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AAT Bioquest/Screen Quest™ Membrane Potential Assay Kit *Orange Fluorescence*/36000/10 plates
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OverviewPrinterFriendlyVersionEx/Em(nm)535/560MWN/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/100µlfor96-wellor10,000to20,000cells/well/25µlfor384-wellplates.1.2 Fornon-adherentcells,centrifugethecellsfromtheculturemediumandthensUSPendthecellpelletsinequalamountofHHBSandMPdye-loADIngsolution(seeSteps2.3below)at125,000to250,000cells/well/100µlfor96-wellor30,000to60,000cells/well/25µlfor384-wellpoly-Dlysineplates. Centrifugetheplatesat800rpmfor2minuteswithbreakoffpriortotheexperimentsNote: Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityforthemembranepotentialchange. 2.PrepareMPsensordye-loadingsolution(for1plate) 2.1 Thaw1vialofComponentA(MPsensor),ComponentB(10XAssaybuffer)andComponentC(HHBS)atroomtemperaturebeforeuse.Note1: 15µlofcomponentA(MPsensor)isenoughfor1plate,un-usedComponentAcanbealiquotedandstoredat<-20oCformorethan6monthsifthetubesaresealedtightly,avoidinglightandrepeatedfreeze-thawcycles.Note2: ComponentBandCcanbestoredat4oCforconvenience.2.2 Make1Xassaybufferbymixing1mLofComponentB(10XAssaybuffer)with9mLofComponentC(HHBS,notincludedinthekit#36001),mixthemwell.Note: 10ml1Xassaybufferisenoughfor1plate,aliquotandstoreun-used1Xassaybufferat<-20oC,avoidlightandrepeatedfreeze-thawcycles. 2.3 MakeMPdye-loadingsolutionforonecellplatebyadding15µlofComponentA(MPsensor)into10mlof1Xassaybuffer(fromStep2.2),mixingthemwell. Thisworkingsolutionisstableforatleast2hoursatroomtemperature. 3. RunMembranePotentialAssay 3.1 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)MPdye-loadingsolutionintothecellplate.Note1: Ifyouscreencompoundsinterferewithgrowthmediumandserumfactors,thenreplacethegrowthmediumwithequalvolumeofHHBSbufferbeforeaddingtheMPdye-loadingbuffer.Alternatively,cellscanbegrowninserum-freeconditions.Note2: DoNOTwashthecellsafterdyeloading.3.2 Incubatethedye-loadingplateatcellincubatorfor30minutes.Note:Insomecases,incubationatroomtemperaturefor30to60minmayworkbetter.3.3 PreparethecompoundplatesbyusingHHBSoryourdesiredbuffer.3.4 RunthemembranepotentialassaybymonitoringthefluorescenceatEx/Em=530/570nm.Note:Itisimportanttorunthesignaltestbeforeyourexperiment. Differentinstrumentshavetheirownintensityrange.Adjustthesignaltestintensitytothelevelof10%to15%ofthemaximuminstrumentintensitycounts. Forexample,themaximumfluorescenceintensitycountforFLIPR-384is65,000,sotheinstrumentsettingsshouldbeadjustedtohaveitssignaltestintensityaround7,000to10,000.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。
1)提供反应探针和发光探针,生物素和端粒酶能够应用于标记药物小分子和生物聚合物,如蛋白、核酸以及其他碳水化合物;
2)研究并生产荧光和发光探针用于检测蛋白,核酸和活细胞;
3)不断推出新型的荧光和发光探针用于检测多种酶,特别是检测水解酶和氧化还原酶类;
4)致力于开发用于信号转导研究的试剂;
5)提供生理和神经探针,特别是钙离子指示剂和膜电位探针。
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