| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 346/617 |
| MW | N/A |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | SuperiorLabelingDyes trFluor™DyesandKits |
| Related | BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Prepareproteinsolution(SolutionA):
Forlabeling50µgprotein(assumingthetargetproteinconcentrationis1mg/mL),mix1.5µL(3%ofthetotalreactionvolume)ofReactionBuffer(ComponentB)with50µLofthetargetproteinsolution.
Note1.Ifyouhaveadifferenceproteinconcentration,adjusttheproteinvolumeaccordinglytomake~50µgproteinavailableforyourlabelingreaction.
Note2:Forlabeling100µgprotein(assumingthetargetproteinconcentrationis1mg/mL),mix3µL(3%ofthetotalreactionvolume)ofReactionBuffer(ComponentB)with100µLofthetargetproteinsolution.
Note3:Theproteinshouldbedissolvedin1Xphosphatebufferedsaline(PBS),pH7.2-7.4;Iftheproteinisdissolvedinglycinebuffer,itmustbedialyzedagainst1XPBS,pH7.2-7.4,oruseAmiconUltra-0.5,Ultracel-10Membrane,10 kDa(cat#UFC501008fromMillipore)toremovefreeaminesorammoniumsalts(suchasammoniumsulfateandammoniumacetate)thatarewidelyusedforproteinprecipitation.
Note4:Impureantibodiesorantibodiesstabilizedwithbovineserumalbumin(BSA)orgelatinwillnotbelabeledwell.
Note5:Theconjugationefficiencyissignificantlyreducediftheproteinconcentrationislessthan1mg/mL.Foroptimallabelingefficiencythefinalproteinconcentrationrangeof1-2mg/mLisrecommended.
2.Runconjugationreaction:
2.1 Addtheproteinsolution(SolutionA)toONEvialoflabelingdye(ComponentA),andmixthemwellbyrepeatedlypipettingforafewtimesorvortexthevialforafewseconds.
Note:Usebothvials(ComponentA)oflabelingdyetolabel100µgproteinbydividingthe100µgproteininto2x50µgproteinandreactingeach50µgproteinwithonevialoflabelingdye.Combinetwovialsforthenextstep.
2.2 Keeptheconjugationreactionmixtureatroomtemperaturefor30-60minutes.
Note:Theconjugationreactionmixturecanberotatedorshakenforlongertimeifdesired.
3.Preparespincolumnforsamplepurification:
3.1 InverttheSpinColumn(ComponentC)severaltimestoresUSPendthesettledgelandremoveanybubbles.
3.2 SnapoffthetipandplacethecolumnintheWashingTube(2mL,ComponentD).Removethecaptoallowtheexcesspackingbuffertodrainbygravitytothetopofthegelbed.Ifcolumndoesnotbegintoflow,pushcapbackintocolumnandremoveitagaintostarttheflow.Discardthedrainedbuffer,andthenplacethecolumnbackintotheWashingTube.However,centrifugeimmediatelyifthecolumnisplacedintoa12x75mmtesttube(notprovided).
3.3 Centrifugefor1mininaswingingbucketcentrifugeat1,000xg(seeCentrifugationNotessection)toremovethepackingbuffer.Discardthebuffer.
3.4 Apply1-2mL1XPBS(pH7.2-7.4)tothecolumn.AftereachapplicationofPBS,letthebufferdrainoutbygravity,orcentrifugethecolumnfor1mintoremovethebuffer.Discardthebufferfromthecollectiontube.Repeatthisprocessfor3-4times.
3.5 Centrifugefor2minutesinaswingingbucketcentrifugeat1,000xg(seeCentrifugationNotessection)toremovethepackingbuffer.Discardthebuffer.
4.Purifytheconjugation:
4.1 Placethecolumn(fromStep3.5)inacleanCollectingTube(1.5mL,ComponentE).Carefullyloadthesample(50–100μL)directlytothecenterofthecolumn.
