Biotium/Caspase-3 DEVD-R110 Fluorometric and Colorimetric Assay Kit/30008-2/100-assays

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.