The Effector Caspases and Substrates Antibody Sampler Kit provides an economical means to evaluate the activation of effector (executioner) caspases. The kit contains enough primary antibody to perform at least four western blots per primary antibody.
Each antibody in the Effector Caspases and Substrates Antibody Sampler Kit recognizes endogenous levels of its respective target. Caspase-3 (8G10) Rabbit mAb recognizes full-length (35 kDa) and the large fragment (17/19 kDa) of caspase-3 resulting from cleavage at Asp175. Caspase-6 Antibody recognizes full length (35 kDa) and the small subunit (15 kDa) of caspase-6 resulting from cleavage at Asp193. Caspase-7 (D2Q3L) Rabbit mAb recognizes full-length (35 kDa) and the large subunit (20 kDa) of caspase-7 resulting from cleavage at Asp198. PARP Antibody recognizes full length (116 kDa) and the large fragment (89 kDa) of PARP1 resulting from caspase cleavage at Asp214. Lamin A/C (4C11) Mouse mAb recognizes full-length lamin A and lamin C proteins, and the larger fragments of lamin A (50 kDa) and lamin C (41 kDa) resulting from caspase cleavage. Lamin B1 (D4Q4Z) Rabbit mAb recognizes endogenous levels of total lamin B1 protein and a 25 kDa fragment resulting from caspase cleavage.
Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to amino-terminal residues adjacent to Asp175 in human caspase-3 protein, residues surrounding Pro158 of human caspase-7 protein, residues surrounding Leu118 of human lamin B1 protein, or with a recombinant fragment of human lamin A protein. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding the cleavage site of caspase-6 or the caspase cleavage site in PARP. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.
Apoptosis is a regulated physiological process leading to cell death. Caspases, a family of cysteine acid proteases, are central regulators of apoptosis. Caspase-3 (CPP-32, Apoptain, Yama, SCA-1), Caspase-6 (Mch2), and Caspase-7 (CMH-1, Mch3, ICE-LAP3) are effector caspases functioning in cellular apoptotic processes (1-6). Upon apoptotic stimulation, initiator caspases such as caspase-9 (ICE-LAP6, Mch6) are cleaved and activated (7). The activated upstream caspases further process downstream executioner caspases by cleaving them into activated large and small subunits, thereby initiating a caspase cascade leading to apoptosis (4,6,8-10).
PARP, a 116 kDa nuclear poly (ADP-ribose) polymerase, appears to be involved in DNA repair in response to environmental stress (11). This protein can be cleaved by many ICE-like caspases in vitro (1,12) and is one of the main cleavage targets of caspase-3 in vivo (10,13). In human PARP, cleavage occurs between Asp214 and Gly215, which separates the PARP amino-terminal DNA binding domain (24 kDa) from the carboxy-terminal catalytic domain (89 kDa) (10,12). PARP helps cells to maintain their viability; cleavage of PARP facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis (14).
Lamins are nuclear membrane structural components that are important in maintaining normal cell functions, such as cell cycle control, DNA replication, and chromatin organization (15-17). Lamins have been subdivided into types A and B. Type-A lamins consist of lamin A and C, which arise from alternative splicing of the lamin A gene LMNA. Lamin A and C are cleaved by caspases into large (41-50 kDa) and small (28 kDa) fragments, which can be used as markers for apoptosis (18,19). Type-B lamins consist of lamin B1 and B2, encoded by separate genes (20-22). Lamin B1 is also cleaved by caspases during apoptosis (23).
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