssayProtocol.................................................14A.PreparationofLuciferaseAssayReagentII...................................................14B.PreparationofStop&Glo®Reagent...............................................................15C.StandardProtocol...............................................................................................15D.ImportantConsiderationsforCleaningReagentInjectors..........................18E.DeterminationofAssayBackgrounds............................................................19VII.References.........................................................................................................21VIII.Appendix...........................................................................................................22A.CompositionofBuffersandSolutions............................................................22B.RelatedProducts.................................................................................................22Dual-Luciferase®ReporterAssaySystemAlltechnicalliteratureisavailableontheInternetat:www.promega.com/tbs/PleasevisitthewebsitetoverifythatyouareusingthemostcurrentversionofthisTechnicalManual.PleasecontactPromegaTechnicalServicesifyouhavequestionsonuseofthissystem.E-mail:techserv@promega.com.tm040.0806.qxp8/11/20062:49PMPage1I.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–e)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,theintegratedformatoftheDLR™Assayprovidesrapidquantitationofbothreporterseitherintransfectedcellsorincell-freetranscription/translationreactions.PromegaoffersthepGL4seriesoffireflyandRenillaluciferasevectorsdesignedforusewiththeDLR™AssaySystems.Thesevectorsmaybeusedtoco-transfectmammaliancellswithexperimentalandcontrolreportergenes.PromegaCorporation·2800WoodsHollowRoad·Madison,WI53711-5399USATollFreeinUSA800-356-9526·Phone608-274-4330·Fax608-277-2516·www.promega.comPart#TM040PrintedinUSA.Page2Revised8/06tm040.0806.qxp8/11/20062:49PMPage2I.A.Dual-Luciferase®ReporterAssayChemistryFireflyandRenillaluciferases,becauseoftheirdistinctevolutionaryorigins,havedissimilarenzymestructuresandsubstraterequirements.Thesedifferencesmakeitpossibletoselectivelydiscriminatebetweentheirrespectivebioluminescentreactions.Thus,usingtheDLR™AssaySystem,theluminescencefromthefireflyluciferasereactionmaybequenchedwhilesimultaneouslyactivatingtheluminescentreactionofRenillaluciferase.Fireflyluciferaseisa61kDamonomericproteinthatdoesnotrequirepost-translationalprocessingforenzymaticactivity(1,2).Thus,itfunctionsasageneticreporterimmediatelyupontranslation.PhotonemissionisachievedthroughoxidationofbeetleluciferininareactionthatrequiresATP,Mg2+andO2(Figure1).Underconventionalreactionconditions,theoxidationoccursthroughaluciferyl-AMPintermediatethatturnsoververyslowly.Asaresult,thisassaychemistrygeneratesa“flash”oflightthatrapidlydecaysafterthesubstrateandenzymearemixed.ManyofourLuciferaseAssayReagentsforquantitatingfireflyluciferaseincorporatecoenzymeA(CoA)toprovidemorefavorableoverallreactionkinetics(3).InthepresenceofCoA,theluciferaseassayyieldsstabilizedluminescencesignalswithsignificantlygreaterintensities(Figure2)thanthoseobtainedfromtheconventionalassaychemistry.Thefireflyluciferaseassayisextremelysensitiveandextendsoveralinearrangecoveringatleastsevenordersofmagnitudeinenzymeconcentration(Figure3).Renillaluciferase,a36kDamonomericprotein,iscomposedof3%carbohydratewhenpurifiedfromitsnaturalsource,Renillareniformis(4).However,likefireflyluciferase,post-translationalmodificationisnotrequiredforitsactivity,andtheenzymemayfunctionasageneticreporterimmediatelyfollowingtranslation.TheluminescentreactioncatalyzedbyRenillaluciferaseutilizesO2andcoelenterate-luciferin(coelenterazine;Figure1).PromegaCorporation·2800WoodsHollowRoad·Madison,WI53711-5399USATollFreeinUSA800-356-9526·Phone608-274-4330·Fax608-277-2516·www.promega.comPrintedinUSA.Part#TM040Revised8/06Page