The CaspGLOW™ Fluorescein Active Caspase-3 Staining Kit contains the reagents to sensitively detect active Caspase-3 in mammalian cells. This assay utilizes the inhibitor specific for Caspase-3, DEVD-FMK, that is directly conjugated to FITC (fluorescein isothiocyanate) for the detection system. This reagent is cell permeable, non-toxic and irreversibly binds to the active enzyme. Detection of the labeled cells can be determined by flow cytometry, fluorescent microscopy or a fluorescent plate reader.
The Caspases are a family of aspartate-specific cysteine proteases that act in a step-wise signaling manner like kinases. Recruitment of these proteases to oligomerized receptors leads to activation and autoproteolytic cleavage. Active caspases can proteolyze additional caspases generating a caspase cascade. The final outcome of this signaling pathway is a form of controlled cell death, termed apoptosis.
Caspase-3, also known as Yama, CPP32, and apopain, cleaves its substrates at the carboxyl terminus of aspartate residues. Active Caspase-3 consists of 2 sets of homodimers (~ 17 and 12 kDa) that are derived from two precursor Caspase-3 polypeptides and has two active sites. Caspase-3 is proteolytically activated by other caspases.
Both subunits contribute to substrate binding and catalysis. The active site cysteine that covalently binds the substrate is located near the C-terminus of the large subunit. Active Caspase-3 has two-fold symmetry, two active site pockets each residing on an opposite side. Caspase-3, together with Caspases 8 and 9, is situated at pivotal junctions in apoptotic pathways. Caspase-3 appears to amplify Caspase 8 and 9 initiation signals into complete commitment to apoptotic disassembly.