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.
AAT Bioquest常用产品报价单
产品名 | 货号 | 公司分类 | 商城分类 | 美金价 |
AAT Bioquest/Fluorescein, disodium salt *CAS 518-47-8*/2/100 mg | 2 | iFluor Rapid Tests | 酸碱缓冲液 | 1088.00 | AAT Bioquest/14-3-3 ζ (Ab-58) Antibody/8B0001/50 ug | 8B0001 | Signaling Intermediate Ab | 重组抗体 | 2828.00 | AAT Bioquest/Opioid Receptor (Phospho-Ser375) Antibody/8A0022/50 ug | 8A0022 | Phospho-Specific Ab | 受体 | 2828.00 | AAT Bioquest/iFluor™ 350 goat anti-mouse IgG (H+L)/16440/200 ug | 16440 | Fluorescent Anti-IgGs | 荧光染料 | 1088.00 | AAT Bioquest/trFluor™ Eu goat anti-mouse IgG (H+L)/16518/100 ug | 16518 | Fluorescent Anti-IgGs | 抗小鼠 | 4278.00 | AAT Bioquest/RPE-streptavidin conjugate/16900/100 ug | 16900 | Streptavidin Conjugates | 其他生物染料 | 1378.00 | AAT Bioquest/GCNT3 Antibody/8C14708/50 ug | 8C14708 | Signaling Intermediate Ab | 功能性抗体 | 2828.00 | AAT Bioquest/Amplite™ Luciferase Reporter Gene Assay Kit *Bright Glow*/12518/1 plate | 12518 | Reporter Gene Enzymes | 其它检测试剂盒 | 2103.00 | AAT Bioquest/ReadiLink™ Rapid mFluor™ Violet 450 Antibody Labeling Kit *Microscale Optimized for Labeling 50 &mi | 1100 | mFluor™ Dyes and Kits | 标记试剂盒 | 2103.00 | AAT Bioquest/Amplite™ Colorimetric Urea Quantitation Kit *Blue Color*/10058/200 Tests | 10058 | Diagnostic Molecules | 定量试剂盒 | 2828.00 | AAT Bioquest/Screen Quest™ CHO-Gqi Chimera Cell line/38101/Each | 38101 | cAMP GPCR Assays | 常规细胞株 | 0.00 |