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当前位置: 首页 > 产品中心 > Other_biological_dyes > AAT Bioquest/Sunnyvale Red™ SE *Superior 6-ROX Replacement*/394/5 mg
商品详细AAT Bioquest/Sunnyvale Red™ SE *Superior 6-ROX Replacement*/394/5 mg
AAT Bioquest/Sunnyvale Red™ SE *Superior 6-ROX Replacement*/394/5 mg
AAT Bioquest/Sunnyvale Red™ SE *Superior 6-ROX Replacement*/394/5 mg
商品编号: 394
品牌: aatbio
市场价: ¥56560.00
美元价: 33936.00
产地: 美国(厂家直采)
公司:
产品分类: 其他生物染料
公司分类: Other_biological_dyes
联系Q Q: 3392242852
电话号码: 4000-520-616
电子邮箱: info@ebiomall.com
商品介绍
Overview
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Ex/Em(nm)576/605
MW~650
CAS#N/A
SolventDMSO
StorageF/D/L
CategoryOligoSynthesis
DyeLabelingReagents
RelatedRhodamines
Amine-ReactiveProbes
AlthoughROXdyeshavestrongfluorescencetheyarenotoriouslyunstable.Somecommercialsamplesonlyhaveafewmonthsshelflife.Comparedto6-ROXlabelingcompoundsourSunnyvaleRed™dyeshavegreatlyenhancedstABIlitywhiletheyhaveessentiallyidenticalspectralpropertiestothoseof6-ROXwhenconjugatedtoaBIOLOGicalsubstrate(suchasoligonucleotides).OurpreliminarystudiesindicatedthatSunnyvaleRed-derivedoligosaremoreeasilypurifiedthanthecorresponding6-ROXoligosinsomecases.
SpectrumAdvancedSpectrumViewer

Sorry,yourbrowserdoesnotsupportinlineSVG.RelativeIntensity(%)100806040200Sorry,yourbrowserdoesnotsupportinlineSVG.
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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

Theprotocolhasbeenoptimizedforlabeling100μgofan5′-amine–modifiedoligonucleotide,15to25basesinlength.Someadjustmentstotheprotocolmaybenecessaryforgreatlyshorterorlongeroligonucleotides.Thereactionmaybescaledupordownaslongastheconcentrationofeachcomponentisnotchanged.

1.      Purifytheamine-modifiedoligonucleotide(ifnecessary).Dissolvetheoligonucleotidein100μLdH2Oandextractthreetimeswithanequalvolumeofchloroform.Precipitatetheoligonucleotidebyaddingone-tenthvolume(10μL)of3MNaClandtwoandahalfvolumes(250μL)ofcoldabsoluteethanol.Mixwellandplaceat≤–20°Cfor30minutes.Centrifugethesolutioninamicrocentrifugeat~12,000gfor30minutes.Carefullyremovethesupernatant,rinsethepelletonceortwicewithcold70%ethanolanddryundervacuum.DissolvethedrypelletindH2Otoachieveafinalconcentrationof25μg/μL.Thisamine-modifiedoligonucleotidestocksolutionmaybestoredfrozenat≤–20°C.

a.      [Note:Toensurethattheoligonucleotideisfreeofinterferingcompounds,especiallyamines(suchastriethylamineorTris)andammoniumsalts,westronglyrecommendextractingandprecipitatingthesamplepriortoinitiatingthelabelingreaction.]

2.      Preparelabelingbuffer.Makea0.1Msodiumtetraboratebufferbydissolving0.038gofsodiumtetraboratedecahydrateforeverymLofwater.AdjustpHwithHClto8.5.

a.      [Note:Thislabelingbuffershouldbemadeascloseaspossibletothetimeoflabeling.Alternatively,itmaybedividedintosmallaliquotsandfrozenimmediatelyforlong-termstorage.Exposureofthissolutiontoairforalongtimewillresultincarbondioxideabsorption,whichwillchangethepHofthebuffer.]