3Figure1.BioluminescentreactionscatalyzedbyfireflyandRenillaluciferases.tm040.0806.qxp8/11/20062:49PMPage3I.A.Dual-Luciferase®ReporterAssayChemistry(continued)IntheDLR™Assaychemistry,thekineticsoftheRenillaluciferasereactionprovideastabilizedluminescentsignalthatdecaysslowlyoverthecourseofthemeasurement(Figure2).Similartofireflyluciferase,theluminescentreactioncatalyzedbyRenillaluciferasealsoprovidesextremesensitivityandalinearrangegenerallyextendingsixordersofmagnitude(Figure3).Notethattheeffectiverangeoftheluminescentreactionsmayvarydependingonthetypeofluminometer(e.g.,96-wellversussingle-sample)used.Aninherentpropertyofcoelenterazineisthatitemitslow-levelautoluminescenceinaqueoussolutions.Originallythisdrawbackpreventedsensitivedeterminationsatthelowerendofenzymeconcentration.Additionally,sometypesofnonionicdetergentscommonlyusedtopreparecelllysates(e.g.,Triton®X-100)greatlyintensifycoelenterazineautoluminescence.TheDLR™AssaySystemsincludeproprietarychemistrythatreducesautoluminescencetoalevelthatisnotmeasurableforallbutthemostsensitiveluminometers.PassiveLysisBufferisformulatedtominimizetheeffectoflysatecompositiononcoelenterazineautoluminescence.Inaddition,theDLR™AssaySystemsincludetworeconstitutedassayreagents,LuciferaseAssayReagentIIandStop&Glo®Reagent,thatcombinetosuppresscoelenterazineautoluminescence.PromegaCorporation·2800WoodsHollowRoad·Madison,WI53711-5399USATollFreeinUSA800-356-9526·Phone608-274-4330·Fax608-277-2516·www.promega.comPart#TM040PrintedinUSA.Page4Revised8/060102030405060708090100024681012Activity(%peak)Time(sec)FireflyRenillaFigure2.LuminescentsignalsgeneratedintheDual-Luciferase®ReporterAssaySystembyfireflyandRenillaluciferases.tm040.0806.qxp8/11/20062:49PMPage4I.B.FormatoftheDual-Luciferase®ReporterAssayQuantitationofluminescentsignalfromeachoftheluciferasereporterenzymesmaybeperformedimmediatelyfollowinglysatepreparationwithouttheneedfordividingsamplesorperformingadditionaltreatments.ThefireflyluciferasereporterassayisinitiatedbyaddinganaliquotoflysatetoLuciferaseAssayReagentII.QuenchingoffireflyluciferaseluminescenceandconcomitantactivationofRenillaluciferaseareaccomplishedbyaddingStop&Glo®Reagenttothesampletubeimmediatelyafterquantitationofthefireflyluciferasereaction.Theluminescentsignalfromthefireflyreactionisquenchedbyatleastafactorof105(to≤0.001%residuallightoutput)within1secondfollowingtheadditionofStop&Glo®Reagent(Figure4).CompleteactivationofRenillaluciferaseisalsoachievedwithinthis1-secondperiod.Whenusingamanualluminometer,thetimerequiredtoquantitatebothluciferasereporteractivitieswillbeapproximately30seconds.Theprocedurecanbesummarizedasfollows:ElapsedTimeStep1:ManuallyaddpreparedlysatetoLuciferaseAssay~3secondsReagentIIpredispensedintoluminometertubes;mix.Step2:Quantifyfireflyluciferaseactivity.12secondsStep3:AddStop&Glo®Reagent;mix.3secondsStep4:QuantitateRenillaluciferaseactivity.12secondsTotalelapsedtimefortheDLR™Assay30secondsPromegaCorporation·2800WoodsHollowRoad·Madison,WI53711-5399USATollFreeinUSA800-356-9526·Phone608-274-4330·Fax608-277-2516·www.promega.comPrintedinUSA.Part#TM040Revised8/06Page54093MA04_3A0.11×10–131×10–141×10–151×10–161×10–171×10–181×10–191×10–201101001,00010,000100,0001,000,00010,000,000Luminescence(RLU)[Luciferase](moles/reaction)FireflyRenillaFigure3.ComparisonofthelinearrangesoffireflyandRenillaluciferases.TheDLR™AssaywasperformedwithamixtureofpurifiedfireflyandRenillaluciferasespreparedinPLBcontaining1mg/mlgelatin.ATurnerDesignsModel20eLuminometerwasusedtomeasureluminescence.AsshowninthisgraphwiththeDLR™AssaySystem,thelinearrangeofthefireflyluciferaseassayissevenordersofmagnitude,providingdetectionsensitivityof≤1femtogram(approximately10–20mole)offireflyluciferasereporterenzyme.TheRenillaluciferaseassayhasalinearrangecoveringsixordersofmagnitudeandallowsforthedetectionofapproximately30femtograms(approximately3×10–19moles)ofRenillaluciferase.tm040.0806.qxp8/11/20062