Overview | PrinterFriendlyVersion |
Ex/Em(nm) | 494/517 |
MW | 909.09 |
CAS# | N/A |
Solvent | Water |
Storage | F/D/L |
Category | GPCR CalciumGPCRAssays |
Related | CalciumChannels pHandIonIndicators BiochemicalAssays |
Spectrum | AdvancedSpectrumViewer |
UseofFluo-8®AMEsters
1.LoadCellswithFluo-8®AMEsters:
AMestersarethenon-polarestersthatreadilycrosslivecellmembranes,andrapidlyhydrolyzedbycellularesterasesinsidelivecells.AMestersarewidelyusedforloadingavarietyofpolarfluorescentprobesintolivecellnon-invasively.However,cautionsmustbeexcisedwhenAMestersareusedsincetheyaresusceptibletohydrolysis,particularlyinsolution.Theyshouldbereconstitutedjustbeforeuseinhigh-quality,anhydrousdimethylsulfoxide(DMSO).DMSOstocksolutionsmaybestoreddesiccatedat–20°Candprotectedfromlight.Undertheseconditions,AMestersshouldbestableforseveralmonths.
FollowingisourrecommendedprotocolforloadingFluo-8®AMestersintolivecells.Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.
a) Preparea2to5mMstocksolutionofFluo-8®AMestersinhigh-quality,anhydrousDMSO.
b) Onthedayoftheexperiment,eitherdissolveFluo-8®inDMSOorthawanaliquotoftheindicatorstocksolutiontoroomtemperature.Prepareaworkingsolutionof1to10µMinHanksandHepesbuffer(HHBS)orthebufferofyourchoicewith0.02%Pluronic®F-127.Formostofcelllines,Fluo-8®reagentswithaconcentrationrangingfrom4-5uMarerecommended.Theexactconcentrationoftheindicatorrequiredforcellloadingmustbedeterminedempirically.Toavoidanyartifactscausedbyoverloadingandpotentialdyetoxicity,itisrecommendedtousetheminimaldyeconcentrationthatcangeneratesufficientsignalstrength.
Note:ThenonionicdetergentPluronic®F-127issometimesusedtoincreasetheaqueoussolubilityofFluo-8®AMesters. AvarietyofPluronic®F-127solutionscanbepurchasedfromAATBioquest.
c) Ifyourcellscontainingtheorganicanion-transports,probenecid(1–2.5mM)orsulfinpyrazone(0.1–0.25mM)maybeaddedtothecellmediumtoreduceleakageofthede-esterifiedindicators.
Note:AvarietyofReadiUse™probenecidincludingwatersolublesodiumsaltandstABIlizedsolutioncanbepurchasedfromAATBioquest.
d) Addequalvolumeofthedyeworkingsolution(fromStepborc)intoyourcellplate.
e) Incubatethedye-loadingplateatacellincubatororroomtemperaturefor20minutestoonehour.
Note:Decreasingtheloadingtemperaturemightreducethecompartmentalizationoftheindictor.
f) ReplacethedyeworkingsolutionwithHHBSorbufferofyourchoice(containingananiontransporterinhibitor,suchas2.5mMprobenecid,ifapplicable)toremoveexcessprobes.
g) RuntheexperimentsatEx/Em =490/525nm
UseofScreenQuest™Fluo-8NWCalciumAssayKitsforHTSApplications
GPCRactivationcanbedetectedbydirectmeasurementofthereceptormediatedcAMPaccumulation,orchangesinintracellularCa2+concentration.GPCRtargetsthatcoupleviaGqproduceanincreaseinintracellularCa2+thatcanbemeasuredusingacombinationofFluo-8®reagentsandafluorescencemicroplatereader.Thefluorescenceimagingplatereaders(suchas,FLIPR™,FDSSorBMGNovoStar™)haveacooledCCDcameraimagingsystemwhichcollectsthesignalfromeachwellofamicroplate(both96and384-well)simultaneously.Theseplatereaderscanreadatsub-secondintervals,whichenablesthekineticsoftheresponsetobecaptured,andhasanintegratedpipettorthatmaybeprogrammedforsuccessiveliquidadditions.BesidestheirrobustapplicationsforGPCRtargets,ourScreenQuest™Fluo-8CalciumAssayKitscanbealsousedforcharacterizingcalciumionchannelsandscreeningcalciumionchannel-targetedcompounds.