3.      PrepareSunnyvaleRed™SEDMSOStockSolution.AllowSunnyvaleRed™SEtocometoroomtemperaturebeforeopeningthevial.Dissolve300μgofSunnyvaleRed™SEin10μLDMSObypipettingupanddown,washingthesidesofthevial.Use200μgSunnyvaleRed™SEforlabeling100μgofoligonucleotide.

a.      [Note:ItisimportantthatSunnyvaleRed™SEbefreshlypreparedforeachlabelingreactionasitisnotstableinsolution.]

4.       Starttheconjugation.TothevialcontainingSunnyvaleRed™SElabelingreagentinDMSO,add7μLdH2O,75μLlabelingbuffer(stepb),4μLofa25μg/μLoligonucleotidestocksolution(stepa).Placethevialonashakeroscillatingatlowspeedorgentlyvortexmixortapthevialeveryhalfhourforthefirsttwohourstoensurethatthereactionremainswellmixed.Donotmixviolently,asmaterialmaybeleftonthesidesofthevial.Aftersixhours,50–90%oftheamine-modifiedoligonucleotidemoleculesshouldbelabeled.

a.      [Note:Thereactionmixturemayhaveagrainyappearance,butthisshouldnotadverselyaffecttheconjugation.Thereactionmaybescaledupordownaslongastheconcentrationofeachcomponentisnotchanged.Donotaddmoredyethanrecommended,asexcessdyewillnotimprovethelabelingefficiencyandmaymakethepurificationmoredifficult.]

5.      PurifyingtheSunnyvaleRed™-LabeledOligonucleotide.Followingthereaction,thelabelingmixturecontainslabeledoligonucleotide,unlabeledoligonucleotide,andunincorporateddye.Precipitatethereactionmixturewithethanolasfollows:Addonetenthvolumeof3MNaClandtwoandahalfvolumesofcoldabsoluteethanoltothereactionvial.Mixwellandplaceat≤–20°Cfor30minutes.Centrifugethesolutioninamicrocentrifugeat~12,000×gforafull30minutes.Thelabeledoligonucleotidecanbefurtherpurifiedbypreparativegelelectrophoresisorreverse-phaseHPLC.

a.      [Note:Lossofsamplemayoccurifthecentrifugationisnotlongenough.Carefullyremovethesupernatant,rinsethepelletonceortwicewithcold70%ethanolanddrybriefly.Ifthelabeledoligonucelotidebecomescompletelydry,itwillbedifficulttoredissolve.]

6.      HPLCPurification:Useastandardanalytical(4.6×250mm)C8column.Dissolvethepelletfromtheethanolprecipitationin0.1MTEAA(triethylammoniumacetate).Loadthedissolvedpelletontothecolumnin0.1MTEAAandrunalinear5–65%acetonitrilegrADIentover30minutes.Thisgradientisa2%increaseinacetonitrileperminute.Theunlabeledoligonucleotidewillmigratefastest,followedbythelabeledoligonucleotideandfinallythefreedye.

7.      GelEelectrophoresisPurification:Topurifythelabeledoligonucleotidebygelelectrophoresis,poura0.5mm–thickpolyacrylamideslabgel.Foroligonucleotideslessthan25basesinlength,use19%acrylamide,foroligonucleotides25–40bases,15%acrylamide,andforoligonucleotides40–100bases,12%acrylamide.ResUSPendthepelletfromethanolprecipitationin200μLof50%formamide,andincubateat55°Cfor5minutestodisruptanysecondarystructure.Loadthewarmedoligonucleotideontothegel(youmayneedtouseseveralwells)andloadanadjacentwellwith50%formamideplus0.05%bromophenolblue.Thebromophenolbluewillmigrateatapproximatelythesamerateastheoligonucleotide.Runthegeluntilthebromophenolblueindicatordyeistwo-thirdsofthewaydownthegel.RemovethegelfromtheglassplatesandplaceonSaranWrap.LaythegelonafluorescentTLCplate.LocatethelabeledandunlabeledoligonucleotidesbyIlluminationwithahandheldUVsource.Fluorophore-labeledoligonucleotideswillshowfluorescencewhenilluminatedwithUVlight.Cutoutthebandcontainingthelabeledoligonucleotideandpurifybythe“crush-and-soak”methodorothersuitablemethod.

