FITC-solution-生命科学试剂-MCE

Hotline:400-820-3792Inhibitors•ScreeningLibraries•Proteinswww.MedChemEFITC(solution)Cat.No.:HY-DY1007CASNo.:3326-32-7分⼦式:C₂₁H₁₁NO₅S分⼦量:389.38作⽤靶点:FluorescentDye作⽤通路:Others储存⽅式:PleasestoretheproductundertherecommendedconditionsintheCertificateofAnalysis.BIOLOGICALACTIVITY⽣物活性FITC(solution)，英⽂全称FluoresceinIsothiocyanate，中⽂名称异硫氰酸荧光素，⽣物学中应⽤最为⼴泛的⼀种绿⾊荧光素衍⽣物。FITC具有⾼吸收率和优良的荧光量⼦产率等特点。FITC的异硫氰酸团可与蛋⽩质上的氨、巯、咪唑、酪氨酰、羰等团相结合，从⽽实现包括抗体，凝集素在内的蛋⽩标记。FITC除了⽤作蛋⽩质标记物，还可⽤作蛋⽩质荧光⽰踪剂，通过标记抗体⽽快速鉴定病原体，或⽤于蛋⽩质和多肽(HPLC)的微量测序。FITC的最⼤激发波长为494nm。⼀旦激发，在最⼤发射波长520nm处呈-绿⾊荧光。此外，FITC也⼀种能诱发接触超敏反应(CHS)的半抗原，并诱导特应性⽪炎模型。溶剂及浓度：DMSO：20mM体外研究Protocol1.ProteinPreparetion1)Inordertoobtainthebestlabelingeffect,pleasepreparetheprotein(antibody)concentrationas2mg/mL.2)ThepHvalueofproteinsolutionshallbe8.5±0.5.IfthepHislowerthan8.0,1msodiumbicarbonateshallbeusedforadjustment.3)Iftheproteinconcentrationislowerthan2mg/ml,thelabelingefficiencywillbegreatlyreduced.Inordertoobtainthebestlabelingefficiency,itisrecommendedthatthefinalproteinconcentrationrangeis2-10mg/mL.4)Theproteinmustbeinthebufferwithoutprimaryamine(suchasTrisorglycine)andammoniumion,otherwisethelabelingefficiencywillbeaffected.2.DyePreparationAddanhydrousDMSOintothevialofFITCtomakea10mMstocksolution.Mixwellbypipettingorvortex.3.Calculationofdyedosage1/2MasterofBioactiveMolecules—您⾝边的抑制剂⼤师www.MedChemETheamountofFITCrequiredforreactiondependsontheamountofproteintobelabeled,andtheoptimalmolarratioofFITCtoproteinisabout10.Example:assumingtherequiredmarkerproteinis1mL2mg/mLIgG(MW=150,000),use1mLDMSOdissolve1mgFITC,therequiredFITCvolumeis40μL.4.Runconjugationreaction1)Agoodvolumeoffreshlyprepared50μLFITCisslowlyaddedto0.5mLproteinsample.Insolution,gentlyshaketomix,thencentrifugebrieflytocollectthesampleatthebottomofthereactiontube.Don'tmixwelltopreventproteinsamplesfromdenaturationandinactivation.2)Thereactiontubuleswereplacedinadarkplaceandincubatedgentlyatroomtemperaturefor60minutesatintervals.For10-15minutes,gentlyreversethereactiontubulesseveraltimestofullymixthetworeactantsandraisethebarefficiency.5.PurifytheconjugationThefollowingprotocolisanexampleofdye-proteinconjugatepurificationbyusingaSephadexG-25column.1)PrepareSephadexG-25columnaccordingtothemanufactureinstruction.2)Loadthereactionmixture(From"Runconjugationreaction")tothetopoftheSephadexG-25column.3)AddPBS(pH7.2-7.4)assoonasthesamplerunsjustbelowthetopresinsurface.4)AddmorePBS(pH7.2-7.4)tothedesiredsampletocompletethecolumnpurification.Combinethefractionsthatcontainthedesireddye-proteinconjugate.Note1.FITCissensitivetolightandhumidity.ImmediatelyaddFITCsolutionanddiscardtheunusedpart.2.Lowconcentrationsofsodiumazide(≤3mMor0.02%)orthiomersal(≤0.02mMor0.01%)didnotsignificantlyinterferewithproteinlabeling;However,20-50%glycerolwillreducelabelingefficiency.3.Avoidbufferingwithprimaryamines(e.g.,Tris,glycine)orammoniumions,Itcompetewithlabeledproteins.4.Thisproductisonlyforscientificresearchbyprofessionals,andshallnotbeusedinclinicaldiagnosisortreatment,foodormedicine.5.Foryoursafetyandhealth,pleasewearlabcoatanddisposablegloves.REFERENCES[1].GaoSG,etal.Phosphorylationofosteopontinhasproapoptoticandproinflammatoryeffectsonhumankneeosteoarthritischondrocytes.

ExpTherMed.2016Nov;12(5):3488-3494.Epub2016Oct5.[2].ZhuX,etal.Ratiometric,visual,dual-signalfluorescentsensingandimagingofpH/copperionsinrealsamplesbasedoncarbondots-

fluoresceinisothiocyanatecomposites.Talanta.2017Jan1;162:65-71[3].ShigenoT,etal.Phthalateester‐inducedthymicstromallymphopoietinmediatesallergicdermatitisinmice[J].Immunology,2009,128(1pt2):e849-e857.McePd