Preparation And Storage
Recommended Assay Procedures
Applications include western blot analysis ( 0.125 - 0.5 µg /ml) and immunoprecipitation (4 µg /200µg cell lysate ). Jurkat T cells ( ATCC CRL-1573) are suggested as a positive control. BD Biosciences Pharmingen offers several caspase-8 antibodies. A Jurkat model cell system was used to evaluate these antibodies; these results are summarized in the following table. However, actual bands observed could vary according to the cell model system or treatment used.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
Caspase-8 (FLICE/MACH-1) is a 55 kDa cytosolic protein with homology to the CD95/Fas-associated signal transducer, FADD/MORT-1, as well as to other caspase (ICE/Ced-3) cysteine proteases. The N-terminal region of caspase-8 contains an amino acid sequence, termed the death domain, that facilitates caspase-8-FADD direct interaction. FADD therefore acts as an adapter molecule, allowing caspase-8 to become recruited to the cytoplasmic region of Fas following receptor activation. Viral proteins (v-FLIPS) which inhibit recruitment and activation of caspase-8 have been isolated. Caspase-8 is produced as a proenzyme (55/50 kDa doublet) which upon receptor aggregation is proteolytically cleaved into smaller subunits of 40/36 (doublet), and 23 kDa. Overexpression of caspase-8 is sufficient to induce apoptosis in certain cell lines (e.g., MCF-7) and this phenotype is blocked by overexpression of the caspase-3 protease inhibitor, CrmA. The antibody recognizes both the proform of human caspase-8 (55/50 kDa doublet) as well as the cleaved forms which migrate at 40/36 (doublet) and 23 kDa in SDS/PAGE. It also immunoprecipitates the full-length human caspase-8. Full-length recombinant human caspase-8 protein was used as immunogen.
Development References (4)
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Boesen-de Cock JG, Tepper AD, de Vries E, van Blitterswijk WJ, Borst J. Common regulation of apoptosis signaling induced by CD95 and the DNA-damaging stimuli etoposide and gamma-radiation downstream from caspase-8 activation. J Biol Chem. 1999; 274(20):14255-14261. (Biology). View Reference
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Cock JG, Tepper AD, de Vries E, van Blitterswijk WJ, Borst J. CD95 (Fas/APO-1) induces ceramide formation and apoptosis in the absence of a functional acid sphingomyelinase. J Biol Chem. 1998; 273(13):7560-7565. (Biology). View Reference
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Muzio M, Chinnaiyan AM, Kischkel FC, et al. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death--inducing signaling complex. Cell. 1996; 85(6):817-827. (Biology).
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Thome M, Schneider P, Hofmann K, et al. Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors. Nature. 1997; 386(6624):517-521. (Biology).
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