| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 346/617 |
| MW | 1219.09 |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | SuperiorLabelingDyes trFluor™DyesandKits |
| Related | LabelingviaAminoGroups BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
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 |  PrinterFriendlyVersion |
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.
AAT Bioquest常用产品报价单
| 产品名 | 货号 | 公司分类 | 商城分类 | 美金价 |
| AAT Bioquest/Fluorescein, disodium salt *CAS 518-47-8*/2/100 mg | 2 | iFluor Rapid Tests | 酸碱缓冲液 | 1088.00 | AAT Bioquest/14-3-3 ζ (Ab-58) Antibody/8B0001/50 ug | 8B0001 | Signaling Intermediate Ab | 重组抗体 | 2828.00 | AAT Bioquest/Opioid Receptor (Phospho-Ser375) Antibody/8A0022/50 ug | 8A0022 | Phospho-Specific Ab | 受体 | 2828.00 | AAT Bioquest/iFluor™ 350 goat anti-mouse IgG (H+L)/16440/200 ug | 16440 | Fluorescent Anti-IgGs | 荧光染料 | 1088.00 | AAT Bioquest/trFluor™ Eu goat anti-mouse IgG (H+L)/16518/100 ug | 16518 | Fluorescent Anti-IgGs | 抗小鼠 | 4278.00 | AAT Bioquest/RPE-streptavidin conjugate/16900/100 ug | 16900 | Streptavidin Conjugates | 其他生物染料 | 1378.00 | AAT Bioquest/GCNT3 Antibody/8C14708/50 ug | 8C14708 | Signaling Intermediate Ab | 功能性抗体 | 2828.00 | AAT Bioquest/Amplite™ Luciferase Reporter Gene Assay Kit *Bright Glow*/12518/1 plate | 12518 | Reporter Gene Enzymes | 其它检测试剂盒 | 2103.00 | AAT Bioquest/ReadiLink™ Rapid mFluor™ Violet 450 Antibody Labeling Kit *Microscale Optimized for Labeling 50 &mi | 1100 | mFluor™ Dyes and Kits | 标记试剂盒 | 2103.00 | AAT Bioquest/Amplite™ Colorimetric Urea Quantitation Kit *Blue Color*/10058/200 Tests | 10058 | Diagnostic Molecules | 定量试剂盒 | 2828.00 | AAT Bioquest/Screen Quest™ CHO-Gqi Chimera Cell line/38101/Each | 38101 | cAMP GPCR Assays | 常规细胞株 | 0.00 |
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