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主营:研究并生产荧光和发光探针,信号转导研究的试剂
℡ 4000-520-616
℡ 4000-520-616
AAT Bioquest/Amplite™ Renilla Luciferase Reporter Gene Assay Kit *Bright Glow*/12537/100 plates
产品编号:12537
市  场 价:¥79000.00
场      地:美国(厂家直采)
联系QQ:1570468124
电话号码:4000-520-616
邮      箱: info@ebiomall.com
美  元  价:$3950.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
公司分类:
AAT Bioquest/Amplite™ Renilla Luciferase Reporter Gene Assay Kit *Bright Glow*/12537/100 plates
商品介绍
Overview
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Ex/Em(nm)429/466
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategoryCellBIOLOGy
ReporterGeneEnzymes
RelatedTagEnzymes
BiochemicalAssays
Commonreportergenesincludebeta-galactosidase,beta-glucuronidaseandluciferase.Themostversatilereportergeneisthefireflyluciferase.RecentlythereissteADIlyincreasinguseofotherluciferases,suchasRenillaluciferasesincethesereportersaresmalleranddonotrequirethepresenceofATP.OurAmplite™RenillaLuciferaseReporterGeneAssayKitisdesignedtoprovideafastandsensitivemethodtodetecttheluciferasefromseapansy(Renillareniformis).ItusesaproprietaryluminogenicformulationtoquantifyRenillaluciferaseactivityincell-basedassays.OurformulationgeneratesaluminescentproductthatgivesstrongluminescenceuponinteractionwithRenillaluciferase.Thekitprovidesalltheessentialcomponents.Ithashighsensitivityandcanbeperformedinaconvenient96-welland384-wellmicrotiter-plateformat.The"glow-type"signalwithahalf-lifeofonehourprovidesaconsistentsignalacrosslargenumberofassayplates.TheassayiscompatIBLewithstandardcellgrowthmedia.ThiskitenablesthemeasurementofprimaryexpressionorgeneexpressionwithwildtypeandthesynthetichRlucgenes.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

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
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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.


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