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AAT Bioquest/D-Luciferin, potassium salt *CAS#: 115144-35-9*/12507/1 g
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 328/533 |
| MW | 318.41 |
| CAS# | 115144-35-9 |
| Solvent | Water |
| Storage | F/D/L |
| Category | CellBIOLOGy ReporterGeneEnzymes |
| Related | BiochemicalAssays |
Note1:TheD-luciferinsaltsarereADIlysolubleinaqueousbuffersupto100mM.StocksolutionscanbemadeinATP-freewaterandstoredat-20°C,protectfromlight.Thefreeacidmustbeneutralizedwithanappropriatebasetosolubilize.
Note2:TheD-luciferincanbeusedwithanyexistingreporterassayorATPassaysystem.
Note3:IftestingforATP,minimizeallpossIBLesourcesofATPcontaminationbywearingglovesandusingATP-freecontainers.UseonlysterileATP-freewaterandreagents.Useautoclavedwaterforallreagentpreparations.
Thefollowingprotocolisanexampleforpotassiumandsodiumsaltpreparation,itcanbeadaptedformostcelltypesandinvivoanimaluse.
1. Exampleprotocolforinvitrobioluminescentimageassays
1.1. Preparea100mM(100-200X)Luciferinstocksolutioninsterilewater.Mixwell.Useimmediately,ormakesingleusealiquots,andstoreat-20°C,avoidfreeze-thawcycles,avoidexposuretothelight.
1.2. Preparea0.5-1mMworkingsolutionofD-Luciferininpre-warmedtissueculturemedium.
1.3. Aspiratemediafromculturedcells.
1.4. AddLuciferinworkingsolutiontocells,andincubatethecellsfor5-10minutesat37°Cjustpriortoimaging.
2. Exampleprotocolforinvivobioluminescentimageassays
2.1. Preparea15mg/mLLuciferinstocksolutioninDPBS,w/oMg2+andCa2+.Mixwell.
2.2. Filtersterilizesthesolutionthrougha0.2μmfilter.Useimmediately,ormakesingleusealiquots,andstoreat-20°C,avoidfreeze-thawcycles,avoidexposuretothelight.
2.3. Injecttheluciferinintra-peritoneally(i.p.)10-15minutesbeforeimagingat150mg/kg(or10μL/gofluciferinstocksolution)oftheanimalbodyweight.
Note:Akineticstudyofluciferinshouldbeperformedforeachanimalmodeltodeterminepeaksignaltime.
3. Exampleprotocolforluciferinreporterassays
3.1. Preparea100mMLuciferinstocksolutioninsterilewater.Useimmediately,ormakesingleusealiquots,andstoreat-20°C,avoidfreeze-thawcycles,avoidexposuretothelight.
3.2. Preparea1mMworkingsolutionofD-Luciferinwith3mMATP,1mMDTTand15mMMgSO4in25mMtricinebufferpH7.8.
3.3. Pipette5-10μlofcelllysateintoamicroplate.Uselysisreagentorbufferwithoutlysateasablank.
3.4. Primeluminometerwithluciferinworkingsolutionaccordingtomanufacturer’sinstructions.
3.5. Inject200μlofluciferinworkingsolutionwithnodelayanda10secondintegrationtime.
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