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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(1)、分子内含有发射荧光的基团,如羰基、氮氮双键、碳氮双键等。
(2)、分子内含有助色基团。助色基团使光谱红移并增大荧光效率,如伯胺基、仲胺基、羟基、醚键、酰胺基等。
(3)、分子内含有刚性平面结构的共轭π键。分子内共轭体系愈大平面性愈强其荧光强度愈高。一些能提高共轭度的因素能提高荧光效率,并使荧光波长向长波方向移动。 就是荧光染料的附着物,主要作用有帮助荧光染料展色、提高荧光染料与下游树酯的相溶性、保护荧光染料的性能。通常载体树脂是强极性树脂,分子中含有胺基、羟基、醚键、酰胺基等强极性基团,一方面有助色作用,增大荧光效率;另一方面与荧光染料有很好的相溶性,有助于染料的均匀分散。
荧光颜料常用的载体树脂有胺基树脂、苯代三聚氰胺一甲醛树脂、聚丙烯酸酯树脂、聚酰胺树酯、聚酯树脂、聚氨酯树脂等。 (1)、热塑性荧光颜料:线型
(2)、热固性荧光颜料: 体型
(3)、可溶解色精荧光颜料
(4)、水乳型荧光颜料 (1)、胺基树酯
(2)、聚酰胺树酯
(3)、聚酯树酯
(4)、丙烯酸乳液 (1)、塑胶类
低温型
中温型
高温型
(2)、涂料类
水性涂料
油性涂料
粉末涂料 (1)、含甲醛
(2)、不含甲醛
荧光增白剂是一种荧光染料,也是一种复杂的有机化合物,其特性就是能产生蓝色荧光,可使肉眼看到的物质很白。
测试面膜是否含有荧光增白剂,打开面膜的包装,然后放到“ZF-C型三用紫外分析仪”下,使用紫外光照射面膜,看其是否会发出亮蓝色光,如果有,说明面膜上含有荧光剂。 如果没有紫外分析仪,可以用紫外手电筒,紫外验钞机/笔等。
含荧光剂的面膜在紫外光照射下,显现出非常大面积的亮蓝色。稍微接触面膜液体,就会粘附上荧光剂成分,拿了面膜的手,也有荧光。用清水冲洗手3遍后, 手上的荧光剂在紫外光照射下依然发出强烈的亮蓝色光,几乎没有洗掉。改用洗手液清洗3遍,照射后手上也发出蓝光,只是比清水的效果略好一些。用洗面奶清洗,与洗手液的效果差不多,都无法彻底清洗掉残留在手上的荧光...
荧光剂对人体的危害:
荧光剂不像一般化学成分那样容易被分解,而是在人体内蓄积,产生许多有害的作用,大大削减人体免疫力;荧光剂与伤口外的蛋白质结合,还会阻碍伤口的愈合;荧光剂能使人体细胞出现变异性倾向,其毒性累积在肝脏或其他重要**,会成为潜在的致癌因素。造成血液系统受损:化学物质容易污染人体血液,虽然血液具有一定的自净能力,微量的有害物质进入其中,会被稀释、分解、吸附和排出,但长期、大量的有毒物质倾注而入,必致其发生质的变化;进入血液循环,会破坏红细胞的细胞膜,引起溶血现象。
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