Apoptosis is an evolutionarily conserved form of cell suicide, which follows a specialized cellular process. The central component of this process is a cascade of proteolytic enzymes called caspases. These enzymes participate in a series of reactions that are triggered in response to pro-apoptotic signals and result in cleavage of protein substrates, causing the disassembly of the cell. Caspases have been identified in organisms ranging from C. elegans to humans. The mammalian caspases play distinct roles in apoptosis and inflammation. In apoptosis, caspases are responsible for proteolytic cleavages that lead to cell disassembly (effector caspases), and are involved in upstream regulatory events (initiator caspases). An active caspase consists of two large (~20 kD) and two small (~10 kD) subunits to form two heterodimers which associate in a tetramer. As is common with other proteases, caspases are synthesized as precursors that undergo proteolytic maturation, either autocatalytically or in a cascade by enzymes with similar specificity. Caspase enzymes specifically recognize a 4 amino acid sequence (on their substrate) which necessarily includes an aspartic acid residue. This residue is the target for the cleavage reaction, which occurs at the carbonyl end of the aspartic acid residue. Caspases can be detected via immunoprecipitation, immuno-blotting techniques using caspase specific antibodies, or by employing fluorogenic substrates which become fluorescent upon cleavage by the caspase.
Our FAM caspase detection kits detect active caspases in living cells through the use of a carboxyfluorescein ( FAM) labeled peptide fluoromethyl ketone (FMK) caspase inhibitor (FAM-Peptide-FMK). These FAM-peptide-FMK compounds are both cell permeable and non-cytotoxic during the course of the assay and thus allow the detection of active caspases in living cell systems. The FAM-peptide inhibitor irreversibly binds to active caspases. Caspase positive cells are distinguished from caspase negative cells with the aid of flow cytometry or fluorescence microscopy. Adaptable to HTS in microplate format.
FAM700-2: 100 Tests