| Apoptosis or programmed cell death is a fundamental cellular process that is essential for normal tissue development and abnormal growth. Apoptosis is driven by two classes of specialized proteases known as caspases (Cysteine Aspartase). Several key factors are released from the mitochondria that regulate apoptosis. The first such factor (Cytochrome-C) to be described binds to a cytoplasmic scaffolding protein called Apaf-1 (Apoptosis Protease activating factor-1), a homolog of C. elegans Ced-4. Both Apaf-1 and Ced-4 are composed of an N-terminal Caspase Recrutiment domain (CARD) linked to a Nuceoltide-bidning domain (NBD), also known as NB-ARC or NOD domain. Ced-4 and Apaf-1 self-associate via the NBD and activate Casp-3 and -9. In response to certain apoptotic stimuli, Cytochrome-C is released from the mitochondria and binds to Apaf-1 to form a ternary complex with, and activate, the initiator pro-caspase-9. Active caspase-9 then turns on downstream effector caspases, initiating apoptosis. Recently, Additional members of Apaf-1 family, NOD1/CARD4 and NOD2 have been cloned and characterized. In C. elegans, Ced4 binds and activates Ced3, an apoptotic initiator caspase, via caspase-associated recruitment domains (CARDs). Human Ced4 homologs include APAF1, NOD1 (CARD4), and NOD2 (CARD15). These proteins have at least 1 N-terminal CARD domain followed by a centrally located nucleotide-binding domain (NBD or NACHT) and a C-terminal regulatory domain, found only in mammals, that contains either WD40 repeats or leucine-rich repeats (LRRs). Ice Protease-Activating Factor (IPAF) or CARD12 is a member of the Ced4 family and can induce apoptosis. Full length IPAF or CLANA, (human 1,024-aa) contains the CARD, LRR, and NACHT domains, including 4 LRRs, whereas the 359-residue protein, termed CLANB, has the CARD spliced directly to the LRRs. The 156- and 92-amino acid proteins, termed CLANC and CLAND, contain only the CARD and regions with no homology to known motifs. CARDB is ubiquitous expressed in lung and spleen, CLANA was detected primarily in lung. CLANC and CLAND were detected only in an embryonic kidney cell line and brain, respectively. Applications: Suitable for use in ELISA, Western Blotting. Other applications not tested. Recommended Dilution: Western Blotting Antiserum at 1:1K-1:3K, affinity purified at 1-5ug/ml using ECL. ELISA: 1:10,000-50,000 for neat serum and 0.5-1ug/ml for affinity pure. Control peptide can be used to coat ELISA plates at 1ug/ml. Optimal dilutions to be determined by the researcher. Storage and Stability: May be stored at 4C for short-term only. For long-term storage and to avoid repeated freezing and thawing, aliquot and add glycerol (40-50%). Freeze at -20C. Aliquots are stable for at least 12 months at -20C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer. |
