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AAT Bioquest/Screen Quest™ No Wash Potassium Channel Assay Kit/36551/10 Plates
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OverviewPrinterFriendlyVersionEx/Em(nm)490/525MWN/ACAS#N/ASolventN/AStorageF/D/LCategoryIonChannelsRelatedPotassium(K+)ionchannelplaysanimportantroleinregulatingfundamentalBIOLOGicalprocessesincludingheartrate,hormoneandneurotransmittersecretion,waterandelectrolytebalance.Potassiumionchannelhasbeenconsideredasdrugtargetsfordiseaseindicationsincludingarrhythmia,pain,diabetes,neurologicaldysfunctionsetc.ThepermeABIlityofTl+throughK+channelhasbeenwidelyusedtoassayK+channel.ThecellsthatexpressK+channelofinterests(e.g.hERG,Kv1.3,Kir2.1,KATP)arepre-loadedwithaTl+sensitivedye.Thedyeisnon-fluorescentandispermeabletocellmembrane.Onceinsidethecell,thenon-fluorescentAMesterdyeiscleavedbyendogenousesteraseintoanegativelychargesdyethatstaysinsidecells.WhenastimulusbuffercontaininglowdoseofTl+isaddedtocells,theTl+flowsacrosstheK+channelandbindstoTl+sensitivedye,generatingafluorescentsignal.ThissignalisproportionaltotheactivityofK+channel.IfanantagoNISTorantagonistisaddedtothecells,thefluorescentsignaldecreasesorincreasesrespectively,toreflecttheinhibitedorstimulatedactivityofK+channel.SpectrumAdvancedSpectrumViewer #xAxis div{ display: inline-block; position: relative; left: 0; margin: 0px 35.5px; padding: 5px; } #graphContainer{ display: inline-block; white-space: nowrap; margin: 0 auto; } #visIBLeSpectrum{display:block;margin-left:50px;padding-right:150px;background-color:#000;height:10px;width:653px;}#yAxis{display:inline-block;height:220px;width:50px;}#graphArea{display:inline-block;border-left:solid3pxblack;border-bottom:solid2pxblack;position:relative;height:220px;width:650px}#graphGrid{position:absolute;top:0;left:0;height:220px;width:650px;overflow:hidden;}#graphGridpath{shape-rendering:crispEdges;}#graphContent{position:absolute;top:0;left:0;height:220px;width:650px;}#hoverText{white-space:normal;background-color:#EFEFEF;display:inline-block;width:150px;height:222px;border-left:solid1px#AFAFAF;border-bottom:solid2pxblack;border-right:solid1px#AFAFAF;vertical-align:top;font-size:14px;text-align:center;padding:5px15px;box-sizing:border-box;line-height:150%;}.noselect{-webkit-touch-callout:none;/*iOSSafari*/-webkit-user-select:none;/*Safari*/-khtml-user-select:none;/*KonquerorHTML*/-moz-user-select:none;/*Firefox*/-ms-user-select:none;/*InternetExplorer/Edge*/user-select:none;/*Non-prefixedversion,currentlysupportedbyChromeandOpera*/}Sorry,yourbrowserdoesnotsupportinlineSVG.RelativeIntensity(%)100806040200Sorry,yourbrowserdoesnotsupportinlineSVG. #grid{ stroke: #DFDFDF; stroke-width: 1; } Sorry,yourbrowserdoesnotsupportinlineSVG. .graphLine{ fill-opacity:0.5; stroke-opacity:0.75; } .graphLine:hover{ fill-opacity:0.75; stroke-opacity:1; } Sorry,yourbrowserdoesnotsupportinlineSVG.Movemouseovergridtodisplaywavelength&intensityvalues.300400500600700800900Wavelength(nm)Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds. ol { counter-reset: item; } ol > li { counter-increment: item; } ol ol > li { display: block; } ol ol > li:before { content: counters(item, ".") ". "; margin-left: -35px; } .tableProtocol {border-collapse:collapse;width:100%;} .tableProtocol td{padding:5px; border:solid 1px black;width:8.3%} Preparecells:Foradherentcells:Platecellsovernightingrowthmediumat40,000to80,000cells/well/100µLfora96-wellpolyD-lysineplateor10,000to20,000cells/well/25µLfora384-wellpoly-D-lysineplate.Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletinequalamountofHHBSandTl-520AMdye-loADIngsolution(seeStep2.3below)at125,000to250,000cells/well/100µLfora96-wellpoly-D-lysineplateor30,000to60,000cells/well/25µLfora384-wellpoly-D-lysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortotheexperiments.Note1:Eachcelllineshouldbeevaluatedontheindividualbasistodeterminetheoptimalcelldensityfortheintracellularthalliummobilization.Note2:ForHEK293-KCNH2cellsthatweusedinthisassay,werecommendtoplatecellsat20,000cells/well/100µLina96-wellpoly-Dlysineplateforovernight.Thecellsshouldreach80-90%confluencyontheexperimentday.Preparedye-loadingsolution:Make500XTl-520AMstocksolution:Add20µL(forCat#36550)or200µL(forCat#36551and36552)ofDMSOintothevialofTl-520AM(ComponentA),andmixwell.Note:TheunusedTl-520AMstocksolutioncanbestoredat-20°Cinasingle-usealiquots.Make1Xassaybuffer:Add1mlof10XPluronic®F127Plus(ComponentB)to9mlofHHBS(ComponentC).Makedye-loadingsolution:Add20µLofdyestocksolution(fromStep2.2)into10mlof1Xassaybuffer(fromStep2.3),andmixwell.Incubatecellswithdye-loadingsolution:Addequalvolume(suchas100µL/wellfora96-wellplateor25µL/wellfora384-wellplate)ofDye-LoadingSolution(fromStep2.3)toyourcellplatewithoutremovingthecellmedium.Incubatethedye-ladingplateinthecellincubatorfor1hour.Prepare5Xstimulussolution:Make1Xchloridefreebuffer(ComponentD):Dilute5Xchloridefreebufferto1XchloridefreebufferinddH2O.MakeTl2SO4solution(Notprovided,SigmaCat#208191):DissolveTl2SO4inultrapureH2Otofinalconcentrationof80mM.Note:Tl2SO4istoxic.TakenecessaryprecautionstopreventinhalationandskincontactPreparea5XStimulussolutionbydilutingligands(fornon-voltagegatedpotassiumchannels)orK2SO4(forvoltagegatedpotassiumchannels)withTl2SO4in1XChloride-freebuffer.Pleaseusethefollowingtableasareference(forvoltagegatedhERGchannelinHEK293-KCNH2cells),concentrationofTl2SO4andK2SO4(forvoltagegatedpotassiumchannels)orligands(fornon-voltagegatedpotassiumchannels)usedfortheassayshouldbeoptimizedforeachtargetchannelandcelltype.ComponentVolume5XConcentrationFinalConcentration5XChloride-freeAssayBuffer(ComponentD)2mL250mMPotassiumSulfate(K2SO4)(ComponentE)1mL12.5mM2.5mM80mMThalliumSulfate(Tl2SO4)(notprovided)0.5mL2.5mM0.5mMddH2O5.5mLTotal:10mLRunthepotassiumassay:PreparetheK+channelantagonistsoragonist(from4.3)inHHBS.Incubatetheplatewithantagonists(forinhibitorystudy)inthecellincubatorfor30minutes.Add50µL/well(fora96-wellplate)or12.5µL/well(fora384-wellplate)of5XstimulusbufferwithFLIPR,FDSSorFlexstation.RuntheexperimentwithafiltersetofEx/Em=490/525nm.Readtheplateevery1–2secondsfor3minutes.

美国AAT Bioquest Inc.(前身是ABD BioquestInc.)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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