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AAT Bioquest/Tide Fluor™ 3WS succinimidyl ester [TF3WS SE] *Superior replacement for Cy3*/2346/5 mg
![AAT Bioquest/Tide Fluor™ 3WS succinimidyl ester [TF3WS SE] *Superior replacement for Cy3*/2346/5 mg](https://www.ebiomall.cn/images/AAT201712/hq-figure-1-for-tide-fluor-3ws-succinimidyl-ester-tf3ws-se-superior-replacement-for-cy3.png)
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 555/565 |
| MW | 1024.31 |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | SuperiorLabelingDyes TideFluor™DyesandKits |
| Related | PeptideLabelingReagents QuencherCPGs BiochemicalAssays |
LabelAmino-ModifiedOligonucleotideswithTideFluor™Dyes
Thefollowingprotocolhasbeenoptimizedforlabeling200µg(~6A260nmunits)ofaproprietaryoligonucleotide.Youneedmodifytheprotocoltogetthebestresultsforyourparticularapplicationbymultipleexperimentations.YOURAMINO-MODIFIEDOLIGOMUSTBETREATEDTOREMOVEAMMONIATHATRAPIDLYREACTSANDCONSUMESDYESUCCINIMIDYLESTERS.
1. PrepareOligoSolution(SolutionA)
a. Dissolveyouramino-modifiedoligo(~200µg)inatetraboratebuffer(100µL,pH8.5±0.5).
b. Note1:Theoligonucleotidemustbesynthesizedwithanaminegrouponthe5’end.SeeAppenxidx1forthepurificationofamino-modifiedoligos.
c. Note2:Avoidbuffersthatcontainprimaryamines,suchasTris,asthesecompeteforconjugationwiththeamine-reactivecompound.
2. PrepareDyeSolution(SolutionB)
a. Dissolve1mgdyeSEin100µLDMSO(>10mg/mLifpossIBLe)bypipettingupanddown.Centrifugethesolutionstockonthesidesofthevialtothevialbottom.
b. Note:preparetheDMSOdyesolutionbeforestartingtheconjugation.Extendedstorageofthedyesolutionmayreducethedyeactivity.Anysolutionscontainingthedyeshouldbekeptfromlight.WedonotrecommendthatyoustoretheDMSOdyesolutionforfutureuse.
3. RunConjugationReaction
a. Tothedyesolution(B,20-50µL)addtheoligosolution(A,100µL)withstirringorshaking(keepingthereactionmixturefromlight).
b. Rotateorshakethereactionmixturefor4-6hoursatroomtemperatureonarotatororshaker.
c. Note:Gentlyvortextapthevialevery10minutesforthefirsthourtoensurethatthereactionsolutionremainswellmixed.Donotmixviolently,asmaterialmaybeleftonthesidesofthevial.Aftersixhours,50–90%oftheamine-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)thatcontainsonlyasinglefreeaminogroup.YOUNEEDMODIFYTHEPROTOCOLTOARCHIETHEBESTRESULTSFORYOURPARTICULARAPPLICATIONBYMULTIPLEEXPERIMENTATIONS.
1. PreparePeptideSolution(SolutionA)
a. Dissolveyourpeptide(~10mg)inDMF(~1ml).
b. Note1:Thepeptidemustbeneutralizedwithabasesuchastriethylamineorpotassiumcarbonate.
c. Note2:Avoidbuffersthatcontainprimaryamines,suchasTris,asthesecompeteforconjugationwiththeamine-reactivecompound.
2. PrepareDyeSolution(SolutionB)
a. Dissolve5mgdyeSEin500µLDMF(>10mg/mLifpossible)bypipettingupanddown.
b. Note:preparetheDMFdyesolutionbeforestartingtheconjugation.Extendedstorageofthedyesolutionmayreducethedyeactivity.Anysolutionscontainingthedyeshouldbekeptfromlight.WedonotrecommendthatyoustoretheDMFdyesolutionforfutureuse.
3. RunConjugationReaction
a. Tothedyesolution(B,500µL)addthepeptidesolution(A,1mL)withstirringorshaking(keepingthereactionmixturefromlight).
b. Stirthereactionmixturefor4-6hoursatroomtemperature.
4. 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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