Figure2.CarbacholDoseResponsewasmeasuredinHEK-293cellswithScreenQuest™Fluo-8NWAssaykitandFluo-4NWAssayKit.HEK-293cellswereseededovernightat40,000cells/100µL/wellina96-wellblackwall/clearbottomcostarplate.Thegrowthmediumwasremoved,andthecellswereincubatedwith,respectively,100µLoftheScreenQuest™Fluo8-NWcalciumassaykitandFluo-4NWkit(accordingtothemanufacturer’sinstructions)for1houratroomtemperature.Carbachol(25µL/well)wasaddedbyNOVOstar(BMGLabTech)toachievethefinalindicatedconcentrations.TheEC50ofFluo-8NWisabout1.2uM.
ComparedtoothercommercialcalciumassaykitsthateitherbasedonFluo-3orFluo-4,ourScreenQuest™CalciumAssayKitshavethefollowingadvantagesforHTSapplications:
•BroadApplications:workwithbothGPCRandcalciumchanneltargets.
•ConvenientSpectralWavelengths:maximumexcitation@~490nm;maximumemission@~514nm.
•FlexibleDyeLoading:dyeloadingatroomtemperature(ratherthan37ºCrequiredforFluo-4AM).
•NoWashRequiredandNoQuencherInterferencewithYourTargets.
•RobustPerformance:enablecalciumassaysthatareimpossiblewithFluo-4AMorFluo-3AM.
•StrongestSignalIntensity:2timesbrighterthanthatofFluo-4AM;4timesbrighterthanthatofFluo-3AM.
UseofFluo-8®Salts
Calciumcalibrationcanbecarriedoutbymeasuringthefluorescenceintensityofthesaltform(25to50µMinfluorescencemicroplatereaders)oftheindicatorsinsolutionswithpreciselyknownfreeCa2+concentrations.Calibrationsolutionscanbeusedbasedon30mMMOPSEGTACa2+buffer.Ingeneral,watercontainstraceamountofcalciumion.Itishighlyrecommendedtouse30mMMOPS+100mMKCl,pH7.2asbuffersystem.Onecansimplymakea0and39µMcalciumstocksolutionsaslistedbelow,andthese2solutionsareusedtomakeaserialsolutionofdifferentCa2+concentrations
A.0µMcalcium:30mMMOPS+100mMKCl,pH7.2buffer+10mMEGTA
B.39µMcalcium:30mMMOPS+100mMKCl,pH7.2buffer+10mMEGTA+10mMCaCl2
TodetermineeitherthefreecalciumconcentrationofasolutionortheKdofasingle-wavelengthcalciumindicator,thefollowingequationisused:
[Ca]free=Kd[F─Fmin]/Fmax─F]
WhereFisthefluorescenceintensityoftheindicatorataspecificexperimentalcalciumlevel,FministhefluorescenceintensityintheabsenceofcalciumandFmaxisthefluorescenceintensityofthecalcium-saturatedprobe.
Thedissociationconstant(Kd)isameasureoftheaffinityoftheprobeforcalcium.Thecalcium-bindingandspectroscopicpropertiesoffluorescentindicatorsvaryquitesignificantlyincellularenvironmentscomparedtocalibrationsolutions.InsituresponsecalibrationsofintracellularindicatorstypicallyyieldKdvaluessignificantlyhigherthaninvitrodeterminations.InsitucalibrationsareperformedbyexposingloadedcellstocontrolledCa2+buffersinthepresenceofionophoressuchasA-23187,4-bromoA-23187andionomycin.Alternatively,cellpermeabilizationagentssuchasdigitoninorTriton®X-100canbeusedtoexposetheindicatortothecontrolledCa2+levelsoftheextracellularmedium.TheKdvaluesofFluo-8®reagentsarelistedinTable1foryourreference.
References&Citations | CitationExplorer |
Below,youmayfindasmallsamplingofspecificFluo-8®AMapplicationssortedbyfieldofstudy.ToinquireaboutapotentialapplicationofFluo-8®AM,ortoconsultwithourfluorescentdyespecialists,pleasecontactusatsupport@aatbio.comor1-800-990-8053.