References&Citations
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1.   SeoTS,BaiX,KimDH,MengQ,ShiS,RuparelH,LiZ,TurroNJ,JuJ.(2005)Four-colorDNAsequencingbysynthesisonachipusingphotocleavablefluorescentnucleotides.ProcNatlAcadSciUSA,102,5926.

2.   PrazeresTJ,SantosAM,MartinhoJM,ElaissariA,PichotC.(2004)AdsorptionofoligonucleotidesonPMMA/PNIPAMcore-shelllatexes:polarityofthePNIPAMshellprobedbyfluorescence.Langmuir,20,6834.

3.   SeoTS,BaiX,RuparelH,LiZ,TurroNJ,JuJ.(2004)PhotocleavablefluorescentnucleotidesforDNAsequencingonachipconstructedbysite-specificcouplingchemistry.ProcNatlAcadSciUSA,101,5488.

4.   FauldsK,SmithWE,GrahamD.(2004)Evaluationofsurface-enhancedresonanceRamanscatteringforquantitativeDNAanalysis.AnalChem,76,412.

5.   YangJH,BasingerSF,GrossRL,WuSM.(2003)Bluelight-inducedgenerationofreactiveoxygenspeciesinphotoreceptorellipsoidsrequiresmitochondrialelectrontransport.InvestOphthalmolVisSci,44,1312.

6.   SlatevaK,ElsnerHA,Albis-CampsM,BlasczykR.(2001)HLA-DRBfluorotypingbydarkquenchingandautomatedanalysis.TissueAntigens,58,250.

7.   HahnM,WilhelmJ,PingoudA.(2001)Influenceoffluorophordyelabelsonthemigrationbehaviorofpolymerasechainreaction--amplifiedshorttandemrepeatsduringdenaturingcapillaryelectrophoresis.Electrophoresis,22,2691.

8.   LiY,GlazerAN.(1999)Design,synthesis,andspectroscopicpropertiesofpeptide-bridgedfluorescenceenergy-transfercassettes.BioconjugChem,10,241.

9.   YoshikawaY,MukaiH,AsadaK,HinoF,KatoI.(1998)Differentialdisplaywithcarboxy-X-rhodamine-labeledprimersandtheselectionofdifferentiallyamplifiedCDNAfragmentswithoutcloning.AnalBiochem,256,82.

10.   HungSC,MathiesRA,GlazerAN.(1998)Comparisonoffluorescenceenergytransferprimerswithdifferentdonor-acceptordyecombinations.AnalBiochem,255,32.


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品牌介绍
美国AAT Bioquest Inc.(前身是ABD Bioquest,Inc.)是一家为从事生命科学研究、诊断研发及药物开发的科学家研发、生产和销售生物分析研究试剂和试剂盒的公司。公司致力于光谱学检测领域,包括吸收(颜色)、荧光和发光技术。AAT Bioquest的产品帮助全世界的科学家和生物医药研究者更好的了解生物化学、免疫学、细胞生物学和分子生物学等领域。AAT Bioquest会不断研发新产品,快速地丰富各个领域的产品。 1)提供反应探针和发光探针,生物素和端粒酶能够应用于标记药物小分子和生物聚合物,如蛋白、核酸以及其他碳水化合物; 2)研究并生产荧光和发光探针用于检测蛋白,核酸和活细胞; 3)不断推出新型的荧光和发光探针用于检测多种酶,特别是检测水解酶和氧化还原酶类; 4)致力于开发用于信号转导研究的试剂; 5)提供生理和神经探针,特别是钙离子指示剂和膜电位探针。
品牌分类
淋巴细胞信号传导 GPCR抗体 羰基活性生物素 细胞代谢 胺反应生物素 链霉亲和素结合物 生物素化抗IgGs 乙酰基特异性抗体 细胞骨架抗体
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