Caspase-3isanactivecell-deathproteaseinvolvedintheexecutionphaseofapoptosis,duringwhichcellsundergomorphologicalchangessuchasDNAfragmentation,chromatincondensationandapoptoticbodyformation.Caspase-3DEVD-R110FluorometricandColorimetricAssayKitprovidesasimpleassaysystemforfastandhighlysensitivedetectionofcaspase-3activityeitherbyfluorescenceorabsorbanceinmammaliancells.Thefluorogenicandchromogenicsubstrate(Ac-DEVD)2-R110containstwoDEVDtetrapeptidesandiscompletelyhydrolyzedbytheenzymeintwosuccessivesteps.CleavageofthefirstDEVDpeptideresultsinthemonopeptideAc-DEVD-R110intermediate,whichhasabsorptionandemissionwavelengthssimilartothoseofR110(λ_Ex/λ_Em=496/520nm)buthasonlyabout10%ofthefluorescenceofthelatter.HydrolysisofthesecondDEVDpeptidereleasesthedyeR110,leADIngtoasubstantialfluorescenceincrease.

Althoughfluorometricdetectionoftheendproductsispreferredbecauseofthesuperiorsensitivity,detectionbyabsorbanceisalsopossIBLe.Infact,theextinctioncoefficientofR110is10timeshigherthanthatofp-nitroaniline(pNA),adyecommonlyusedinchromogenicsubstrates,makingR110-basedsubstratessignificantlymoresensitivethanpNA-basedsubstrates,evenbycolorimetricdetection.

TheassaykitincludesAc-DEVD-CHO,whichisacaspase-3inhibitorandcanbeusedasanegativecontrol.Also,R110isprovidedinthekitforgeneratingastandardcurve,whichcanbeusedforquantifyingcaspase-3activity.

Note:Whilecaspase-3preferentiallycleavestheconsensussequenceDEVDcomparedtoothersubstratesequences,othercaspasesalsocancleaveDEVDefficiently.Overlappingcaspasesubstraterecognitionlimitstheusefulnessofcaspasesubstratepeptidesfordistinguishingbetweendifferentcaspaseactivitiesincelllysates.

KitComponents:

• Celllysisbuffer
• Assaybuffer
• Enzymesubstrate(Ac-DEVD)2-R110
• EnzymeinhibitorAc-DEVD-CHO
• R110

Features:

• Fast:Fastenzymekinetics.
• Sensitive:Theenzymaticreactionformsintenselyyellowcoloredandhighlygreenfluorescentrhodamine110(R110)product.
• Versatile:Compatiblewithbothfluorometricandcolorimetricdetectionsystems.

BiotiumalsoofferstheCaspase-3DEVD-R110FluorometricHTSAssayKit(catalog#30009),aonestephomogenousassayforhighthroughputscreening(HTS)usingfluorescent-basedmeasurement.Forreal-timedetectionofcaspase-3activityinintactcells,seeournovelNucView™488Caspase-3Substrate.

Biotium公司是一家在荧光技术领域独树一帜的生物技术公司，公司位于美国加利福尼亚的BAY AREA------世界上首要的生物科技中心。该公司一直致力于开发和生产用于生物医学研究的荧光指示剂及其他特殊生化产品。其产品被广泛应用于细胞生物学、蛋白质组学、分子生物学、免疫学、微生物学、诊断学、生物化学、神经科学、血液流动检测及大规模药物筛选等众多领域。Biotium公司专利产品众多，包括已经被广泛使用的实时PCR定量荧光染料EvaGreen、具有划时代意义的凝胶核酸染料GelRed和GelGreen，以及最新推出的Cheetah Taq热启动酶个CF系列蛋白质标记染料等等。