InOncology,Fluo-8®AMhasbeenusedtostudy:
»BreastcancercellsbymonitoringintracellularCa2+fluxassociatedwithapoptosisandinhibitionby2-aminoethoxydiphenylborate[1]
»Antitumoractivitybywayofthioredoxin-bindingprotein2anditsdependenceonintracellularcalciumconcentration[2]
»Bcl-1andBcl-2regulationthroughcharacterizationofcytosolictransportasquantifiedbycalciumflux[3]
»Ca2+influxandCa2+channelactivityinNCI-H460cellsasaparameterformonitoringprogressionofnon-smallcelllungcancer[4]
»Ca2+releasebyHN4cellsandCLIC4upregulationofapoptosisthroughmitochondrialandendoplasmicreticulumpathways[5]
InCardiology,Fluo-8®AMhasbeenusedtostudy:
»Low-energyfar-fieldstimulationasatherapyfortachycardiaandfibrillation[6]
»Calciumfluxduringcalciumsparksinventricularmyocytes[7]
»Cardiacconductionasafunctionofcellrigidityinthecontextofcardiovasculardisease[8]
»DiastolicCa2+transientsincardiacmyocytesandSR-luminalandfreecytoplasmicCa2+concentrations[9]
»Sphingosine-1-phosphate(S1P)receptoractivationinvalvularinterstitialcellsasdetectedbycytosolicCa2+flux[10]
InNeurobiology,Fluo-8®AMhasbeenusedtostudy:
»HippocampalCA1neurons,visualizatingneuronstoinvestigatetheroleofamyloid-βintheprogressionofAlzheimer"sdisease[11]
»CytosolicCa2+concentrationsinHEK293cellsanditsregulatoryeffectonAβ1-42andhAmylinandassociatedsignalingpathways[12]
»Gprotein-coupledreceptors(GPRs)inresponsetocannabinoidsinpresynapticCA3orpostsynapticCA1pyramidalcells[13]
»Medullaryinterneuronsanddendriticcalciumactivityinregardstoinspiratorybursts[14]
»N2acellactivationbyhistamine,asmonitoredbyincreasesinintracellularCa2+concentrations[15]
InStemCells,Development&Differentiation,Fluo-8®AMhasbeenusedtostudy:
»Inductionofpluripotentstemcells(iPSCs)intofunctioningcardiaccells,asvalidatedbyCa2+fluxandmembranepotential[16]
»CXCR4andCXCR7receptorsinTcellsandtheirroleincellsurvivalandchemotaxis[17]
»Ca2+uptakebymyocytesderivedfromhumaninducedpluripotentstemcellsduringpathogenesisofDuchennemusculardystrophy[18]
»AgoNIST-inducedcalciumtransientsindifferentiationofratbonemarrowmesenchymalstemcellsintosmoothmusclecells[19]
»CalciumchannelblockadesandtheireffectoncardiacProgenitorcellproliferationanddifferentiation[20]
AAT Bioquest AAT Bioquest是一家位于美国的生物公司,前身为ABD Bioquest,总部位于加利福尼亚州。专门从事光学检测技术十多年,一直致力于光谱学检测领域技术的创新和突破。其独特的光学检测技术,综合了化学、生物学和信息学等各个领域的研究,引领了比色、荧光和发光技术新一代光学探针的浪潮。AAT Bioquest在全球拥有强大的经验丰富的专业分销商网络,为从小型研究机构到《财富》500强企业的各类客户提供卓越的产品和定制服务。
美国AATBioquestInc.(前身是ABDBioquest,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。AATBioquest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。AATBioquest会不断介绍新产品,快速的丰富各个领域的产品。
1)我们提供反应荧光探针和发光探针,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)我们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)我们不断的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)我们致力于开发用于信号转导研究的试剂;5)我们提供生理和神经探针,特别是钙离子指示剂和膜电位探针。
作为AATBioquestInc.的中国区域代理,艾美捷科技为中国客户提供光谱学检测领域,包括吸收(颜色),荧光和发光技术等全系列解决方案。我们也将一如既往更加努力为国内用户提供快捷、方便的高质量产品,同时更为您售前售后全面技术支持。
AATBioquest,Inc.(formerlyABDBioquest,Inc.)develops,manufacturesandmarketsbioanalyticalresearchreagentsandkitstoscientistsengagedinlifesciencesresearch,diagnosticR&Danddrugdiscovery.Wespecializeintheareaofphotometricdetectionsincludingabsorption(color),fluorescenceandluminescencetechnologies.TheCompany"sproductsenablescientistsandbiomedicalresearcherstobetterunderstandbiochemistry,immunology,cellBIOLOGyandmolecularbiology.AATBioquestconstantlyintroducesnewproducts,andoffersarapidlyexpandinglistofproductsthataregroupedintoseveralproductlines.