4.2 Afterloadingthesample,add1XPBS(pH7.2-7.4)tomakethetotalvolumeof110μL.Centrifugethecolumnfor5minat1,000xg,andcollectthesolutionthatcontainsthedesireddye-labeledprotein.
| References&Citations |  PrinterFriendlyVersion |
1. SavilleL,SpaisC,MasonJL,AlbomMS,MurthyS,MeyerSL,AtorMA,AngelesTS,HustenJ.(2012)Time-ResolvedFluorescenceResonanceEnergyTransferasaVersatileToolintheDevelopmentofHomogeneousCellularKinaseAssays.AssayDrugDevTechnol.
2. LoMC,NgoR,DaiK,LiC,LiangL,LeeJ,EmkeyR,EksterowiczJ,VenturaM,YoungSW,XiaoSH.(2012)Developmentofatime-resolvedfluorescenceresonanceenergytransferassayforcyclin-dependentkinase4andidentificationofitsATP-noncompetitiveinhibitors.AnalBiochem,421,368.
3. PailaYD,KombrabailM,KrishnamoorthyG,ChattopadhyayA.(2011)OligomerizationoftheSEROtonin(1A)receptorinlivecells:atime-resolvedfluorescenceanisotropyapproach.JPhysChemB,115,11439.
4. MartikkalaE,Rozwandowicz-JansenA,HanninenP,Petaja-RepoU,HarmaH.(2011)Ahomogeneoussingle-labeltime-resolvedfluorescencecAMPassay.JBiomolScreen,16,356.
5. GaboritN,LarbouretC,VallagheJ,PeyrussonF,Bascoul-MolleviC,CrapezE,AzriaD,ChardesT,PoulMA,MathisG,BazinH,PelegrinA.(2011)Time-resolvedfluorescenceresonanceenergytransfer(TR-FRET)toanalyzethedisruptionofEGFR/HER2dimers:anewmethodtoevaluatetheefficiencyoftargetedtherapyusingmonoclonalantibodies.JBiolChem,286,11337.
6. LeyrisJP,RouxT,TrinquetE,VerdieP,FehrentzJA,OueslatiN,DouzonS,BourrierE,LamarqueL,GagneD,GalleyrandJC,M"KadmiC,MartinezJ,MaryS,BaneresJL,MarieJ.(2011)Homogeneoustime-resolvedfluorescence-basedassaytoscreenforligandstargetingthegrowthhormonesecretagoguereceptortype1a.AnalBiochem,408,253.
7. PosokhovYO,KyrychenkoA,LadokhinAS.(2010)Steady-stateandtime-resolvedfluorescencequenchingwithtransitionmetalionsasshort-distanceprobesforproteinconformation.AnalBiochem,407,284.
8. Alvarez-CurtoE,WardRJ,PedianiJD,MilliganG.(2010)LigandregulationofthequaternaryorganizationofcellsurfaceM3muscarinicacetylcholinereceptorsanalyzedbyfluorescenceresonanceenergytransfer(FRET)imagingandhomogeneoustime-resolvedFRET.JBiolChem,285,23318.
9. KotaS,ScampaviaL,SpicerT,BeelerAB,TakahashiV,SnyderJK,PorcoJA,HodderP,StrosbergAD.(2010)Atime-resolvedfluorescence-resonanceenergytransferassayforidentifyinginhibitorsofhepatitisCviruscoredimerization.AssayDrugDevTechnol,8,96.
10. VisserAJ,LaptenokSP,VisserNV,vanHoekA,BirchDJ,BrochonJC,BorstJW.(2010)Time-resolvedFRETfluorescencespectroscopyofvisiblefluorescentproteinpairs.EurBiophysJ,39,241.
