Caspase-9 Fluorimetric Assay Kit provides a simple procedure for assaying the activity of caspases that recognize the sequence LEHD. Consequently, the user can detect the fluorophore 7-amino-4-trifluoromethylcoumarin (AFC), after cleavage from the labeled substrate LEHD-AFC. The excitation and emission wavelength range for monitoring AFC in a spectrofluorometer are 400 nm and 480-520 nm, respectively. Comparison of AFC fluorescence from an apoptotic sample vs AFC fluorescence of control allows determination of the fold increase in caspase-9 activity.
There are two classes of caspases that involve apoptosis; initiators and effectors. Proapoptotic signals autocatalytically activate initiator caspases, such as Caspase 8 and Caspase 9. As a consequence, activated initiator caspases then process effector caspases, such as Caspase 3 and Caspase 7, which in turn cause cell collapse. As a resume, initiator caspases (caspase 2, 8, 9, and 10) initiate the apoptosis signal and the executioner caspases (caspase 3, 6, and 7) carry out the mass proteolysis that leads to apoptosis.