| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 535/560 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | IonChannels MembranePotentials |
| Related | BiochemicalAssays |
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. Centrifugetheplatesat800rpmfor2minuteswithbreakoffpriortotheexperiments
Note: 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&Citations |  PrinterFriendlyVersion |
1. 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.
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我用Promega公司的双荧光素酶检测试剂盒（E2920）检测到的firefly萤光素酶活性很低，只有4*10的3次方；海肾萤光素酶活性有10的6次方。
我用Ad293细胞做了转染，fireflyluciferase质粒：RanillaLuciferase质粒=0.1ug：0.025ug/一个孔（96孔板），共转染了二天，再进行双萤光素酶检测。
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我做了3次重复实验，每次firefly萤光素酶活性很低，只有4*10的3次方；而海肾萤光素酶活性有10的6次方。
求指教？fireflyluciferase活性低？会是什么原因呢？
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牛胰岛素,是一种多肽
在1965年9月17日我国完成了结晶牛胰岛素的全合成。经过严格鉴定，它的结构、生物活力、物理化学性质、结晶形状都和天然的牛胰岛素完全一样。这是世界上第一个人工合成的蛋白质，为人类认识生命、揭开生命奥秘迈出了可喜的一大步。这项成果获1982年中国自然科学一等奖。
1953年，英国人F. SangerSanger由于测定了牛胰岛素的一级结构而获得1958年诺贝尔化学奖。
