Caspase-8isthemostupstreamcaspaseintheCD95/FasapoptoticpathwayandisactivatedbythesignalingpathwaysforCD95/FasandTNF.Caspase-8FluorometricandColorimetricAssayKitprovidesasimpleassaysystemforfastandsensitivedetectionofcaspase-8activityeitherbyfluorescenceorabsorbance.Thekitincludesthefluorogenicsubstrate(Ac-IETD)2-R110,theenzymeinhibitorAc-IETD-CHOasanegativecontrol,R110asastandardandoptimizedlysisandassaybuffers.Thesubstrate(Ac-IETD)2-R110containstwoIETDtetrapeptidesandiscompletelyhydrolyzedbytheenzymeinatwo-stepprocess.CleavageofthefirstIETDresultsinthemonopeptideAc-IETD-R110intermediate,whichhasabsorptionandemissionwavelengthssimilartothoseofR110(λEx/λEm=496/520nm)buthasonlyabout10%ofthefluorescenceofthelatter.HydrolysisofthesecondIETDpeptidereleasesthedyeR110,leADIngtoasubstantialfluorescenceincrease.
Althoughfluorometricdetectionoftheendproductsispreferredbecauseofthesuperiorsensitivity,detectionbyabsorbanceisalsopossIBLe.Infact,theextinctioncoefficientofR110is10timeshigherthanthatofp-nitroaniline(pNA),adyecommonlyusedinchromogenicsubstrates,makingR110-basedsubstratessignificantlymoresensitivethanpNA-basedsubstrates,evenbycolorimetricdetection.
Note:Whilecaspase-8preferentiallycleavestheconsensussequenceIETDcomparedtoothersubstratesequences,othercaspasessuchascaspase-3alsocancleaveIETDefficiently.Overlappingcaspasesubstraterecognitionlimitstheusefulnessofcaspasesubstratepeptidesfordistinguishingbetweendifferentcaspaseactivitiesincelllysates.
KitComponents:
- Celllysisbuffer
- Assaybuffer
- Enzymesubstrate(Ac-IETD)2-R110
- EnzymeinhibitorAc-IETD-CHO
- R110
Features:
- Fast:Fastenzymekinetics.
- Sensitive:Theenzymaticreactionformsintenselyyellowcoloredandhighlygreenfluorescentrhodamine110(R110)product.
- Versatile:Compatiblewithbothfluorometricandcolorimetricdetectionsystems.
References
1)Biochemistry38,13906(1999).