AAT Bioquest AAT Bioquest是一家位于美国的生物公司，前身为ABD Bioquest，总部位于加利福尼亚州。专门从事光学检测技术十多年，一直致力于光谱学检测领域技术的创新和突破。其独特的光学检测技术，综合了化学、生物学和信息学等各个领域的研究，引领了比色、荧光和发光技术新一代光学探针的浪潮。AAT Bioquest在全球拥有强大的经验丰富的专业分销商网络，为从小型研究机构到《财富》500强企业的各类客户提供卓越的产品和定制服务。
美国AATBioquestInc.(前身是ABDBioquest，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。AATBioquest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。AATBioquest会不断介绍新产品，快速的丰富各个领域的产品。
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Besidesthestandardcatalogproductswealsooffercustomservicetomeetyourspecialresearchneeds.Ourcurrentservicesincludecustomsynthesisofcolorimetric,fluorescentandluminescentprobes,customdevelopmentofbiochemical,cell-basedanddiagnosticassaysandcustomscreeningofyourcompoundlibrariesagainstyourdefinedtargetsusingourvalidatedHTSassays.
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TUNEL的缺点是：1），操作要求高，组织样本需要固定（即使是培养细胞，也需要固定），不恰当的固定方法对实验结果影响很大，导致背景过高或者信号过弱，因此实验结果重复性不好;有的人花上半年做一个体内的TUNEL是一点都不奇怪的。2），大部分诱导凋亡的药物也引起DNA损伤，从而产生DNA断裂，易引入假阳性;3）凋亡晚期细胞基因组大量降解，导致TUNEL标记反而减少，因此此法反应的是早期凋亡比例，不能严格反应凋亡比例，属于半定量研究。
尽管有如此多缺点，但由于其是目前为数不多的能原位标记凋亡细胞的方法，因此用于组织体内凋亡研究仍然是首选。但体外细胞实验研究，很少用此法。
凋亡检测中，TUNEL并不是过时的方法，现在研究凋亡的文献仍然常见。而且相反，还比以前多一点，因为现在体内实验越来越多了，甚至线虫的凋亡研究，都用TUNEL。
凋亡研究中，的确有几种方法过时了，当然是因为有替代方案了，比如DNAladder，电镜。至于AnnexinV是不能用来和TUNEL一起评论的，前者用于细胞，而且主要是悬浮细胞，后者主要用于组织。虽然有人也做镜下的AnnexinV观察，TUNEL的细胞staining，但这都不是主流。
回到lz的原帖，的确如大家所言，做贴壁细胞，不推荐用TUNEL。如果是经典凋亡途径，只是确定比例，用最经典，最常用的subG1法即可，如果是不确定是否是凋亡，用AnnexinV，不过贴壁细胞用此法，要注意消化时间。
想请问下那个公司有专门的蛋白质荧光标记试剂盒出售。最好的是CY5的，我准备做三标。性价比越高越好
那些做过的前辈们指导一下。
那为什么SFDA不批准CA199CEAAFP等检测试剂盒作为癌症检测的手段呢？
在弱碱性（pH 8~9）、暗处、室温或40℃条件下，氨基酸的α-氨基很容易与2,4-二硝基氟苯（缩写为FDNB或DNFB）反应，生成黄色的2,4-二硝基苯氨基酸（dinitrophenyl amino acid，简称DNP-氨基酸）。多肽或蛋白质的N-末端氨基酸的α-氨基也能与FDNB反应，生成一种二硝基苯肽（DNP-肽）。由于硝基苯与氨基结合牢固，不易被水解，因此当DNP-多肽被酸水解时，所有肽键均被水解，只有N-末端氨基酸仍连在DNP上，所以产物为黄色的DNP-氨基酸和其它氨基酸的混合液。混合液中只有DNP-氨基酸溶于乙酸乙酯，所以可以用乙酸乙酯抽提并将抽提液进行色谱分析，再以标准的DNP-氨基酸作为对照鉴定出此氨基酸的种类。因此2,4-二硝基氟苯法可用于鉴定多肽或蛋白质的N-末端氨基酸。
