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当前位置: 首页 > 产品中心 > Fluorescent_dyes > AAT Bioquest/trFluor™ Tb succinimidyl ester/1443/1 mg
商品详细AAT Bioquest/trFluor™ Tb succinimidyl ester/1443/1 mg
AAT Bioquest/trFluor™ Tb succinimidyl ester/1443/1 mg
AAT Bioquest/trFluor™ Tb succinimidyl ester/1443/1 mg
商品编号: 1443
品牌: aatbio
市场价: ¥433560.00
美元价: 260136.00
产地: 美国(厂家直采)
公司:
产品分类: 荧光染料
公司分类: Fluorescent_dyes
联系Q Q: 3392242852
电话号码: 4000-520-616
电子邮箱: info@ebiomall.com
商品介绍
Overview
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Ex/Em(nm)330/544
MW1226.05
CAS#N/A
SolventDMSO
StorageF/D/L
CategorySuperiorLabelingDyes
trFluor™DyesandKits
RelatedLabelingviaAminoGroups
BiochemicalAssays
ManyBIOLOGicalcompoundspresentincells,serumorotherbiologicalfluidsarenaturallyfluorescent,andthustheuseofconventional,promptfluorophoresleadstoseriouslimitationsinassaysensitivityduetothehighbackgroundcausedbytheautofluorescenceofthebiologicalmoleculestobeassayed.Theuseoflong-livedfluorophorescombinedwithtime-resolveddetection(adelaybetweenexcitationandemissiondetection)minimizespromptfluorescenceinterferences.OurtrFluor™Tbprobesenabletime-resolvedfluorometry(TRF)fortheassaysthatrequirehighsensitivity.ThesetrFluor™TbprobeshavelargeStokesshiftsandextremelylongemissionhalf-liveswhencomparedtomoretrADItionalfluorophoressuchasAlexaFluororcyaninedyes.ComparedtotheotherTRFcompounds,ourtrFluor™TbprobeshaverelativelyhighstABIlity,highemissionyieldandabilitytobelinkedtobiomolecules.Moreover,ourtrFluor™Tbprobesareinsensitivetofluorescencequenchingwhenconjugatedtobiologicalpolymerssuchasantibodies.
SpectrumAdvancedSpectrumViewer

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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.      Prepareproteinstocksolution(SolutionA):

a.      Mix100µLofareactionbuffer(e.g.,1Msodiumcarbonatesolutionor1MphosphatebufferwithpH~9.0)with900µLofthetargetproteinsolution(e.g.antibody,proteinconcentration>2mg/mlifpossible)togive1mLproteinlabelingstocksolution.

b.     Note1:ThepHoftheproteinsolution(SolutionA)shouldbe8.5±0.5.IfthepHoftheproteinsolutionislowerthan8.0,adjustthepHtotherangeof8.0-9.0using1Msodiumbicarbonatesolutionor1MpH9.0phosphatebuffer.

c.       Note2:Theproteinshouldbedissolvedin1Xphosphatebufferedsaline(PBS),pH7.2-7.4.IftheproteinisdissolvedinTrisorglycinebuffer,itmustbedialyzedagainst1XPBS,pH7.2-7.4,toremovefreeaminesorammoniumsalts(suchasammoniumsulfateandammoniumacetate)thatarewidelyusedforproteinprecipitation

d.     Note3:Impureantibodiesorantibodiesstabilizedwithbovineserumalbumin(BSA)orgelatinwillnotbelabeledwell.Thepresenceofsodiumazideorthimerosalmightalsointerferewiththeconjugationreaction.Sodiumazideorthimerosalcanberemovedbydialysisorspincolumnforoptimallabelingresults.

e.     Note4:Theconjugationefficiencyissignificantlyreducediftheproteinconcentrationislessthan2mg/mL.Foroptimallabelingefficiencythefinalproteinconcentrationrangeof2-10mg/mLisrecommended.

2.      Preparedyestocksolution(SolutionB):

a.      AddanhydrousDMSOintothevialoftrFluor™EuSEtomakea10-20mMstocksolution.Mixwellbypipettingorvortex.

b.     Note:Preparethedyestocksolution(SolutionB)beforestartingtheconjugation.Usepromptly.Extendedstorageofthedyestocksolutionmayreducethedyeactivity.SolutionBcanbestoredinfreezerfortwoweekswhenkeptfromlightandmoisture.Avoidfreeze-thawcycles.

3.      Determinetheoptimaldye/proteinratio(optional):

a.      Note:Eachproteinrequiresdistinctdye/proteinratio,whichalsodependsonthepropertiesofdyes.Overlabelingofaproteincoulddetrimentallyaffectsitsbindingaffinitywhiletheproteinconjugatesoflowdye/proteinratiogivesreducedsensitivity.Werecommendyouexperimentallydeterminethebestdye/proteinratiobyrepeatingSteps4and5usingaserialdifferentamountoflabelingdyesolutions.Ingeneral4-6dyes/proteinarerecommendedformostofdye-proteinconjugates.

b.     Use10:1molarratioofSolutionB(dye)/SolutionA(protein)asthestartingpoint:Add5µlofthedyestocksolution(SolutionB,assumingthedyestocksolutionis10mM)intothevialoftheproteinsolution(95µlofSolutionA)witheffectiveshaking.Theconcentrationoftheproteinis~0.05mMassumingtheproteinconcentrationis10mg/mLandthemolecularweightoftheproteinis~200KD.

