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LabelThiol-ModifiedOligonucleotideswithTideFluor™Dyes

Thefollowingprotocolhasbeenoptimizedforlabeling200µg(~6A260nmunits)ofaproprietaryoligonucleotide.Youneedmodifytheprotocoltogetthebestresultsforyourparticularapplicationbymultipleexperimentations.YOURTHIOL-MODIFIEDOLIGOMUSTBETREATEDTOREMOVESMALLTHIOLCOMPOUNDS(SUCHASDTT)THATRAPIDLYREACTSANDCONSUMESDYEMALEIMIDES.

1.      PrepareOligoSolution(SolutionA)

a.      Dissolveyourthiol-modifiedoligo(~200µg)inaPBSbuffer(100µL,pH7.2).

b.     Note1:Theoligonucleotidemustbesynthesizedwithathiolgrouponthe5’end.SeeAppenxidx1forthepurificationofthiolmodifiedoligos.

c.      Note2:Avoidbuffersthatcontainthiolcompounds,suchasDTT,asthesecompeteforconjugationwiththethiol-reactivecompound.

2.      PrepareDyeSolution(SolutionB)

a.      Dissolve1mgdyemaleimidein100µLDMSO(>10mg/mLifpossible)bypipettingupanddown.Centrifugethesolutionstockonthesidesofthevialtothevialbottom.

b.     Note:preparetheDMSOdyesolutionbeforestartingtheconjugation.Extendedstorageofthedyesolutionmayreducethedyeactivity.Anysolutionscontainingthedyeshouldbekeptfromlight.

3.      RunConjugationReaction

a.      Tothedyesolution(B,20-50µL)addtheoligosolution(A,100µL)withstirringorshaking(keepingthereactionmixturefromlight).

b.     Rotateorshakethereactionmixturefor4-6hoursatroomtemperatureonarotatororshaker.

c.      Note:Gentlyvortextapthevialevery10minutesforthefirsthourtoensurethatthereactionsolutionremainswellmixed.Donotmixviolently,asmaterialmaybeleftonthesidesofthevial.Aftersixhours,50–90%ofthethiol-modifiedoligonucleotidemoleculesshouldbelabeled.Thereactionmightbeincubatedovernightifitismoreconvenient.However,overnightincubationwillnotresultinagreaterlabelingefficiencyinmostcases.

4.      PurifyDye-OligoConjugate

a.      Preliminarypurificationbyethanolprecipitationoflabeledoligonucleotide

                                                              i.     Add20µL(one-tenthreactionsolutionvolumeingeneral)of3MNaCland300µLcoldabsoluteethanol(twoandhalfreactionsolutionvolumevolumesingeneral)tothereactionvial.

                                                            ii.     Mixthesolutionwellandplaceitat–20°Cfor30minutes.

                                                           iii.     Centrifugethesolutioninamicrocentrifugeat10,000to15,000×gfor30minutes.

                                                          iv.     Note:Lossofsamplemayoccurifthecentrifugationisnotlongenough.

                                                            v.     Carefullyremovethesupernatant,rinsethepellet1-3timeswithcold70%ethanolanddrybriefly.

                                                          vi.     Note:Someunreactedlabelingreagentmayhaveprecipitatedoverthecourseofthereactionormaybestuckonthewallsofthereactionvial.Thismaterialshouldbecompletelyredissolvedbyextensivevortexmixingbeforecentrifugation.Redissolvingthelabelingreagentensuresthattheprecipitatedoligonucleotidewillbeminimallycontaminatedwithunreactedlabel.

b.     FinalPurificationbyHPLCorbygelelectrophoresis

                                                              i.     SeeAppendixI

LabelPeptideswithTideFluor™Dyes

Thefollowingprotocolhasbeenoptimizedforlabeling10mgofaproprietarypeptide(MW~2000)thatcontainsonlyasinglefreethiolgroup.YOUNEEDMODIFYTHEPROTOCOLTOARCHIETHEBESTRESULTSFORYOURPARTICULARAPPLICATIONBYMULTIPLEEXPERIMENTATIONS.

1.      PreparePeptideSolution(SolutionA)

a.      Dissolvethepepetide(tobelabeled)inDMF,DMSOorwater(>10mg/mLifpossible).

b.     Note:Foreffectivelabeling,higherpeptideconcentrationispreferable.

2.      PrepareDyeSolution(SolutionB)

a.      DissolvethedyemaleimideinDMForDMSO(>5mg/mLifpossible).

b.     Note:Foreffectivelabeling,higherdyeconcentrationispreferable.

3.       Mixthepeptideanddyesolutions(fromSteps1and2)inroughlyequalmolarratio.

a.      Note:Eitherpeptideordyemaleimidecanbeusedinexcessdependingonthecostofpeptideanddye.Goodseparationoffreedye,freepeptideanddye-peptideconjugateisanotherimportantfactorthatneedbeconsideredaswelltodecidethateitherpeptideordyeneedbeusedinslightexcess.

4.      Stirthereactionmixtureatroomtemperaturefor12-24hours.

a.       Note:Thisreactionisquiteefficient.Itisgenerallycompletedwithinafewhours.Ifthereactiondoesnotgenerateaconsiderableamountofdesiredconjugatewithin2hours,youneedcheckthereactionpH,averycriticalfactor.TheoptimalpHis4-6.ToolowpHsignificantlydecreasesthereactionratewhileinhighpHsolutionspeptidestendtobeoxidizedtogivethedisulfides.PeptidesoftencontainTFA,thereforeitisimportanttocheckthereactionmixturepHandadjustittopH4-6usingNaHCO3ifnecessary.

5.      PurifyDye-PeptideConjugate

a.      ThereactionsolutionwasconcentratedandpurifiedonaC18columntoaffordthedesiredconjugate.ThefractionswereanalyzedbyHPLC,andthefractionsof>97%puritywerepooledandlyophilized.

b.     Note1:HPLCPurificationConditions:TEABbuffer(triethylammoniumbicarbonate,0.25mmol,pH=7.0-8.0)wasusedasbufferAandacetonitrileasbufferB.TheHPLCwasrunfrom0%Bto30%Bin60min(flowrate:100mL/min).

c.      Note2:Avoidstronglightduringtheoperation.

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