Abcam's Caspase 10 Assay Kit (Fluorometric) (ab65662) provides a simple and convenient means for assaying the activity of caspases that recognize the sequence AEVD such as caspase 10. The assay is based on detection of cleavage of substrate AEVD-AFC (AFC: 7-amino-4-trifluoromethyl coumarin). AEVD-AFC emits blue light (lmax = 400 nm); upon cleavage of the substrate by caspase 10 or related caspases, free AFC emits a yellow-green fluorescence (lmax = 505 nm), which can be quantified using a fluorometer or a fluorescence microtiter plate reader. Comparison of the fluorescence of AFC from an apoptotic sample with an uninduced control allows determination of the fold increase in AEVD-dependent caspase activity.
Store kit at –20oC. Store Cell Lysis Buffer and 2X Reaction Buffer at 4oC after opening. Protect AEVD-AFC from light. All reagents are stable for 6 months under proper storage conditions.
RelevanceCaspases are a family of intracellular proteases that mediate cell death and are the principal effectors of apoptosis. Caspase 10 (Mch4, ICE-LAP4, FLICE2) plays an important role in apoptosis induced by a variety of inducers such as TNF alpha. It is a large prodomain caspase classified together with caspases 2, 8, and 9 as a signaling caspase. Four isoforms of caspase 10 (caspase 10a, 10b, 10c, and 10d) having the same prodomain but different mature large and small subdomain, have been described. Caspase 10 contains two death domains (DED) involved in linking to the death effector domain of the adapter protein FADD and recruiting the complex to TNFR1 and Fas. The inactive procaspase 10 is variably expressed in many tissues and cell lines as a cytosolic protein. The mature form of caspase 10 comprises two subunits, p23/p17 (splice isoforms) and p12. Caspase 10 can cleave and activate caspases 3, 4, 6, 7, 8, and 9. This is followed by cleavage of numerous key proteins, including the nuclear protein PARP.