                                                              i.     Note:TheconcentrationoftheDMSOintheproteinsolutionshouldbe<10%

c.      Runconjugationreaction(seeStep4below).

d.     Repeat#3.2withthemolarratiosofSolutionB/SolutionAat5:1;15:1and20:1respectively.

e.     Purifythedesiredconjugatesusingpremadespincolumns.

f.       Calculatethedye/proteinratio(DOS)fortheabove4conjugates(seenextpage).

g.      Runyourfunctionaltestsoftheabove4conjugatestodeterminethebestdye/proteinratiotoscaleupyourlabelingreaction.

4.      Runconjugationreaction:

a.      Addtheappropriateamountofdyestocksolution(SolutionB)intothevialoftheproteinsolution(SolutionA)witheffectiveshaking.

b.     Note:ThebestmolarratioofSolutionB/SolutionisdeterminedfromStep3.6.IfStep3isskipped,werecommendtouse10:1molarratioofSolutionB(dye)/SolutionA(protein).4.2Continuetorotateorshakethereactionmixtureatroomtemperaturefor30-60minutes.

5.      PurifytheconjugationThefollowingprotocolisanexampleofdye-proteinconjugatepurificationbyusingaSephadexG-25column.

a.       PrepareSephadexG-25columnaccordingtothemanufactureinstruction.

b.     Loadthereactionmixture(directlyfromStep4)tothetopoftheSephadexG-25column.

c.      AddPBS(pH7.2-7.4)assoonasthesamplerunsjustbelowthetopresinsurface.

d.     AddmorePBS(pH7.2-7.4)tothedesiredsampletocompletethecolumnpurification.Combinethefractionsthatcontainthedesireddye-proteinconjugate.

e.     Note1:Forimmediateuse,thedye-proteinconjugateneedbedilutedwithstainingbuffer,andaliquotedformultipleuses.

f.       Note2:Forlongertermstorage,dye-proteinconjugatesolutionneedbeconcentratedorfreezedried(seebelow).

References&Citations
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1.   SavilleL,SpaisC,MasonJL,AlbomMS,MurthyS,MeyerSL,AtorMA,AngelesTS,HustenJ.(2012)Time-ResolvedFluorescenceResonanceEnergyTransferasaVersatileToolintheDevelopmentofHomogeneousCellularKinaseAssays.AssayDrugDevTechnol.

2.   LoMC,NgoR,DaiK,LiC,LiangL,LeeJ,EmkeyR,EksterowiczJ,VenturaM,YoungSW,XiaoSH.(2012)Developmentofatime-resolvedfluorescenceresonanceenergytransferassayforcyclin-dependentkinase4andidentificationofitsATP-noncompetitiveinhibitors.AnalBiochem,421,368.

3.   PailaYD,KombrabailM,KrishnamoorthyG,ChattopadhyayA.(2011)OligomerizationoftheSEROtonin(1A)receptorinlivecells:atime-resolvedfluorescenceanisotropyapproach.JPhysChemB,115,11439.

4.   MartikkalaE,Rozwandowicz-JansenA,HanninenP,Petaja-RepoU,HarmaH.(2011)Ahomogeneoussingle-labeltime-resolvedfluorescencecAMPassay.JBiomolScreen,16,356.

5.   GaboritN,LarbouretC,VallagheJ,PeyrussonF,Bascoul-MolleviC,CrapezE,AzriaD,ChardesT,PoulMA,MathisG,BazinH,PelegrinA.(2011)Time-resolvedfluorescenceresonanceenergytransfer(TR-FRET)toanalyzethedisruptionofEGFR/HER2dimers:anewmethodtoevaluatetheefficiencyoftargetedtherapyusingmonoclonalantibodies.JBiolChem,286,11337.

6.   LeyrisJP,RouxT,TrinquetE,VerdieP,FehrentzJA,OueslatiN,DouzonS,BourrierE,LamarqueL,GagneD,GalleyrandJC,M"KadmiC,MartinezJ,MaryS,BaneresJL,MarieJ.(2011)Homogeneoustime-resolvedfluorescence-basedassaytoscreenforligandstargetingthegrowthhormonesecretagoguereceptortype1a.AnalBiochem,408,253.

7.   PosokhovYO,KyrychenkoA,LadokhinAS.(2010)Steady-stateandtime-resolvedfluorescencequenchingwithtransitionmetalionsasshort-distanceprobesforproteinconformation.AnalBiochem,407,284.

8.   Alvarez-CurtoE,WardRJ,PedianiJD,MilliganG.(2010)LigandregulationofthequaternaryorganizationofcellsurfaceM3muscarinicacetylcholinereceptorsanalyzedbyfluorescenceresonanceenergytransfer(FRET)imagingandhomogeneoustime-resolvedFRET.JBiolChem,285,23318.

9.   KotaS,ScampaviaL,SpicerT,BeelerAB,TakahashiV,SnyderJK,PorcoJA,HodderP,StrosbergAD.(2010)Atime-resolvedfluorescence-resonanceenergytransferassayforidentifyinginhibitorsofhepatitisCviruscoredimerization.AssayDrugDevTechnol,8,96.

10.   VisserAJ,LaptenokSP,VisserNV,vanHoekA,BirchDJ,BrochonJC,BorstJW.(2010)Time-resolvedFRETfluorescencespectroscopyofvisiblefluorescentproteinpairs.EurBiophysJ,39,241.


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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。
品牌分类
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
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