:49PMPage5I.B.FormatoftheDual-Luciferase®ReporterAssay(continued)I.C.PassiveLysisBufferPassiveLysisBuffer(PLB)isspecificallyformulatedtopromoterapidlysisofculturedmammaliancellswithouttheneedtoscrapeadherentcellsorperformadditionalfreeze-thawcycles(activelysis).Furthermore,PLBpreventssamplefoaming,makingitideallysuitedforhigh-throughputapplicationsinwhicharraysoftreatedcellsareculturedinmultiwellplates,processedintolysatesandassayedusingautomatedsystems.AlthoughPLBisformulatedforpassivelysisapplications,itsrobustlyticperformanceisofequalbenefitwhenharvestingadherentcellsculturedinstandarddishesusingactivelysis.Regardlessofthepreferredlysismethod,thereleaseoffireflyandRenillaluciferasereporterenzymesintothecelllysateisbothquantitativeandreliableforculturedmammaliancells(Figure5).PromegaCorporation·2800WoodsHollowRoad·Madison,WI53711-5399USATollFreeinUSA800-356-9526·Phone608-274-4330·Fax608-277-2516·www.promega.comPart#TM040PrintedinUSA.Page6Revised8/061,000,000100,00010,0001,00010010.1010FireflyLuciferaseActivityRenillaLuciferaseActivityQuenchedReporter#1Luminescence80,6000.28116,800Reporter#1Reporter#20.0004%ResidualActivityLuminescence(RLU)Figure4.MeasurementofluciferaseactivitiesbeforeandaftertheadditionofStop&Glo®Reagent.TheDLR™Assayallowssequentialmeasurementoffireflyluciferase(Reporter#1),followedbyRenillaluciferaseactivity(Reporter#2)onadditionofStop&Glo®Reagenttothereaction.BothreporteractivitieswerequantitatedwithinthesamesampleoflysatepreparedfromCHOcellsco-transfectedwithpGL3ControlVector(Cat.#E1741)andpRL-SV40Vector(Cat.#E2231).TodemonstratetheefficientquenchingofReporter#1byStop&Glo®Reagent,anequalvolumeofStop&Glo®Buffer(whichdoesnotcontainthesubstrateforRenillaluciferase)wasadded.Fireflyluciferaseluminescencewasquenchedbygreaterthan5ordersofmagnitude.tm040.0806.qxp8/11/20062:49PMPage6Inadditiontoitslyticproperties,PLBisdesignedtoprovideoptimumperformanceandstabilityofthefireflyandRenillaluciferasereporterenzymes.AnimportantfeatureofPLBisthat,unlikeothercelllysisreagents,itelicitsonlyminimalcoelenterazineautoluminescence.Hence,PLBisthelyticreagentofchoicewhenprocessingcellsforquantitationoffireflyandRenillaluciferaseactivitiesusingtheDLR™AssaySystem.Otherlysisbuffers(e.g.,GloLysisBuffer,CellCultureLysisReagentandReporterLysisBuffer)eitherincreasebackgroundluminescencesubstantiallyorareinadequateforpassivelysis.Ifdesired,theproteincontentofcelllysatespreparedwithPLBmaybereadilyquantitatedusingavarietyofcommonchemicalassaymethods.Determinationofproteincontentmustbeperformedusingadequatecontrols.DilutinglysateswitheitherwaterorabufferthatisfreeofdetergentsorreducingagentsisrecommendedinordertoreducetheeffectsthatPassiveLysisBuffermayhaveonbackgroundabsorbance.AstandardcurvewithBSAmustbegeneratedinparallelunderthesamebufferconditions.PromegaCorporation·2800WoodsHollowRoad·Madison,WI53711-5399USATollFreeinUSA800-356-9526·Phone608-274-4330·Fax608-277-2516·www.promega.comPrintedinUSA.Part#TM040Revised8/06Page7PassiveLysisActiveLysis1201101009080706050403020100%FireflyLuciferaseActivityLysisMethodCHOCV-1HeLaNIH/3T3A.FireflyLuciferaseAssay1201101009080706050403020100%RenillaLuciferaseActivityLysisMethodCHOCV-1HeLaNIH/3T3B.RenillaLuciferaseAssay1403MA03_6AFigure5.ComparisonoffireflyandRenillaluciferasereporteractivitiesincelllysatespreparedwithPassiveLysisBufferusingeitherthepassiveoractivelysisprocedure.Fourdifferentmammaliancelltypeswereco-transfectedwithfireflyandRenillaluciferaseexpressionvectors.LysateswerepreparedbyeitherexposingadherentcellstoPassiveLysisBufferfor15minutes(passivelysis),orscrapingadherentcellsinthepresenceofPassiveLysisBufferfollowedbyonefreeze-thawcycle(activelysis).Forcomparativepurposes,reporteractivitieswerenormalizedtothoseobtainedwiththeactivelysismethodforeachcelltype.tm040.0806.qxp8/11/20062:49PMPage7II.ProductComponentsandStorageConditionsProductSizeCat.