BD Pharmingen™ Ac-IETD-CHO, Caspase 8 Inhibitor
Activity of recombinant human caspase-8. Ac-IETD-AFC is a synthetic tetrapeptide substrate that is cleaved by active human caspase-8. This substrate is cleaved between D and AFC, releasing the fluorogenic AFC, which is detected by spectrofluorometry. When coupled to an aldehyde group (CHO), the IETD tetrapeptide functions as a potent inhibitor of caspase activity and can be used to block caspase-8 mediated cleavage of Ac-IETD-AFC. In the presence of active caspase-8, fluorogenic AFC is released from Ac-IETD-AFC, demonstrating the activity of caspase-8 enzyme (left panel). In the presence of both active caspase-8 and Ac-IETD-CHO, fluorogenic AFC is not released, indicating that Ac-IETD-AFC was not cleaved and that caspase-8 activity was blocked by Ac-IETD-CHO (right panel).
Activity of recombinant human caspase-8. Ac-IETD-AFC is a synthetic tetrapeptide substrate that is cleaved by active human caspase-8. This substrate is cleaved between D and AFC, releasing the fluorogenic AFC, which is detected by spectrofluorometry. When coupled to an aldehyde group (CHO), the IETD tetrapeptide functions as a potent inhibitor of caspase activity and can be used to block caspase-8 mediated cleavage of Ac-IETD-AFC. In the presence of active caspase-8, fluorogenic AFC is released from Ac-IETD-AFC, demonstrating the activity of caspase-8 enzyme (left panel). In the presence of both active caspase-8 and Ac-IETD-CHO, fluorogenic AFC is not released, indicating that Ac-IETD-AFC was not cleaved and that caspase-8 activity was blocked by Ac-IETD-CHO (right panel).
Product Details
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.
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.
Recommended Assay Procedures
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).
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.
Companion Products
Development References (3)
-
Mashima T, Naito M, Kataoka S, Kawai H, Tsuruo T. Aspartate-based inhibitor of interleukin-1 beta-converting enzyme prevents antitumor agent-induced apoptosis in human myeloid leukemia U937 cells. Biochem Biophys Res Commun. 1995; 209(3):907-915. (Biology: Fluorescence quantitation). View Reference
-
Nicholson DW, Ali A, Thornberry NA, et al. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature. 1995; 376(6535):17-18. (Biology: Fluorescence quantitation). View Reference
-
Thornberry NA, Lazebnik Y. Caspases: enemies within. Science. 1998; 281(5381):1312-1316. (Biology: Fluorescence quantitation). View Reference
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
