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NEB/BioLux® Gaussia Luciferase Assay Kit/E3300S/100 assays
  • NEB/BioLux® Gaussia Luciferase Assay Kit/E3300S/100 assays

NEB/BioLux® Gaussia Luciferase Assay Kit/E3300S/100 assays

价格: ¥1092.00 市场价: 1820.00
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货号: E3300S-100assays
品牌: NEB
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    • Description:

      TheBioLux®GaussiaLuciferaseAssayKitcontainsthereagentsnecessaryforassayingGaussiaLuciferase(GLuc)activity,mostcommonlyfromcellculturesupernatants.GaussiaLuciferaseisareporterluciferasefromthemarinecopepodGaussiaprinceps(1,2).GaussiaLuciferasecanbeexpressedinmammaliancellsusingreporterplasmidsavailablefromNEB(RefertotheCompanionProducts).Thisluciferase,whichdoesnotrequireATP,catalyzestheoxidationofthesubstratecoelenterazineinareactionthatproduceslight(Figure1),andhasconsiderableadvantagesoverotherluminescentreportergenes.

      ThiskitnowincludesanadditionalstABIlizercomponent,whichallowstheuseoftheassayinhighthroughputformatorwithouttherequirementofaninjector-equippedluminometer.Thisthree-componentassaysystemprovidestheuserwith2options:(a)usetheassaywithoutstabilizerforenhancedlightoutputor(b)usewiththedesiredamountofstabilizerforenhancedsignalstability.Thestabilizercomponentallowstheuseoftheassayinhighthroughputformatorwithouttherequirementofaninjector-luminometer. Forstandardassaysgivingthehighestactivity,thekitcanbeusedwiththeGLucsubstratemixedintheassaybuffer.Withthestabilizedassayprotocols,thelightemissiondecaysslowlywithahalf-lifeofapproximately25minutes.Theadditionofstabilizerdecreasestheabsolutevalueoflightoutputbutconferssignalstabilityovertime(Figure2).

      TheluminescencemeasuredfromthesupernatantofculturedcellstransfectedwithaplasmidexpressingGLucisproportionaltotheamountofenzymeproduced,whichinturn,reflectstheleveloftranscription.Alternatively,acelllysatesamplecanbeusedfortheassay.AlthoughmostoftheGLucissecreted,thehighsensitivityofGLucallowsmeasurementsfromthecellularfraction.


      Figure1:ThePhoto-oxidationcatalyzedbyGaussiaLuciferase.Figure 1: The Photo-oxidation catalyzed by Gaussia Luciferase.

      Haddock,S.H.D.,McDougall,C.M.andCase,J.F.,TheBioluminescenceWebPage,http://Lifesci.ucsb.edu/~biolum/(created1997;updated2005).
      Figure2:GLuckineticsusingtheBioLuxGLucAssayKitineitherstandardorstabilizedassay.Figure 2: GLuc kinetics using the BioLux GLuc Assay Kit in either standard or stabilized assay.

      Assaysweresetupusingassaysolutionwithoutstabilizerorwiththeindicatedamountsofstabilizer(5µL,8µLor10µLofstabilizerper50µLGLucassaysolution).
      Figure3:StabilityofGaussiaLuciferaseatvarioustemperatures.Figure 3: Stability of Gaussia Luciferase at various temperatures.

      GrowthmediafromGLuc-expressingcells(GLuc-sup)wereincubatedat95°Cand55°Cfor30minutesandallowedtocooltoroomtemperature(25°C)beforeassayingforGLucactivity.
      Figure4:StabilityofGaussiaLuciferaseat37°Coveraperiod ofsevendays.Figure 4: Stability of Gaussia Luciferase at 37°C over a period of seven days.

      GrowthmediafromGLuc-expressingcellsgrownin±β-mercaptoethanol-containingmedia(GLuc-sup&GLuc-sup,β-mercaptoEtOH)wereplacedat37°Candassayedeverydayforaperiodofsevendays.
      Figure5:GaussiaLuciferaseactivityafteraddingGLucassaysolutioncontainingstabilizertoasample.
      Figure 5: Gaussia Luciferase activity after adding GLuc assay solution containing stabilizer to a sample.
      TheGLucassaysolutioncontainingstabilizer(i.e.8μlofstabilizerper50μlGLucassaysolution)wasaddedtoaGLucsampleandthemeasurementsweretakenat1-secondincrements(seeUsageNotes).

      KitComponents

      Thefollowingreagentsaresuppliedwiththisproduct:

      Storeat(°C)Concentration
      BioLux® GLucAssayBuffer41X
      BioLux® GLucSubstrate-20100X
      BioLux® GLucStabilizer4100X

      Notes:

      BecauseofthestabilityofGLuc,theactivitymeasuredinthegrowthmediaofGLuc-expressingculturereflectstheproteinthathasaccumulateduptothetimeofsampling.Forthestandardassaysolution,i.e.solutionthatdoesnotcontainstabilizer,equilibrationoftheassaysolutionisnotnecessary.AfteraddingtheGLucassaysolutiontothesample,werecommendadelayof1-5secondsbeforetakingameasurement.Keepingthedelaytimeconsistentacrossexperimentswillensurereproducibility.Forthestabilizedassaysolution,i.e.,thestabilizer-containingGLucassaysolution,thesolutionshouldbeequilibratedatroomtemperaturefor25minutes(protectfromlightinatightlycappedtube/bottle)beforeaddingtothesample.AfteraddingtheequilibratedGLucassaysolutiontothesample,werecommendadelaytimeof35-40secondsbeforetakingameasurementinordertoreachmaximumlevelofdetection.ThisisespeciallyimportantwhentheGLucactivitylevelislow(e.g.<e4RLU).Forexample,thereadoutobtainedafter35-40secondsofdelayis~e4;whencompareto30,20and10secondsofdelay,thereadoutsareasfollows:~2%decrease(for30secondsofdelay),~7%decrease(for20secondsofdelay),&~20%decrease(for10secondsofdelay)inRLU(refertoFigure5).UsethepreparedGLucassaysolutionwithin24hours.Theunusedportionoftheassaysolutionshouldtightlycappedandstoredat-20˚C.Itshouldbecompletelythawed(inthedark)atroomtemperaturebeforeuse.Thelinearrangeoftheluminometerusedfortheassaymustbeestablished.Thisiseasilydonebyassayingserialdilutionsofasample.Inaddition,theassaysolutionitself,aswellastheconditionedmedia(i.e.growthmediafromuntransfectedcells)shouldbeincludedintheassaytoestablishthebackgroundsignalintheassay.Ifexcessactivityfortheinstrumentrangeisfound,thesampleshouldbedilutedineitherPBSor10%serum-containingmedia.Theintegrationtimecanalsobereduced.Whenassayingtheserialdilutionofasample,itisbesttoassaythemostdilutedsamplesfirstandthemostconcentratedsampleslast.ThiswillhelptominimizefalsereADIngs,i.e.,cross-talkeffect(signalsfromsamplesofhighRLUcrossintothatofthenextsample).Thecross-talkeffectseemstobemorepronouncedwhenplates(whiteorblack)withclear-bottomsareused.TheBioLuxGLucAssayBufferandtheBioLuxGLucStabilizercanbestoredat4˚CwhiletheBioLuxGLucSubstratemustbestoredat-20˚C.
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