Heat-mediated antigen retrieval is recommended prior to staining, using a 10mM citrate buffer, pH 6.0, for 10 minutes followed by cooling at room temperature for 20 min. Following antigen retrieval, incubate samples with primary antibody for 30 min at room temperature. A suggested positive control is breast carcinoma or stomach tissue.
Caspase 8 (CASP8) is a member of the cysteine-aspartic acid protease (caspase) family. Sequential activation of caspases play a central role in the execution-phase of cell apoptosis. Caspases exist as inactive proenzymes composed of a pro-domain, a large protease subunit, and a small protease subunit. Activation of caspases requires proteolytic processing at conserved internal aspartic residues to generate a heterodimeric enzyme consisting of the large and small subunits. Caspase 8 is involved in the programmed cell death induced by Fas and various apoptotic stimuli. The N-terminal FADD-like death effector domain of Caspase 8 suggests that it may interact with Fas-interacting protein FADD. Caspase 8 was detected in the insoluble fraction of the affected brain region from Huntington disease patients but not in those from normal controls, which implicated the role in neurodegenerative diseases. Caspase 8 binds to the death effector domain (DED) of FADD through an analogous DED domain present in tandem in the pro-form of the Caspase 8 protein. Activated Caspase 8 then activates other downstream caspases including Caspase 9, thereby committing the cell to undergo apoptosis. In addition, Caspase 8 also reacts with Jurkat cells and Tonsil. Overexpression of Caspase 8 induces apoptosis, which can be blocked by inhibitors specific for the ICE family. Many alternatively spliced transcript variants encoding different isoforms have been described for Caspase 8, however, not all variants have had their full-length sequences determined.
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