Bioquochem’s Caspase-9 Fluorimetric Assay Kit Principle
Bioquochem’s 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.
Caspases are also known as cysteine-aspartic proteases, cysteine aspartases or cysteine-dependent aspartate-directed proteases. They are a family of protease enzymes that play essential roles in programmed cell death. Caspases have the ability to cleave after aspartic acid residues.
There are two classes of caspases that involve in apoptosis; initiators (activation by receptor cluster) and effectors (activation by mitochondrial permeability transition). 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.
Caspase 9 is also known as ICE like apoptotic protease 6 (ICE LAP6), apoptotic protease Mch6, and apoptotic protease activating factor 3 (Apaf3)). Specifically,iIt is a member of the peptidase family C14 that contains a CARD domain. It is active as a heterotetramer and has two isoforms, according to literature. ProCaspase 9 is approximately 47 kD. It is present in the cytosol and, upon activation, translocates to the mitochondria. Caspase 9 takes part in the caspase activation cascade, responsible for apoptosis execution, and cleaves/activates Caspase 3 and Caspase 6. It becomes activated when recruited to the Apaf1/cytochrome c complex.
Bioquochem’s Caspase-9 Fluorimetric Assay Kit bases in a short peptide that contains specific cleavage sequences recognized by the caspase. As a result, caspase-9 cleaves the reagent and releases the colorimetric or fluorogenic detection probe.