Caspase-3isanactivecell-deathproteaseinvolvedintheexecutionphaseofapoptosis,duringwhichcellsundergomorphologicalchangessuchasDNAfragmentation,chromatincondensationandapoptoticbodyformation.Caspase-3DEVD-R110FluorometricandColorimetricAssayKitprovidesasimpleassaysystemforfastandhighlysensitivedetectionofcaspase-3activityeitherbyfluorescenceorabsorbanceinmammaliancells.Thefluorogenicandchromogenicsubstrate(Ac-DEVD)2-R110containstwoDEVDtetrapeptidesandiscompletelyhydrolyzedbytheenzymeintwosuccessivesteps.CleavageofthefirstDEVDpeptideresultsinthemonopeptideAc-DEVD-R110intermediate,whichhasabsorptionandemissionwavelengthssimilartothoseofR110(λEx/λEm=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.
References
1.CellDeathDiff.6,99(1999).
2.JBiolChem274,11549(1999).
3.JBiolChem275,288(2000).
4.Biochemistry,38,13906(1999).
