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NEB/BioLux? Gaussia Luciferase Assay Kit/E3300S/1,000 assays

價格: ￥5724.00 市場價: 9540.00

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貨號: E3300S-1,000assays

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.
  
  Haddock,S.H.D.,McDougall,C.M.andCase,J.F.,TheBioluminescenceWebPage,http://Lifesci.ucsb.edu/~biolum/(created1997;updated2005).
  Figure2:GLuckineticsusingtheBioLuxGLucAssayKitineitherstandardorstabilizedassay.
  
  Assaysweresetupusingassaysolutionwithoutstabilizerorwiththeindicatedamountsofstabilizer(5μL,8μLor10μLofstabilizerper50μLGLucassaysolution).
  Figure3:StabilityofGaussiaLuciferaseatvarioustemperatures.
  
  GrowthmediafromGLuc-expressingcells(GLuc-sup)wereincubatedat95°Cand55°Cfor30minutesandallowedtocooltoroomtemperature(25°C)beforeassayingforGLucactivity.
  Figure4:StabilityofGaussiaLuciferaseat37°Coveraperiod?ofsevendays.
  
  GrowthmediafromGLuc-expressingcellsgrownin±β-mercaptoethanol-containingmedia(GLuc-sup&GLuc-sup,β-mercaptoEtOH)wereplacedat37°Candassayedeverydayforaperiodofsevendays.
  Figure5:GaussiaLuciferaseactivityafteraddingGLucassaysolutioncontainingstabilizertoasample.
  TheGLucassaysolutioncontainingstabilizer(i.e.8μlofstabilizerper50μlGLucassaysolution)wasaddedtoaGLucsampleandthemeasurementsweretakenat1-secondincrements(seeUsageNotes).
  KitComponents
  Thefollowingreagentsaresuppliedwiththisproduct:
  Storeat(°C) Concentration
  BioLux??GLucAssayBuffer 4 1X
  BioLux??GLucSubstrate -20 100X
  BioLux??GLucStabilizer 4 100X
  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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NEB/BioLux? Gaussia Luciferase Assay Kit/E3300S/1,000 assays

Description:

KitComponents

Notes:

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	Storeat(°C)	Concentration
BioLux??GLucAssayBuffer	4	1X
BioLux??GLucSubstrate	-20	100X
BioLux??GLucStabilizer	4	100X