1)Ourreactivefluorescentandluminescentprobes,biotinsandtagenzymesareusedforlabelingsmalldrugmoleculesandbiopolymers,e.g,proteins,nucleicacidsandcarbohydrates;2)Wedevelopfluorescentandluminescentprobesfordetectingproteins,nucleicacidsandlivecells;3)Weconstantlyintroducenovelfluorescentandluminescentprobesfordetectingvariousenzymes,inparticular,hydrolyticandredoxenzymes;4)Wefocusondevelopingreagentsforsignaltransductionresearch;and5)Wealsoofferphysiologicalandneurologicalprobes,e.g.,calciumindicatorsandmembranepotentialprobes.
Besidesthestandardcatalogproductswealsooffercustomservicetomeetyourspecialresearchneeds.Ourcurrentservicesincludecustomsynthesisofcolorimetric,fluorescentandluminescentprobes,customdevelopmentofbiochemical,cell-basedanddiagnosticassaysandcustomscreeningofyourcompoundlibrariesagainstyourdefinedtargetsusingourvalidatedHTSassays.
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前一阵子实验室开始使用一种新的核酸染料-goldview,取代原先用的溴化乙锭-EB.goldview现在是由赛百盛出售.以下摘自官方网站:
GoldViewTM核酸染料——使用说明
概述
GoldViewTM是一种可代替溴化乙锭(EB)的新型核酸染料,采用琼脂糖电泳检测DNA时,GoldViewTM与核酸结合后能产生很强的荧光信号,其灵敏度与EB相当,使用方法与之完全相同。在紫外透射光下双链DNA呈现绿色荧光,而单链DNA呈红色荧光。GoldView不仅能染DNA,也可用于染RNA。
通过Ames试验、小鼠骨髓嗜多染红细胞微核试验、小鼠睾丸精母细胞染色体畸变试验,致突变性结果均为阴性;而溴化乙锭(EB)是一种强致癌剂。因此用GoldviewTM代替EB不失为一种明智的选择。
使用方法
1.将100ml琼脂糖凝胶溶液(浓度一般为0.8%~2%)在微波炉中融化。
2.加入5µlGoldView,轻轻摇匀,避免产生气泡。
3.冷却至不烫手时倒胶,待琼脂糖凝胶完全凝固后上样电泳。
4.电泳完毕在紫外灯下观察。若使用数码相机照像记录,则关闭相机的闪光灯,放在自动档即可;若使用凝胶成像系统照相,通过调节光圈、曝光时间,选择合适的滤光片,可得到成像清晰、背景较低的照片。
注意事项
1.胶厚度不宜超过0.5cm,胶太厚会影响检测的灵敏度。
2.加入GoldView的琼脂糖凝胶反复融化可能会对核酸检测的灵敏度产生一定影响,但不明显。
3.通过凝胶电泳回收DNA片段时,建议使用GoldView染色,在自然光下切割DNA条带,避免紫外线与EB对目的DNA产生的损伤,可明显提高克隆、转化、转录等分子生物学下游操作的效率。
4.虽然未发现GoldView有致癌作用,但对皮肤、眼睛会有一定的刺激,操作时应戴上手套。
电泳结果显示GV灵敏度与EB相当
问题1.Goldview到底是不是丫啶橙?
2.赛百盛不公布其成分的原因是害怕其为丫啶橙还是出于技术保密?
