| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 330/544 |
| MW | 1226.05 |
| 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.
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(1)、分子内含有发射荧光的基团，如羰基、氮氮双键、碳氮双键等。
(2)、分子内含有助色基团。助色基团使光谱红移并增大荧光效率，如伯胺基、仲胺基、羟基、醚键、酰胺基等。
(3)、分子内含有刚性平面结构的共轭π键。分子内共轭体系愈大平面性愈强其荧光强度愈高。一些能提高共轭度的因素能提高荧光效率，并使荧光波长向长波方向移动。 就是荧光染料的附着物，主要作用有帮助荧光染料展色、提高荧光染料与下游树酯的相溶性、保护荧光染料的性能。通常载体树脂是强极性树脂，分子中含有胺基、羟基、醚键、酰胺基等强极性基团，一方面有助色作用，增大荧光效率;另一方面与荧光染料有很好的相溶性，有助于染料的均匀分散。
荧光颜料常用的载体树脂有胺基树脂、苯代三聚氰胺一甲醛树脂、聚丙烯酸酯树脂、聚酰胺树酯、聚酯树脂、聚氨酯树脂等。 （1）、热塑性荧光颜料：线型
（2）、热固性荧光颜料： 体型
（3）、可溶解色精荧光颜料
（4）、水乳型荧光颜料 （1）、胺基树酯
（2）、聚酰胺树酯
（3）、聚酯树酯
（4）、丙烯酸乳液 （1）、塑胶类
低温型
中温型
高温型
（2）、涂料类
水性涂料
油性涂料
粉末涂料 （1）、含甲醛
（2）、不含甲醛
希望同路人多多交流啊!
想请问一下，DAPI这个染料到底有没有膜通透性，我通过百度搜索查询关于DAPI染料的，基本上是说它能透过细胞膜对活细胞和死细胞均能染上蓝色；但是也有人说DAPI只可以透过死细胞膜，不能对活细胞进行染色，用以区分活死细胞，到底哪个是对的啊，蒙了！！！！！！
