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AAT Bioquest/PacBlue succinimidyl ester [equivalent to Pacific Blue succinimidyl ester]/570/5 mg
![AAT Bioquest/PacBlue succinimidyl ester [equivalent to Pacific Blue succinimidyl ester]/570/5 mg](https://www.ebiomall.cn/images/AAT201712/chemical-structure-for-pacblue-succinimidyl-ester-pacific-blue-succinimidyl-ester.png)
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 416/451 |
| MW | 339.21 |
| CAS# | 215868-33-0 |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | SuperiorLabelingDyes mFluor™DyesandKits |
| Related | Generalproteins LabelingviaAminoGroups |
| 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. AddanhydrousDMSOintothevialofmFluor™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.
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).
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.
Note1:Forimmediateuse,thedye-proteinconjugateneedbedilutedwithstainingbuffer,andaliquotedformultipleuses.
Note2:Forlongertermstorage,dye-proteinconjugatesolutionneedbeconcentratedorfreezedried(seebelow).
| References&Citations |  PrinterFriendlyVersion |
1. Lee,M.M.;Peterson,B.R.,QuantificationofSmallMolecule-ProteinInteractionsusingFRETbetweenTryptophanandthePacificBlueFluorophore.ACSOmega2016,1(6),1266-1276.
2. Cohen,J.D.;Thompson,S.;Ting,A.Y.,Structure-guidedengineeringofaPacificBluefluorophoreligaseforspecificproteinimaginginlivingcells.Biochemistry2011,50(38),8221-5.
3. Chiesl,T.N.;Chu,W.K.;Stockton,A.M.;Amashukeli,X.;Grunthaner,F.;Mathies,R.A.,EnhancedamineandaminoacidanalysisusingPacificBlueandtheMarsOrganicAnalyzermicrochipcapillaryelectrophoresissystem.AnalChem2009,81(7),2537-44.
4. Lukomska,J.;Gryczynski,I.;Malicka,J.;Makowiec,S.;Lakowicz,J.R.;Gryczynski,Z.,One-andtwo-photoninducedfluorescenceofPacificBlue-labeledhumanserumalbumindepositedondifferentcoresizesilvercolloids.Biopolymers2006,81(4),249-55.
