Description
Members of the caspase family have key roles in inflammation and mammalian apoptosis. Caspase-8 (FLICE/MACH-1) is a 55 kDa cytosolic protein with homology to the CD95/Fas-associated signal transduction molecule, FADD, in addition to its homology with other caspases. Caspase-8 is activated early in apoptosis and is involved in the proteolysis and activation of pro-caspase-3. The upstream sequence of the site recognized by active caspase-8, IETD (Ile-Glu-Thr-Asp), is utilized as a basis for the highly specific caspase-8 substrate, Ac-IETD-AFC, and the caspase-8 inhibitor, Ac-IETD-CHO. Ac-IETD-CHO (502 Daltons) is a synthetic tetrapeptide inhibitor of caspase-8 and contains the amino acid sequence that is the target for caspase-8 mediated proteolysis. The tetrapeptide inhibitor can be used as a negative (blocking) control, in parallel with Ac-IETD-AFC, to study caspase-8 activity in apoptotic cell lysates.
Format
- FormatPurified
Suggested Companion Products
Recombinant Human Active Caspase-8 RUO 5µgCat No: 556481
Ac-IETD-AFC Caspase-8 Fluorogenic Substrate RUO 1mgCat No: 556552
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Preparation and Storage
Preparation and StorageStore the lyophilized Ac-IETD-CHO inhibitor at -20°C.Reconstitute the Ac-IETD-CHO inhibitor with 1 ml DMSO before use.Store the reconstituted Ac-IETD-CHO inhibitor at -20°C for up to two months and avoid repeated freeze-thaw cycles, which can greatly alter product stability.
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.
The Ac-IETD-CHO inhibitor may be used in protease assays like those described in the literature. When the caspase-8 fluorogenic substrate Ac-IETD-AFC is treated with apoptotic cell lysates or with purified, active caspase-8, AFC is released. AFC release can be monitored in a spectrofluorometer at an excitation wavelength of 400 nm and an emission wavelength range of 480-520 nm. Apoptotic cell lysates yield a considerable emission as compared to non-apoptotic cell lysates, or apoptotic lysates, which also contain Ac-IETD-CHO. The amount of cell lysate required for protease assays will vary between experimental systems and should be optimized by the user. A suggested protease assay protocol follows.
Ac-IETD-CHO PROTEASE ASSAY
Materials Required
1. Purified, Active Recombinant Caspase-8 (Cat. No. 556481). Not Included.5 µg enzyme in 25 µl.The enzyme is buffered with 50 mM Tris, pH 8.0, with 100 mM NaCl and 50 mM imidazole.
2. Ac-IETD-AFC (Cat. No. 556552). Not Included. 1 mg peptide in DMSO; lyophilized. Reconstitute in 1 ml DMSO to yield 1 mg/ml peptide.
3. Ac-IETD-CHO (Cat. No. 556554). Included.1 mg peptide, lyophilized. Reconstitute in 1 ml DMSO to yield 1 mg/ml peptide.
4. Protease assay buffer: Not Included.20 mM PIPES, 100 mM NaCl, 10mM DTT, 1mM EDTA, 0.1% (w/v) CHAPS, 10% sucrose; pH 7.2.
Procedure
1. To one tube, add 10 µl of Ac-IETD-AFC into 1 ml of assay buffer. In a separate tube, add 10 µl of Ac-IETD-AFC and 1 µl Ac-IETD-CHO into 1 ml assay buffer.
2. Add 1 µg purified, active caspase-8 to each tube.
3. Incubate for 1 hr at 37°C.
4. Measure the AFC liberated from the Ac-IETD-AFC using a spectrofluorometer with an excitation wavelength of 400 nm and an emission wavelength of 480-520 nm (peak at 505 nm).