产品主要应用:点击化学(Clickchemistry)、蛋白质组学研究中的双向荧光差异凝胶电泳(2DDIGE)和实时荧光定量PCR(RealtimePCR)。
菁染料是性能优良的荧光标记染料,摩尔吸光系数在荧光染料中是最高的。N-羟基琥珀酰亚胺酯是最常用的脂肪氨基标记试剂,广泛用于蛋白质、氨基酸多肽、抗体、核酸及其他生物分子的标记和检测。通过改变次甲基链的长度,可改变其荧光发射波长,每增加一个双键,按照Huoffman规则正好红移约100nm。
菁染料Cy3和Cy5已成为基因芯片的首选荧光标记物;另外,Cy5,Cy5.5和Cy7,Cy7.5的吸收在近红外区背景非常低,是荧光强度最高、最稳定的长波长染料,特别适合于活体小动物体内成像。但由于菁染料,尤其是不对称菁染料的合成副反应多,副产物极性相近,产物的分离提纯相当困难。菁染料特别是水溶性菁染料分子极性大,分离提纯越加困难。Lumiprobe供应脂溶性和水溶性菁染料。
相关产品:
产品分子结构可替代染料编号:AGF1371A
6-ROX-N-羟基琥珀酰亚胺酯
ROXNHSester,pure6-isomer[img]/KindEditor_4.0.1/attached/image/20130704/20130704102819_7906.jpg[/img]AlexaFluor568编号:AGF1326A
Cy3-N-羟基琥珀酰亚胺酯
Cy3NHSester[img]/KindEditor_4.0.1/attached/image/20130704/20130704103024_8531.jpg[/img]AlexaFluor546NHSester
DyLight549NHSester
编号:AGF1330A
Cy3.5-N-羟基琥珀酰亚胺酯
Cy3.5NHSester[img]/KindEditor_4.0.1/attached/image/20130704/20130704103242_2437.jpg[/img]AlexaFluor594NHSester
DyLight594NHSester
编号:AGF1338A
Cy5-N-羟基琥珀酰亚胺酯
Cy5NHSester[img]/KindEditor_4.0.1/attached/image/20130704/20130704103422_4468.jpg[/img]AlexaFluor647NHSester
DyLight649NHSester
编号:AGF1345A
Cy5.5-N-羟基琥珀酰亚胺酯
Cy5.5NHSester[img]/KindEditor_4.0.1/attached/image/20130704/20130704103557_5875.jpg[/img]AlexaFluor680NHSester
DyLight680NHSester
编号:AGF1349A
Cy7-N-羟基琥珀酰亚胺酯
Cy7NHSester[img]/KindEditor_4.0.1/attached/image/20130704/20130704103713_8843.jpg[/img]编号:AGF1356A
Cy7.5-N-羟基琥珀酰亚胺酯
Cy7.5NHSester[img]/KindEditor_4.0.1/attached/image/20130704/20130704103756_5406.jpg[/img]编号:AGF1374A
磺酸基-Cy3-N-羟基琥珀酰亚胺酯
Sulfo-Cy3NHSester[img]/KindEditor_4.0.1/attached/image/20130704/20130704103830_1656.jpg[/img]AlexaFluor546
DyLight549
编号:AGF1377A
磺酸基-Cy5-N-羟基琥珀酰亚胺酯
Sulfo-Cy5NHSester[img]/KindEditor_4.0.1/attached/image/20130705/20130705095752_6656.jpg[/img]AlexaFluor647
DyLight649
编号:AGF1379A
磺酸基-Cy7-N-羟基琥珀酰亚胺酯
Sulfo-Cy7NHSester[img]/KindEditor_4.0.1/attached/image/20130704/20130704104047_6968.jpg[/img]相似系列产品:
羰基活性荧光染料
氨基类染料
巯基反应性染料
羧酸类染料
二苯胺
甲基绿派洛宁
还有一种忘了。
假期没法联系老师呵呵。
这三种的作用都是什么。
而且样品中的无水硫酸钠未变色,而做标准曲线的五个和空白对照的变为蓝色了,请高手指教,多谢!
还有,是否变蓝对测定结果有影响吗?
谢了哈
欢迎你!请下次规范发贴:)
健那绿染液是一种活体染液,实验对象必须是活细胞,健那绿可以使活细胞中的线粒体呈现蓝绿色,而细胞质接近无色。
请各位大侠给予帮助!!
谢谢!!
健那绿——高中唯一一个活体染色剂。染线粒体的,染成蓝绿色
2、苏丹三 脂肪 橙红
3、苏丹四 脂肪 红
4、双缩脲 蛋白质 紫
5、龙胆紫 染色质 紫
6、碘 淀粉 蓝
7、健那绿 线粒体 绿
8、甲基绿 DNA 绿
9、吡罗红 RNA 红
10、溴麝香草酚蓝 CO2 由蓝变绿再变黄
11、重铬酸钾 酒精 酸性条件下由橙色变成灰绿
12、醋酸洋红(龙胆紫、改良苯酚品红) 染色质 红
13、台盼蓝 检验活死细胞 死细胞会被染成蓝色(不常用)