Active caspase recognizes 4 amino acid recidues in a substrate, which are from the aspartate residue next to 3 amino acid recidues, and specifically cleaves the substrate at the C-terminal side of the aspartate. Using this property, caspase activity can be measured.
In this method, the C-terminal ends of the tetrapeptides recognized with by activated caspases are conjugated with p-nitroanilide (pNA), 7-amino-4-methyl coumarin (AMC), and 7-amino-4-trifluoromethyl coumarin (AFC) at the C-terminal side and they are used as substrates. When After the caspases cleaves the substrates, pNA, AMC, and AFC are released. By The caspase activity can be quantified by measurement of the released emitted fluorescences with a microplate reader, the caspase activities can be quantified (pNA; Abs. 400 nm or 405 nm, AMC; Ex. 380 nm Em. 460 nm, AFC; Ex. 400 nm Em. 505 nm).
Amino acid sequence of substrates which are used in each kits
Activity of Caspases measured by APOPCYTO Caspase Colorimetric Assay Kits