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AAT Bioquest/Amplite™ Renilla Luciferase Reporter Gene Assay Kit *Bright Glow*/12536/10 plates
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 429/466 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy ReporterGeneEnzymes |
| Related | TagEnzymes BiochemicalAssays |
1.Preparecells(orsamples):
1.1 Foradherentcells:Platecellsovernightingrowthmediumat1,000-10,000cells/90µL/well(96-wellplate)or250-2,000cells/20µL/well(384-wellplate).
1.2 Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletsinculturemediumat20,000-200,000cells/90µL/wellfora96-wellpoly-Dlysineplateor5000-50,000cells/20µL/wellfora384-wellpoly-Dlysineplate.Centrifugetheplatesat800rpmfor2minuteswithbrakeoffpriortotheexperiment.
Note1:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.Cellsmaybeseededthedaybeforeoronthedayoftheexperimentdependinguponthecelltypeand/ortheeffectofthetestcompounds.
Note2:Forallluminescentexperiments,itisrecommendedtousewhiteplatestogetthebestresults.
Note3:ItishighlyrecommendusephenolredfreegrowthmediumespeciallyDMEMandMEMwhichisknowntoabsorbthelightemittedfromluciferasesandhencequenchthesignalobserved.
2.PrepareRenillaluciferaseassaysolution:
2.1 Make100XRenillaluciferaseassaystocksolution:Transfer100µL(for#12535),1mL(for#12536and #12537)ofLuciferaseSubstrateBuffer(ComponentA2)into1vialofLuciferaseSubstrate(ComponentA),andmixthemwell.
Note:Storetheunused100XRenillaLuciferasesubstratestocksolutionat-20°C,andkeepfromlight.
2.2 MakeRenillaluciferaseassaysolution:Addonevolumeof100XRenillaLuciferaseassaystocksolution(fromStep2.1)to100volumesofAssayBuffer(ComponentB).
Note1:ThereconstitutedRenillaluciferaseassaysolutionisverysensitivetolight,shouldbekeptfromlight.Inaddition,itisnotstable,shouldbepreparedfresh,keptoniceandusedwithin2hours.
Note2:Alternatively,make2XRenillaluciferaseassaysolutionbyaddingonevolumeof100XRenillaLuciferaseassaystocksolution(fromStep2.1)to50volumesofAssayBuffer(ComponentB).
3.RunRenillaluciferaseassay:
3.1 Treatcells(orsamples)withtestcompoundsbyadding10µLof10Xtestcompounds(96-wellplate)or5µLof5Xtestcompounds(384-wellplate)indesiredcompoundbuffer.
3.2 Incubatethecellplatesina37oC,5%CO2incubatorfordesiredperiodoftime,typically4hourstoovernight.
3.3 Removethemediumcompletely.
3.4 Add100µL(96-wellplate)or25µL(384-wellplate)perwellofRenillaluciferaseassaysolution(fromStep2)andincubatetheplateatroomtemperaturefor5-10minutes.Keepitfromlight.
Note1:Alternatively,onecanadd50µL(96-wellplate)or12.5µL(384-wellplate)perwellofAssaybuffer(ComponentB),andincubatetheplateatroomtemperaturefor15minutes.Andthenadd2XRenillaluciferaseassaysolution(fromStep2,Note2)andincubatetheplateatroomtemperaturefor5-10minutes.Keepitfromlight.
3.5 Monitorluminescenceintensitywithaluminometer.
| References&Citations |  PrinterFriendlyVersion |
1. AndouT,EndohT,MieM,KobatakeE.(2009)RNAdetectionusingpeptide-insertedRenillaluciferase.AnalBioanalChem,393,661.
2. CissellKA,RahimiY,ShresthaS,DeoSK.(2009)ReassemblyofabioluminescentproteinRenillaluciferasedirectedthroughDNAhybridization.BioconjugChem,20,15.
3. HerbstKJ,AllenMD,ZhangJ.(2009)ThecAMP-dependentproteinkinaseinhibitorH-89attenuatesthebioluminescencesignalproducedbyRenillaLuciferase.PLoSOne,4,e5642.
4. KaiharaA,UmezawaY,FurukawaT.(2008)BioluminescentindicatorsforCa2+basedonsplitRenillaluciferasecomplementationinlivingcells.AnalSci,24,1405.
5. TitushinMS,MarkovaSV,FrankLA,MalikovaNP,StepanyukGA,LeeJ,VysotskiES.(2008)Coelenterazine-bindingproteinofRenillamuelleri:CDNAcloning,overexpression,andcharacterizationasasubstrateofluciferase.PhotochemPhotobiolSci,7,189.
6. WooJ,HowellMH,vonArnimAG.(2008)Structure-functionstudiesontheactivesiteofthecoelenterazine-dependentluciferasefromRenilla.ProteinSci,17,725.
7. WooJ,vonArnimAG.(2008)Mutationaloptimizationofthecoelenterazine-dependentluciferasefromRenilla.PlantMethods,4,23.
8. HansonJ,ReeseJ,GormanJ,CashJ,FraizerG.(2007)HormonetreatmentenhancesWT1activationofRenillaluciferaseconstructsinLNCaPcells.FrontBiosci,12,1387.
9. LoeningAM,FennTD,GambhirSS.(2007)CrystalstructuresoftheluciferaseandgreenfluorescentproteinfromRenillareniformis.JMolBiol,374,1017.
10. LoeningAM,WuAM,GambhirSS.(2007)Red-shiftedRenillareniformisluciferasevariantsforimaginginlivingsubjects.NatMethods,4,641.
