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NEB/BioLux® Cypridina Luciferase Assay Kit/E3309S/100 assays
  • NEB/BioLux® Cypridina Luciferase Assay Kit/E3309S/100 assays

NEB/BioLux® Cypridina Luciferase Assay Kit/E3309S/100 assays

价格: ¥1272.00 市场价: 2120.00
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货号: E3309S-100assays
品牌: NEB
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    • Description:

      TheBioLuxCypridinaLuciferaseAssayKitcontainsthereagentsnecessaryforassayingCypridinaLuciferaseactivity.CypridinaisanewsecretedluciferasereporterfromtheostracodCypridinanoctiluca.ThisluciferasedoesnotrequireATPandcatalyzestheoxidationofitsluciferinsubstrateinaphotochemicalreaction(1,2)(Figure1).ThesubstrateforCypridinaisdifferentthancoelenterazine,whichisthecommonsubstrateofothermarineluciferasesincludingRenillaandGaussia.

      PropertiesofCypridinaLuciferase: 

      CypridinaLuciferaseissecretedfromcellsbyvirtueofitsnaturalsignalpeptideanditsluminescencecanbemeasuredfromthesupernatantoftransfectedcells.Therefore,celllysisisnotnecessary(Figure2). 

      SecretedCLucisaverystableprotein.Becauseofthisproperty,theactivitymeasuredfromthesupernatantreflectstheamountofproteinaccumulateduptothetimeofsampling.Multiplesamplescanthereforebeobtainedfromthesametransfectedcells(Figure2). 

      TheCLucassayisverysensitive,allowingdetectionofverysmallamountsofCypridinaLuciferaseactivity(Figure3). 

      SecretedCLucisthermallystableat55°C(Figure4A),whichisthetypicalinactivationtemperatureofmostviruses.SecretedCLucremainsactiveinthepresenceofβ-mercaptoethanol,whichiscommonlypresentinthecompleteculturemediumofmouseEScells(Figure4B). 

      AlthoughthelightreactioncatalyzedbyCypridinahasaninitialemissionhalf-lifeofapproximately5minutes,lightproductioncontinuestodecayslowly,andisreADIlydetectable25minutesaftersubstrateaddition(Figure5).

      The0.5mlBioLuxCypridina LuciferaseSubstrateSolventmustbecompletelythawedanddilutedwithabsoluteethanol(notincluded)beforeusingittomakethe100Xsubstratesolution(pleaserefertothesectionofReconstitutionofBioLuxCypridinaLuciferaseSubstrate).

      Figure1:ThephotochemicalreactioncatalyzedbyCypridinaLuciferase.Figure 1: The photochemical reaction catalyzed by Cypridina Luciferase.

      Figure2:ActivityofsecretedCLucatdifferenttimepoints.Figure 2: Activity of secreted CLuc at different time points.

      Supernatantsoftransfectedcellswerecollectedandmediumwasreplacedeachdayfor4days.Thesesupernatantswerestoredat-20°Cuntilthelastsampleswereobtained.RelativeLightUnits,RLU.
      Figure3:SensitivityofCypridinaLuciferaseassay.Figure 3: Sensitivity of Cypridina Luciferase assay.

      TheCLucactivitywasassayedfrom10-foldserialdilutionsofthesupernatantfromastableCLuc-expressingcell line.
      Figure4:StABIlityofCypridinaLuciferase.Figure 4: Stability of Cypridina Luciferase.

      (A)SupernatantobtainedfromstableCLuc-expressingcellswasincubatedat55°Cor95°Cfor30minutesandallowedtocooltoroomtemperature(25°C).Five-foldserialdilutionsofsupernatantwereassayedforCLucactivity.Thecontrolisthesupernatantoftheparentalcellsfromwhichthestablecelllinewascreated.(B)SupernatantsfromstableCLuc-expressingcells,growninmediumwithorwithoutβ-mercaptoethanol,wereincubatedat37°Candassayedeachdayfor7days.
      Figure5:Kineticsoflightemission.Figure 5: Kinetics of light emission.

      TheCypridinaLuciferasekineticassaywasperformedwiththesupernatantfromHeLacellstransiently
      transfectedwithaCLuc-expressingvector.Theassaysolutionwaspreparedandincubatedatroom
      temperaturefor30minutesbeforeuse.
      Figure6:CypridinaLuciferaseAssaysFigure 6: Cypridina Luciferase Assays

      Figure7:ActivityofCypridinaLuciferaseinthesupernatantandlysatefromastableCLuc-expressingcellline.Figure 7: Activity of Cypridina Luciferase in the supernatant and lysate from a stable CLuc-expressing cell line

      TheCLucactivitywasmeasuredfrom20μlofsupernatant(from500μltotalvolume)andfrom20μlofcelllysate(100μltotallysatevolume)ofastableCLuc-expressingcellline.

      KitComponents

      Thefollowingreagentsaresuppliedwiththisproduct:

      Storeat(°C)Concentration
      BioLuxClucAssayBuffer1X
      BioLuxClucSubstrateSolvent
      lyophilizedBioLuxClucSubstrate

      Notes:

      StoretheBioLuxCypridinaLuciferaseAssayBuffer(1X)at-20°Cforupto2years.TheBioLuxCypridinaLuciferaseAssayBuffer(1X),thereconstitutedCLucsubstrate(100Xsolution)andtheCLucassaysolutionmustbeprotectedfromlight.TheCLucassaysolutionshouldbefreshlypreparedandincubatedatroomtemperaturefor30minutes(protectfromlight)beforeuse.Thelinearrangeoftheluminometermustbeestablished.ThiscanbeeasilydonebyassayingserialdilutionsofaCLuccontainingsample(Figure3),suchastheculturemediumofCLuc-expressingcells.Inaddition,theassaysolutionitselfaswellastheconditionedmedium(culturemediumfromuntransfectedcells)shouldbeincludedtoestablishthebackgroundintheassay.Ifexcessactivityfortheinstrumentrangeisfound,thesampleshouldbedilutedineitherPBSor10%serum-containingmedium.Theintegrationtimecanalsobereduced,e.g.2secondsinsteadof5seconds.Thepresenceofserum,i.e.culturemedium,typicallyincreasesthebackgroundsignalintheassay.Forexample,theCLucassaysolutionaloneshows101–102RLU;theCLucassaysolutionin10%FBS-containingmediumresultsin102–104RLU,dependingonthetypeofmediumused.Therecommendedintegrationtimefortheluminescencemeasurementis2–10seconds.Itisimportanttokeeptheintegrationtimeconstant,inordertoobtainconsistentresults.BioLuxCypridinaLuciferaseAssayBuffer(1X)andSubstrate(lyophilizedpowder)mustalwaysbeprotectedfromlight.BioLuxCypridinaLuciferaseSubstrateissuppliedinlyophilizedformtoensurelongshelflifeandmaximumstability.
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