Overview | PrinterFriendlyVersion |
Ex/Em(nm) | 496/524 |
MW | 620.52 |
CAS# | N/A |
Solvent | DMSO |
Storage | F/D/L |
Category | SuperiorLabelingDyes iFluor™DyesandKits |
Related | Amine-ReactiveProbes LabelingviaAminoGroups |
Spectrum | AdvancedSpectrumViewer |
- Prepareproteinstocksolution(SolutionA):Mix100µLofareactionbuffer(e.g.,1Msodiumcarbonatesolutionor1MphosphatebufferwithpH~9.0)with900µLofthetargetproteinsolution(e.g.antibody,proteinconcentration>2mg/mlifpossible)togive1mLproteinlabelingstocksolution.
Note1:ThepHoftheproteinsolution(SolutionA)shouldbe8.5±0.5.IfthepHoftheproteinsolutionislowerthan8.0,adjustthepHtotherangeof8.0-9.0using1Msodiumbicarbonatesolutionor1MpH9.0phosphatebuffer.
Note2:Theproteinshouldbedissolvedin1Xphosphatebufferedsaline(PBS),pH7.2-7.4.IftheproteinisdissolvedinTrisorglycinebuffer,itmustbedialyzedagainst1XPBS,pH7.2-7.4,toremovefreeaminesorammoniumsalts(suchasammoniumsulfateandammoniumacetate)thatarewidelyusedforproteinprecipitation.
Note3:ImpureantibodiesorantibodiesstABIlizedwithbovineserumalbumin(BSA)orgelatinwillnotbelabeledwell.Thepresenceofsodiumazideorthimerosalmightalsointerferewiththeconjugationreaction.Sodiumazideorthimerosalcanberemovedbydialysisorspincolumnforoptimallabelingresults.
Note4:Theconjugationefficiencyissignificantlyreducediftheproteinconcentrationislessthan2mg/mL.Foroptimallabelingefficiencythefinalproteinconcentrationrangeof2-10mg/mLisrecommended. - Preparedyestocksolution(SolutionB):AddanhydrousDMSOintothevialofFITC-xtratomakea10-20mMstocksolution.Mixwellbypipettingorvortex.
Note:Preparethedyestocksolution(SolutionB)beforestartingtheconjugation.Usepromptly.Extendedstorageofthedyestocksolutionmayreducethedyeactivity.SolutionBcanbestoredinfreezerfortwoweekswhenkeptfromlightandmoisture.Avoidfreeze-thawcycles. - Preparethedesiredproteinconjugate:Note:Eachproteinrequiresdistinctdye/proteinratio,whichalsodependsonthepropertiesofdyes.Overlabelingofaproteincoulddetrimentallyaffectsitsbindingaffinitywhiletheproteinconjugatesoflowdye/proteinratiogivesreducedsensitivity.Werecommendyouexperimentallydeterminethebestdye/proteinratiobyrepeatingSteps4and5usingaserialdifferentamountoflabelingdyesolutions.Ingeneral4-6dyes/proteinarerecommendedformostofdye-proteinconjugates.
- Use10:1molarratioofSolutionB(dye)/SolutionA(protein)asthestartingpoint:Add5µlofthedyestocksolution(SolutionB,assumingthedyestocksolutionis10mM)intothevialoftheproteinsolution(95µlofSolutionA)witheffectiveshaking.Theconcentrationoftheproteinis~0.05mMassumingtheproteinconcentrationis10mg/mLandthemolecularweightoftheproteinis~200KD.
Note:TheconcentrationoftheDMSOintheproteinsolutionshouldbe<> - Runconjugationreaction.Addtheappropriateamountofdyestocksolution(SolutionB)intothevialoftheproteinsolution(SolutionA)witheffectiveshaking.Continuetorotateorshakethereactionmixtureatroomtemperaturefor30-60minutes.
- Repeat#3.2withthemolarratiosofSolutionB/SolutionAat5:1;15:1and20:1respectivelyifdesired.
- Purifythedesiredconjugatesusingpremadespincolumnsorothertechniques.
- Calculatethedye/proteinratio(DOS)fortheabove4conjugates(seebelow).
- Use10:1molarratioofSolutionB(dye)/SolutionA(protein)asthestartingpoint:Add5µlofthedyestocksolution(SolutionB,assumingthedyestocksolutionis10mM)intothevialoftheproteinsolution(95µlofSolutionA)witheffectiveshaking.Theconcentrationoftheproteinis~0.05mMassumingtheproteinconcentrationis10mg/mLandthemolecularweightoftheproteinis~200KD.
References&Citations | CitationExplorer |
Roleofreactiveoxygenspecies-mediatedMAPKandNF-$kappa$Bactivationinpolygonatumcyrtonemalectin-inducedapoptosisandautophagyinhumanlungadenocarcinomaA549cells
Authors:TaoLiu,LeiWu,DiWang,HaiyangWang,JinwuChen,ChunlanYang,JinkuBao,ChuanfangWu
Journal:JournalofBiochemistry(2016):mvw040
Determinationofsodiumbenzoateinfoodproductsbyfluorescencepolarizationimmunoassay
Authors:LinlinRen,MengMeng,PengWang,ZhihuanXu,SergeiAEremin,JunhongZhao,YongmeiYin,RimoXi
Journal:Talanta(2014):136--143
ComplexescontainingcationicandanionicpH-sensitiveliposomes:comparativestudyoffactorsinfluencingplasmidDNAgenedeliverytotumors
Authors:YanChen,JiSun,YingLu,ChunTao,JingbinHuang,HeZhang,YuanYu,HaoZou,JingGao,YanqiangZhong
Journal:Internationaljournalofnanomedicine(2013):1573
SwimmingbehaviorofthemonotrichousbacteriumPseudomonasfluorescensSBW25
Authors:LiyanPing,JanBirkenbeil,ShamciMonajembashi
Journal:FEMSmicroBIOLOGyecology(2013):36--44