![AAT Bioquest/Tide Fluor™ 3WS amine [TF3WS amine] *Superior replacement for Cy3*/2347/1 mg](images/AAT201712/hq-figure-1-for-tide-fluor-3ws-amine-tf3ws-amine-superior-replacement-for-cy3.png)
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 555/565 |
| MW | 996.00 |
| 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.
| References&Citations |  CitationExplorer |
AmechaNISTicmodeltopredicteffectsofcathepsinBandcystatinConβ-amyloidaggregationanddegradation
Authors:TylerJPerlenfein,ReginaMMurphy
Journal:JournalofBIOLOGicalChemistry(2017):jbc--M117
Real-TimeDetectionofaSelf-ReplicatingRNAEnzyme
Authors:CharlesOlea,GeraldFJoyce
Journal:Molecules(2016):1310
DevelopmentofMulti-Parametric/MultimodalSpectroscopyApparatusforCharacterizationofFunctionalInterfaces
Authors:LangZhou,MaryArugula,ChristopherJEasley,CurtisShannon,AleksandrSimonian
Journal:ECSTransactions(2015):9--16
Maternalserumglycosylatedfibronectinasapoint-of-carebioMarkerforassessmentofpreeclampsia
Authors:JuhaRasanen,MatthewJQuinn,AmberLaurie,EricBean,CharlesTRoberts,SrinivasaRNagalla,MichaelGGravett
Journal:Americanjournalofobstetricsandgynecology(2015):82--e1
Arrayofbiodegradablemicroraftsforisolationandimplantationofliving,adherentcells
Authors:YuliWang,ColleenNPhillips,GabrielaSHerrera,ChristopherESims,JenJenYeh,NancyLAllbritton
Journal:RSCadvances(2013):9264--9272
DevelopmentofSNAP-TagFluorogenicProbesforWash-FreeFluorescenceImaging
Authors:XiaoliSun,AihuaZhang,BrendaBaker,LuoSun,AngelaHoward,JohnBuswell,DamienMaurel,AnastasiyaMasharina,KaiJohnsson,ChristopherJNoren
Journal:ChemBioChem(2011):2217--2226
FERRAMENTASPARAESTUDODABIOLOGIADEGPCRS(G-PROTEINCOUPLEDRECEPTORS)
Authors:FredericoMarianettiSoriani,RemoCastroRusso
Journal:Unknown
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然后是碱性荧光染料，造纸厂用来增加纸的亮度。
直接和分散的荧光黄，印染厂用来增加棉质和化纤面料的艳度
For principle,look at this site:
碘化丙啶(propiolium iodide,PI)能嵌入DNA双螺旋中,可使荧光强度增加约20倍,以488nm波长激发,DNA/PI复合物最大的发射波长约为615nm.
1.小鼠Lewis肺癌细胞DNA含量测定方法
(1).从C57BL/6小鼠上切除肿块,在培养皿内用PBS冲洗.
(2).去除结缔组织及脂肪,剪碎肿块.
(3).小碎片移入1.20×38mm注射针,加压使其通过,于4℃条件下重悬细胞于HBSS中.
(4).将200～300μL细胞悬液(5×105细胞/mL)中加入3mL PI(50μg/mL),染色3LL细胞,于4℃存放20～30分钟.
(5).测定580～750nm之间的发射荧光,以去除末结合PI产生的激发光与发射光谱线之间的重叠部分.
是欢快活泼的光辉色彩，是暖色系中最温暖的色，它使人联想到金色的秋天，丰硕的果实；
是一种富足、快乐而幸福的颜色。让人感觉成一种稳重、含蓄又明快的温暖；
橙色也会带来一种甜甜的感觉。
