Apoptosis is a regulated physiological process leading to cell death. Caspases, a family of cysteine acid proteases, are central regulators of apoptosis. Initiator caspases (including 2, 8, 9, 10 and 12) are closely coupled to proapoptotic signals, which include FasL, TNF-alpha, and DNA damage. Once activated, these caspases cleave and activate downstream effector caspases (including 3, 6 and 7), which in turn cleave cytoskeletal and nuclear proteins such as PARP, alpha-fodrin, DFF and lamin A; inducing apoptosis. Formation of a death-inducing signaling complex (DISC) around the receptors for death factors, including FasL and TNF-alpha, is essential for receptor-mediated apoptosis. Upon ligand activation, Fas and TNF-R1 associate with death domain (DD) containing adaptor proteins FADD (Fas associated death domain) and TRADD (TNF-R1 associated death domain). In addition to a carboxy-terminal DD, FADD contains an amino-terminal death effector domain (DED) that binds to DEDs and activates initiator caspase 8 (FLICE, Mch5, MACH) and caspase 10 (FLICE2, Mch4). TRADD does not contain a DED and therefore must associate with FADD in response to TNF-R1 driven apoptosis. Caspase-9 (ICE-LAP6, Mch6) is activated through the mitochondrial-mediated pathway. Cytochrome c released from mitochondria associates with procaspase-9 (47kD)/Apaf-1. Apaf-1 mediated activation of caspase-9 involves proteolytic processing resulting in cleavage at Asp315 and producing a p35 subunit. Another cleavage occurs at Asp330 producing a p37 subunit that can amplify the apoptotic response. Caspase-2 (Nedd2/ICH-1) is the nuclear apoptotic respondent to cellular genotoxic stress or mitotic catastrophe. The procaspase is cleaved at Asp316, producing a 14kD fragment and a 32kD prodomain/large subunit. Subsequent processing at Asp152 and Asp330 produces an 18kD large subunit and a 12kD small fragment. Activation occurs upon recruitment to a complex containing a p53-induced death domain protein, PIDD. This suggests that caspase-2 can be a nuclear initiator caspase with a requirement for caspase-9 and caspase-3 activation in downstream apoptotic events. In apoptotic pathways resulting from UV-induced DNA damage, processing of caspase-2 occurs downstream of mitochondrial dysfunction and of caspase-9 and caspase-3 activation, extending a possible role for caspase-2 as a parallel effector caspase. Caspase-3 (CPP-32, Apoptain, Yama, SCA-1) is a critical executioner of apoptosis and caspase-3 cleavage is a key indicator of initiator caspase activation. Caspase-3 is either partially or totally responsible for the proteolytic cleavage of many key proteins including the nuclear enzyme poly (ADP-ribose) polymerase (PARP). Activation of caspase-3 requires proteolytic processing of its inactive zymogen into activated p17 and p12 fragments.
Specificity:Human
Intended Use:The Initiator Caspases Antibody Sampler BioAssay™ Kit provides an economical means of evaluating initiator (apical) caspase proteins. The kit contains enough primary antibody to perform two western blots with each primary antibody.
Applications:Suitable for use in Western Blot. Other applications not tested.
Recommended Dilution:Western Blot: 1:500-1:2000Optimal dilutions to be determined by the researcher.
Storage and Stability:May be stored at 4°C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Aliquots are stable for 12 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
