AAT生物任务/屏幕任务与交易；无洗涤钾通道分析试剂盒/36551/10板-免疫在线蚂蚁淘旗下平台

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商品详细AAT生物任务/屏幕任务与交易；无洗涤钾通道分析试剂盒/36551/10板

AAT生物任务/屏幕任务与交易；无洗涤钾通道分析试剂盒/36551/10板

商品编号： 36551

品牌： aatbio

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产地：美国(厂家直采)

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产品分类：其它检测试剂盒

公司分类： Other_kits

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Ex/Em(nm)	490/525
MW	N/A
CAS#	N/A
Solvent	N/A
Storage	F/D/L
Category	IonChannels
Related

Potassium(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.

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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

Preparecells:
1. Foradherentcells:Platecellsovernightingrowthmediumat40,000to80,000cells/well/100µLfora96-wellpolyD-lysineplateor10,000to20,000cells/well/25µLfora384-wellpoly-D-lysineplate.
2. 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:
1. Make500XTl-520AMstocksolution:Add20µL(forCat#36550)or200µL(forCat#36551and36552)ofDMSOintothevialofTl-520AM(ComponentA),andmixwell.
  Note:TheunusedTl-520AMstocksolutioncanbestoredat-20°Cinasingle-usealiquots.
2. Make1Xassaybuffer:Add1mlof10XPluronic®F127Plus(ComponentB)to9mlofHHBS(ComponentC).
3. Makedye-loadingsolution:Add20µLofdyestocksolution(fromStep2.2)into10mlof1Xassaybuffer(fromStep2.3),andmixwell.

Incubatecellswithdye-loadingsolution:
1. Addequalvolume(suchas100µL/wellfora96-wellplateor25µL/wellfora384-wellplate)ofDye-LoadingSolution(fromStep2.3)toyourcellplatewithoutremovingthecellmedium.
2. Incubatethedye-ladingplateinthecellincubatorfor1hour.

Prepare5Xstimulussolution:

Make1Xchloridefreebuffer(ComponentD):Dilute5Xchloridefreebufferto1XchloridefreebufferinddH2O.
MakeTl2SO4solution(Notprovided,SigmaCat#208191):DissolveTl2SO4inultrapureH2Otofinalconcentrationof80mM.
Note:Tl2SO4istoxic.Takenecessaryprecautionstopreventinhalationandskincontact

Preparea5XStimulussolutionbydilutingligands(fornon-voltagegatedpotassiumchannels)orK2SO4(forvoltagegatedpotassiumchannels)withTl2SO4in1XChloride-freebuffer.

Pleaseusethefollowingtableasareference(forvoltagegatedhERGchannelinHEK293-KCNH2cells),concentrationofTl2SO4andK2SO4(forvoltagegatedpotassiumchannels)orligands(fornon-voltagegatedpotassiumchannels)usedfortheassayshouldbeoptimizedforeachtargetchannelandcelltype.

Component	Volume	5XConcentration	FinalConcentration
5XChloride-freeAssayBuffer(ComponentD)	2mL
250mMPotassiumSulfate(K2SO4)(ComponentE)	1mL	12.5mM	2.5mM
80mMThalliumSulfate(Tl2SO4)(notprovided)	0.5mL	2.5mM	0.5mM
ddH2O	5.5mL
Total:	10mL

Runthepotassiumassay:
1. PreparetheK+channelantagonistsoragonist(from4.3)inHHBS.
2. Incubatetheplatewithantagonists(forinhibitorystudy)inthecellincubatorfor30minutes.
3. Add50µL/well(fora96-wellplate)or12.5µL/well(fora384-wellplate)of5XstimulusbufferwithFLIPR,FDSSorFlexstation.RuntheexperimentwithafiltersetofEx/Em=490/525nm.Readtheplateevery1–2secondsfor3minutes.

品牌介绍

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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淋巴细胞信号传导 GPCR抗体羰基活性生物素细胞代谢胺反应生物素链霉亲和素结合物生物素化抗IgGs 乙酰基特异性抗体细胞骨架抗体

受体结合试验香豆素类经典标记染料神经酶 cAMP-GPCR分析糖蛋白类荧光成像 Boc氨基酸巯基活性生物素淬火CPG 蛋白激酶类解理特异性抗体胺到羧基细胞功能分析 pH和离子指示剂 iFluor快速测试诊断分子氯离子通道潮汐染料蛋白激酶Ab 生物素结合物金属离子葡萄糖转运蛋白脂肪酸转运体 MDR研究水解酶磷酸特异性抗体膜电位经典淬火剂胺和硫醇改性剂一般蛋白质类胆汁酸转运蛋白粘附力Ab trFluor染料和试剂盒 DNA检测 β淀粉样蛋白分析转移酵素潮汐荧光染料和试剂盒 Fmoc氨基酸细胞周期Ab 标记单元格染料CPGs 细胞凋亡菁信号中间体Ab 荧光碳水化合物标签酶标抗IgGs 癌症研究所流式细胞术肽酶和蛋白酶报告基因酶细胞凋亡与细胞毒性磷酸二酯酶活性氧种类 Cytocrhome P450酶重要污点染料标记试剂染色积木标记抗体神经抗体荧光抗IgGs 离子通道Ab 辣根过氧化物酶（HRP）磷酸酶钙GPCR测定猝灭磷酰胺细胞毒性氧化还原酶 RNA检测转录抗体 mFluor染料和试剂盒翻译Ab 染料磷酰胺岩 iFluor染料和试剂盒组蛋白脱乙酰酶（HDAC）肽标记试剂离子通道植物蛋白阴离子

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