#Dual-Luciferase®ReporterAssaySystem100assaysE1910Eachsystemcontainssufficientreagentstoperform100standardDual-Luciferase®ReporterAssays.Includes:•10mlLuciferaseAssayBufferII•1vialLuciferaseAssaySubstrate(LyophilizedProduct)•10mlStop&Glo®Buffer•200µlStop&Glo®Substrate,50X•30mlPassiveLysisBuffer,5X•1ProtocolProductSizeCat.#Dual-Luciferase®ReporterAssaySystem,10-Pack1,000assaysE1960Eachsystemcontainssufficientreagentstoperform1,000standardDual-Luciferase®ReporterAssaysusing96-wellluminometryplates.Includes:•10×10mlLuciferaseAssayBufferII•10×1vialLuciferaseAssaySubstrate(LyophilizedProduct)•10×10mlStop&Glo®Buffer•10×200µlStop&Glo®Substrate,50X•30mlPassiveLysisBuffer,5X•1ProtocolNoteregardingCat.#E1960:Forapplicationsrequiringmorelysisreagent(e.g.,>100µl/well),additionalPassiveLysisBuffermaybepurchasedseparately(Cat.#E1941).StorageConditions:Uponreceipt,storetheDual-Luciferase®ReporterAssaySystemat–20°C.OncetheLuciferaseAssaySubstratehasbeenreconstituted,itshouldbedividedintoworkingaliquotsandstoredat–20°Cforupto1monthorat–70°Cforupto1year.Ideally,Stop&Glo®Reagent(Substrate+Buffer)shouldbepreparedjustbeforeeachuse.Ifnecessary,thisreagentmaybestoredat–20°Cfor15dayswithnodecreaseinactivity.Ifstoredat22°Cfor48hours,thereagent’sactivitydecreasesby8%,andifstoredat4°Cfor15days,thereagent’sactivitydecreasesby13%.TheStop&Glo®Reagentcanbethawedatroomtemperatureupto6timeswith≤15%decreaseinactivity.PromegaCorporation·2800WoodsHollowRoad·Madison,WI53711-5399USATollFreeinUSA800-356-9526·Phone608-274-4330·Fax608-277-2516·www.promega.comPart#TM040PrintedinUSA.Page8Revised8/06tm040.0806.qxp8/11/20062:49PMPage8III.ThepGL4LuciferaseReporterVectorsIII.A.DescriptionofpGL4VectorsThepGL4LuciferaseReporterVectorsarethenextgenerationofreportergenevectorsoptimizedforexpressioninmammaliancells.NumerousconfigurationsofpGL4Vectorsareavailable,includingthosewiththesyntheticfireflyluc2(Photinuspyralis)andRenillahRluc(Renillareniformis;5)luciferasegenes,whichhavebeencodonoptimizedformoreefficientexpressioninmammaliancells.Furthermore,boththereportergenesandthevectorbackbone,includingtheampicillin(Ampr)geneandmammalianselectablemarkergenesforhygromycin(Hygr),neomycin(Neor)andpuromycin(Puror),havebeenengineeredtoreducethenumberofconsensustranscriptionfactorbindingsites,reducingbackgroundandtheriskofanomaloustranscription.ThepGL4Vectorbackboneisprovidedwitheithertheluc2orhRlucgenesand,incertainvectors,oneorbothoftwoRapidResponse™reportergenes.TheproteinlevelsmaintainedbytheseRapidResponse™luciferasegenesrespondmorequicklyandwithgreatermagnitudetochangesintranscriptionalactivitythantheirmorestablecounterparts.FormoreinformationonadvantagesofandimprovementsmadetothepGL4seriesofvectors,pleasevisit:www.promega.com/pgl4/orseethepGL4LuciferaseReportersTechnicalManual#TM259.III.B.ImportantConsiderationsforCo-TransfectionExperimentsFireflyandRenillaluciferasevectorsmaybeusedtogethertoco-transfectmammaliancells.EitherfireflyorRenillaluciferasemaybeusedasthecontrolortheexperimentalreportergene,dependingontheexperimentandthegeneticcontructsavailable.However,itisimportanttorealizethattranseffectsbetweenpromotersonco-transfectedplasmidscanpotentiallyaffectreportergeneexpression(6).Primarily,thisisofconcernwheneitherthecontrolorexperimentalreportervector,orboth,containverystrongpromoter/enhancerelements.Theoccurrenceandmagnitudeofsucheffectswilldependonthecombinationandactivitiesofthegeneticregulatoryelementspresentontheco-transfectedvectors,therelativeratioofexperimentalvectortocontrolvectorintroducedintothecells,andthecelltypetransfected.Tohelpensureindependentgeneticexpressionbetweenexperimentalandcontrolreportergenes,weencourageuserstoperformpreliminaryco-transfectionexperimentstooptimizeboththeamountofvectorDNAandtheratioofco-reportervectorsaddedtothetransfectionmix.TheextremesensitivityofbothfireflyandRenillaluciferaseassays,andtheverylargelinearrangeofluminometers(typically5–6ordersofmagnitude),allowsaccuratemeasurementofevenva