首页 > AAT Bioquest > AAT Bioquest/Screen Quest™ Membrane Potential Assay Kit *Red Fluorescence*/36004/1 plate

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AAT Bioquest/Screen Quest™ Membrane Potential Assay Kit *Red Fluorescence*/36004/1 plate

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OverviewPrinterFriendlyVersionEx/Em(nm)635/660MWN/ACAS#N/ASolventN/AStorageF/D/LCategoryIonChannelsMembranePotentialsRelatedBiochemicalAssaysMembranepotentialisthedifferenceinvoltagebetweentheinteriorandexteriorofacell.Themembranepotentialallowsacelltofunctionasabattery,providingpowertooperateavarietyof"moleculardevices"embeddedinthemembrane.Inelectricallyexcitablecellssuchasneurons,membranepotentialisusedfortransmittingsignalsbetweendifferentpartsofacell.Openingorclosingofionchannelsatonepointinthemembraneproducesalocalchangeinthemembranepotential,whichcauseselectriccurrenttoflowrapidlytootherpointsinthemembrane.Ionchannelshavebeenidentifiedasimportantdrugdiscoverytargets.OurScreenQuest™MembranePotentialAssayKitisahomogeneousassaywithfastreadtime.Itusesourproprietarylongwavelengthmembranepotentialindicatortodetectthemembranepotentialchangethatiscausedbytheopeningandclosingoftheionchannels.Theredfluorescenceofthemembranepotentialindicatorusedinthekithasenhancedfluorescenceuponenteringcellsandminimizestheinterferencesresultedfromthescreeningcompoundsand/orcellularautofluorescence.Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.1.PrepareCells:1.1 Foradherentcells:Platecellsovernightingrowthmediumat40,000to80,000cells/well/100µLfora96-wellplateor10,000to20,000cells/well/25µLfora384-wellplate.1.2 Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletsinequalamountofHHBSandMPdye-loADIngsolution(seeStep2.2below)at125,000to250,000cells/well/100µLfora96-wellpoly-Dlysineplateor30,000to60,000cells/well/25µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortotheexperiments.Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityfortheintracellularcalciummobilization.2.PrepareMPdye-loadingsolution:2.1 Thawonebottleof10XMPSensor(ComponentA),andonebottleofHHBS(ComponentB)atroomtemperaturebeforeuse.Note1:1mLof10XMPSensor(ComponentA)isenoughforoneplate.Unused10XMPSensor(ComponentA)canbealiquotedandstoredat<-20oCforafewmonthsifthebottleissealedtightlyandkeptfromlight.Avoidrepeatedfreeze-thawcycles.Note2:HHBS(ComponentB)canbestoredat4oCforconvenience.2.2 MakeMPdye-loadingsolutionforonecellplatebyadding1mLof10XMPSensor(ComponentA)into9mLofHHBS(CompontB),andmixingthemwell.ThisMPdye-loadingsolutionisstableforatleast2hoursatroomtemperature. 3.RunMembranePotentialAssay:3.1 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofMPdye-loadingsolution(fromStep2.2)intothecellplate.Note1:Ifyourscreeningcompoundsinterferewithgrowthmediumandserumfactors,replacethegrowthmediumwithequalvolumeofHHBSbufferbeforeaddingtheMPdye-loadingsolution.Alternatively,cellscanbegrownunderserum-freeconditions.Note2:DoNOTwashthecellsafterdyeloading.3.2 Incubatethedye-loadingplateina5%CO2,37oCincubatorfor30to60minutes.Note:Insomecases,30to60minutesroomtemperatureincubationmayworkbetter.3.3 PreparethecompoundplatesbyusingHHBS(ComponentB)oryourdesiredbuffer.3.4 MonitorthefluorescenceintensityatEx/Em=620/650nm(bottomread).Note:Itisimportanttorunthesignaltestbeforetheexperiment.Differentinstrumentshavetheirownintensityrange.Adjustthesignaltestintensitytothelevelof10%to15%ofthemaximuminstrumentintensitycounts.References&CitationsPrinterFriendlyVersion1. VasilyevDV,ShanQJ,LeeYT,SolovevaV,NawoschikSP,KaftanEJ,DunlopJ,MayerSC,BowlbyMR.(2009)Anovelhigh-throughputscreeningassayforHCNchannelblockerusingmembranepotential-sensitivedyeandFLIPR.JBiomolScreen,14,1119.2. SollyK,CassadayJ,FelixJP,GarciaML,FerrerM,StruloviciB,KissL.(2008)MiniaturizationandHTSofaFRET-basedmembranepotentialassayforK(ir)channelinhibitors.AssayDrugDevTechnol,6,225.3. WeinglassAB,SwensenAM,LiuJ,SchmalhoferW,ThomasA,WilliamsB,RossL,HashizumeK,KohlerM,KaczorowskiGJ,GarciaML.(2008)Ahigh-capacitymembranepotentialFRET-basedassayforthesodium-coupledglucoseco-transporterSGLT1.AssayDrugDevTechnol,6,255.4. LiuCJ,PriestBT,BugianesiRM,DulskiPM,FelixJP,DickIE,BrochuRM,KnausHG,MiddletonRE,KaczorowskiGJ,SlaughterRS,GarciaML,KohlerMG.(2006)Ahigh-capacitymembranepotentialFRET-basedassayforNaV1.8channels.AssayDrugDevTechnol,4,37.5. BenjaminER,SkeltonJ,HanwayD,OlanrewajuS,PruthiF,IlyinVI,LaveryD,VictorySF,ValenzanoKJ.(2005)Validationofafluorescentimagingplatereadermembranepotentialassayforhigh-throughputscreeningofglycinetransportermodulators.JBiomolScreen,10,365.6. FelixJP,WilliamsBS,PriestBT,BrochuRM,DickIE,WarrenVA,YanL,SlaughterRS,KaczorowskiGJ,SmithMM,GarciaML.(2004)Functionalassayofvoltage-gatedsodiumchannelsusingmembranepotential-sensitivedyes.AssayDrugDevTechnol,2,260.7. JensenAA,Brauner-OsborneH.(2004)PharmacologicalcharacterizationofhumanexcitatoryaminoacidtransportersEAAT1,EAAT2andEAAT3inafluorescence-basedmembranepotentialassay.BiochemPharmacol,67,2115.8. JensenAA,KristiansenU.(2004)Functionalcharacterisationofthehumanalpha1glycinereceptorinafluorescence-basedmembranepotentialassay.BiochemPharmacol,67,1789.9. DavidLS,PlakasSM,ElSaidKR,JesterEL,DickeyRW,NicholsonRA.(2003)Arapidassayforthebrevetoxingroupofsodiumchannelactivatorsbasedonfluorescencemonitoringofsynaptoneurosomalmembranepotential.Toxicon,42,191.10. FitchRW,XiaoY,KellarKJ,DalyJW.(2003)Membranepotentialfluorescence:arapidandhighlysensitiveassayfornicotinicreceptorchannelfunction.ProcNatlAcadSciUSA,100,4909.ViewAllAsPDF

美国AAT Bioquest Inc.(前身是ABD Bioquest，Inc.)是一家为从事生命科学研究、诊断研发及药物开发的科学家研发、生产和销售生物分析研究试剂和试剂盒的公司。公司致力于光谱学检测领域，包括吸收(颜色)、荧光和发光技术。AAT Bioquest的产品帮助全世界的科学家和生物医药研究者更好的了解生物化学、免疫学、细胞生物学和分子生物学等领域。AAT Bioquest会不断研发新产品，快速地丰富各个领域的产品。