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AAT Bioquest/Amplite™ Luciferase Reporter Gene Assay Kit *Bright Glow*/12519/10 plates
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | None/560 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy ReporterGeneEnzymes |
| Related | TagEnzymes |
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.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortotheexperiment.
Note1:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.Cellsmaybeseededthedaybeforeoronthedayoftheexperimentdependinguponthecelltypeand/ortheeffectofthetestcompounds.
Note2:Forallluminescentexperiments,itisrecommendedtousewhiteplatestogetthebestresults.
2.Prepareluciferaseassaysolution:
2.1 Thawallthekitcomponentstoroomtemperaturebeforeuse.
2.2 ForCat.#12518,transferthewholecontentofReactionBuffer(ComponentB)intothebottleofLuciferaseSensor(ComponentA),andmixwell.
ForCat.#12519,add10mLofReactionBuffer(ComponentB)andforCat.#12520,add100mLofReactionBuffer(ComponentB)intothebottleofLuciferaseSensor(ComponentA),andmixwell.TransfertheresultedsolutionbacktothebottleofReactionBuffer(ComponentB).Multiplewashesarenecessarytocompletelytransferthecontents.
Note:Thereconstitutedluciferaseassaysolutionisnotstable.Aliquotandstoretheunusedreconstitutedluciferaseassaysolutionat-80oC.Avoidfreeze/thawcycles.
3.Runluciferaseassay:
3.1 Treatcells(orsamples)withtestcompoundsbyadding10µLof10Xtestcompounds(96-wellplate)or5µLof5Xtestcompounds(384-wellplate)indesiredcompoundbuffer.Forblankwells(mediumwithoutthecells),addthecorrespondingamountofcompoundbuffer.
3.2 Incubatethecellplateina5%CO2incubatorat37oCfordesiredperiodoftime,typically4hourstoovernight.
3.3 Add100µL(96-wellplate)or25µL(384-wellplate)perwellofluciferaseassaysolution(fromStep2.2)andincubatetheplateatroomtemperaturefor10-20minutes.Keepfromlight.
3.4 Monitorluminescenceintensitywithaluminometer.
4.Establishstandardluciferasecalibrationcurve:
Luciferasestandardcurveshouldbegeneratedtogetherwiththeaboveassayiftheabsoluteamountofluciferaseinsamplesneedstobecalculated.
4.1 MakeaseriesdilutionsofluciferaseinPBSbufferwith0.1%BSAbyincludingasamplewithoutluciferase(asacontrol)formeasuringbackgroundluminescence.
Note:Typicallyluciferaseconcentrationsfrom1pg/mLto1ng/mLareappropriate.
4.2 Addthesameamountofthedilutedluciferasesolutionintoanemptyplate(100µLfora96-wellplate,25µLfora384-wellplate).
4.3 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofluciferaseassaysolution(fromStep2.2).
4.4 Incubatethereactionmixtureatroomtemperaturefor10-20minutes,keptfromlight.
4.5 Recordtheluminescenceintensitywithastandardluminometer.
4.6 Generatetheluciferasestandardcurve.
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DiscoveryofML367,inhibitorofATAD5stABIlization
Authors:JasonMRohde,GaneshaRai,YongJunChoi,SrilathaSakamuru,JenniferTFox,RuiliHuang,MenghangXia,KyungjaeMyung,MatthewBBoxer,DavidJMaloney
Journal:(2013)
