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℡ 4000-520-616
AAT Bioquest/D-Luciferin, sodium salt *CAS#: 103404-75-7*/12509/25 mg
产品编号:12509
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AAT Bioquest/D-Luciferin, sodium salt *CAS#: 103404-75-7*/12509/25 mg
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Ex/Em(nm)328/533
MW302.3
CAS#103404-75-7
SolventWater
StorageF/D/L
CategoryCellBIOLOGy
ReporterGeneEnzymes
RelatedBiochemicalAssays
Luciferinisthemostpopularandversatilebioluminescentsubstrate.Thefireflyluciferase/luciferinbioluminescentsystemisfoundinthefirefly(Photinuspyralis)andseveralotherbeetles.LuciferaseoxidizesATP-activatedluciferinthroughadioxetanoneintermediate.FireflyluciferaseproduceslightbytheATP-dependentoxidationofluciferin.The560nmchemiluminescencefromthisreactionpeakswithinseconds,withlightoutputthatisproportionaltoluciferaseactivitywhenluciferinandATParepresentinexcess.Fireflyluciferasehaslongbeenconjugatedtoantibodiesandusedasalabelinimmunoassaysusingluciferinasthesubstratefordetection.ComparedtoHRPandalkalinephosphatase,luciferaseislesstoleranttochemicalmodifications.Oneparticularadvantagetotheenzymeisthatthereislowendogenousluciferaseactivityinmammaliantissuesbesidesitshighsensitivity.Anotherimportantuseofluciferaseisintheareaofhygienemonitoring.Theluciferase/luciferinsystemcanbeusedtodetectcontaminationbecauseATP,presentinalllivingorganisms,isrequiredtoproduceluminescence.ThemainapplicationforthistypeofATPbioluminescenceisqualityassurancebytestingsurfacesinfoodprocessingplantstodeterminewhetherornotthereiscontaminationofeitherequipmentorproducts.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

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.

References&Citations
CitationExplorer

C3-LucCellsAreanExcellentModelforEvaluationofCellularImmunityfollowingHPV16L1Vaccination
Authors:Li-LiLi,He-RongWang,Zhi-YiZhou,JingLuo,Xiao-LiWang,Xiang-QianXiao,Yu-BaiZhou,YiZeng
Journal:PloSone(2016):e0149748

Genome-widemicroRNAanalysisidentifiesmiR-188-3pasnovelprognosticMarkerandmolecularfactorinvolvedincolorectalcarcinogenesis
Authors:MartinPichler,VerenaStiegelbauer,PetraVychytilova-Faltejskova,CristinaIvan,HuiLing,ElkeWinter,XinnaZhang,MatthewGoblirsch,AnnikaWulf-Goldenberg,MasahisaOhtsuka
Journal:AmericanAssociationforCancerResearch(2016):clincanres--0497

IdentificationofaNovelProteinKinaseAInhibitorbyBioluminescence-BasedScreening
Authors:TetsuyaIshimoto,KenjiAzechi,HisashiMori
Journal:BiologicalandPharmaceuticalBulletin(2015):1969--1974

Discoveryofnoveladenylylcyclaseinhibitorbycell-basedscreening
Authors:HirokiMano,TetsuyaIshimoto,TakuyaOkada,NaokiToyooka,HisashiMori
Journal:BiologicalandPharmaceuticalBulletin(2014):1689--1693


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