| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 594/614 |
| MW | ~1200 |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | SuperiorLabelingDyes iFluor™DyesandKits |
| Related | Amine-ReactiveProbes LabelingviaAminoGroups BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
Note:ThislabelingprotocolwasdevelopedfortheconjugateofGoatanti-mouseIgGwithiFluor™647SE.Youmightneedfurtheroptimizationforyourparticularproteins.
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. AddanhydrousDMSOintothevialofiFluor™dyeSEtomakea10-20mMstocksolution.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.
c. 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).
c. Continuetorotateorshakethereactionmixtureatroomtemperaturefor30-60minutes.
5. Purifytheconjugation
a. Thefollowingprotocolisanexampleofdye-proteinconjugatepurificationbyusingaSephadexG-25column.
b. PrepareSephadexG-25columnaccordingtothemanufactureinstruction.
c. Loadthereactionmixture(directlyfromStep4)tothetopoftheSephadexG-25column.
d. AddPBS(pH7.2-7.4)assoonasthesamplerunsjustbelowthetopresinsurface.
e. AddmorePBS(pH7.2-7.4)tothedesiredsampletocompletethecolumnpurification.Combinethefractionsthatcontainthedesireddye-proteinconjugate.
f. Note1:Forimmediateuse,thedye-proteinconjugateneedbedilutedwithstainingbuffer,andaliquotedformultipleuses.
g. Note2:Forlongertermstorage,dye-proteinconjugatesolutionneedbeconcentratedorfreezedried(seebelow).
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