Caspase-3DEVD-R110FluorometricHTSAssayKitisspecificallydesignedforHTS-basedassays.Thekitprovidesahomogenousassaysystemforfastandhighlysensitivedetectionofcaspase-3activitybyfluorescenceinenzymaticreactionormammaliancells.Thefluorogenicsubstrate(Ac-DEVD)2-R110containstwoDEVDtetrapeptidesandiscompletelyhydrolyzedbytheenzymeintwosuccessivesteps.CleavageofthefirstDEVDpeptideresultsinthemonopeptideAc-DEVD-R110intermediate,whichhasabsorptionandemissionwavelengthssimilartothoseofR110(λEx/λEm=496/520nm)buthasonlyabout10%ofthefluorescenceofthelatter.HydrolysisofthesecondDEVDpeptidereleasesthedyeR110,leADIngtoasubstantialfluorescenceincrease.
TheassaykitincludesAc-DEVD-CHO,whichisacaspase-3inhibitorandcanbeusedasanegativecontrol.Also,R110isprovidedinthekitforgeneratingastandardcurve,whichcanbeusedforquantifyingcaspase-3activity.OnemLcelllysis/assaybufferissufficientfor10assaysin96-wellformat.
Note:Whilecaspase-3preferentiallycleavestheconsensussequenceDEVDcomparedtoothersubstratesequences,othercaspasesalsocancleaveDEVDefficiently.Overlappingcaspasesubstraterecognitionlimitstheusefulnessofcaspasesubstratepeptidesfordistinguishingbetweendifferentcaspaseactivitiesincelllysates.
KitComponents:
- Celllysis/assaybuffer
- Enzymesubstrate(Ac-DEVD)2-R110
- EnzymeinhibitorAc-DEVD-CHO
- R110
Features:
- HTS-compatIBLe:Single-stephomogenousassayspecificallydesignedforHTS-baseddetection.
- Fast:Fastenzymekinetics.
- Sensitive:Greenfluorescentreadout
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).
