| 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:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletsinculturemediumat2,000-20,000cells/90µL/wellfora96-wellpoly-Dlysineplateor500-5,000cells/20µL/wellfora384-wellpoly-Dlysineplate.Centrifugetheplatesat800rpmfor2minuteswithbrakeoffpriortotheexperiment.
Note1:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.Cellsmaybeseededthedaybeforeoronthedayoftheexperimentdependinguponthecelltypeand/ortheeffectofthetestcompounds.
Note2:Forallluminescentexperiments,itisrecommendedtousewhiteplatestogetthebestresults.
2.PrepareGaussialuciferaseassaysolution:
2.1 Make100XGaussialuciferaseassaystocksolution:Transfer50µL(for#12530),0.5mL(for#12531)and2.5mL(for#12532)ofReactionBuffer(ComponentB)into1vialofLuciferaseSubstrate(ComponentA),andmixthemwell.
Note:Storetheunused100XGaussiaLuciferasesubstratestocksolutionat-20°C,andkeepfromlight.
2.2 MakeGaussialuciferaseassaysolution:Addonevolumeof100XGaussiaLuciferaseassaystocksolution(fromStep2.1)to100volumesofAssayBuffer(ComponentC).
Note:ThereconstitutedGaussialuciferaseassaysolutionisverysensitivetolight,shouldbekeptfromlight.Anditisnotstable,shouldbepreparedfresh,keptoniceandusedwithin2hours.
3.RunGaussialuciferaseassay:
3.1 Treatcells(orsamples)withtestcompoundsbyadding10µLof10Xtestcompounds(96-wellplate)or5µLof5Xtestcompounds(384-wellplate)indesiredcompoundbuffer.Forblankwells(mediumwithoutthecells),addthecorrespondingamountofcompoundbuffer.
3.2 Incubatethecellplatesina37oC,5%CO2incubatorforadesiredperiodoftime,typically4hourstoovernight.
3.3 RunGaussiaLuciferaseAssay:Pipette50µL/well/96-wellplateor12.5µL/well/384-wellplateoftheserialdilutedGaussialuciferaseorculturesupernatantintoamicrotiterplate,andthenmixwith50µL/well/96-wellplateor12.5µL/well/384-wellplateoftheGaussialuciferaseassaysolution(fromStep2.2).Incubatetheplateatroomtemperaturefor10to15minutes,keptfromlight.
3.4 Readluminescenceintensitywithaluminometer.
| References&Citations |  PrinterFriendlyVersion |
1. ChungE,YamashitaH,AuP,TannousBA,FukumuraD,JainRK.(2009)SecretedGaussialuciferaseasabioMarkerformonitoringtumorprogressionandtreatmentresponseofsystemicmetastases.PLoSOne,4,e8316.
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3. KimSB,SatoM,TaoH.(2009)SplitGaussialuciferase-basedbioluminescencetemplatefortracingproteindynamicsinlivingcells.AnalChem,81,67.
4. MaguireCA,DeliolanisNC,PikeL,NiersJM,Tjon-Kon-FatLA,Sena-EstevesM,TannousBA.(2009)Gaussialuciferasevariantforhigh-throughputfunctionalscreeningapplications.AnalChem,81,7102.
5. PatelKG,NgPP,KuoCC,LevyS,LevyR,SwartzJR.(2009)Cell-freeproductionofGaussiaprincepsluciferase--antibodyfragmentbioconjugatesforexvivodetectionoftumorcells.BiochemBiophysResCommun,390,971.
6. SantosEB,YehR,LeeJ,NikhaminY,PunzalanB,LaPerleK,LarsonSM,SadelainM,BrentjensRJ.(2009)SensitiveinvivoimagingofTcellsusingamembrane-boundGaussiaprincepsluciferase.NatMed,15,338.
7. TannousBA.(2009)Gaussialuciferasereporterassayformonitoringbiologicalprocessesincultureandinvivo.NatProtoc,4,582.
8. GoerkeAR,LoeningAM,GambhirSS,SwartzJR.(2008)Cell-freemetabolicengineeringpromoteshigh-levelproductionofbioactiveGaussiaprincepsluciferase.MetabEng,10,187.
9. InouyeS,SaharaY.(2008)IdentificationoftwocatalyticdomainsinaluciferasesecretedbythecopepodGaussiaprinceps.BiochemBiophysResCommun,365,96.
10. RueckerO,ZillnerK,Groebner-FerreiraR,HeitzerM.(2008)Gaussia-luciferaseasasensitivereportergeneformonitoringpromoteractivityinthenucleusofthegreenalgaChlamydomonasreinhardtii.MolGenetGenomics,280,153.
