Recombinant Human Caspase-8 Protein, CF

Catalog #: 705-C8/CF
2 Citations Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
705-C8-010/CF

R&D Systems Recombinant Proteins and Enzymes

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Citations (2)

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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Human Caspase-8 Protein, CF Summary

Product Specifications

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by silver stain

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

Activity

Measured by its ability to cleave a fluorogenic peptide substrate Ac-IETD-AFC. The specific activity is >1,000 pmol/min/μg, as measured under the described conditions.

Source

E. coli-derived human Caspase-8 protein
Ser217-Asp384 (Asp285His) (p18 subunit) & Leu385-Asp479 (p10 subunit), with a C-terminal 6-His tag

Accession #

Q14790

N-terminal Sequence
Analysis

Ser217 (p18 subunit) & Leu385 (p10 subunit)

Predicted Molecular Mass

18 kDa & 10 kDa

SDS-PAGE

18 kDa and 10 kDa, reducing conditions

Product Datasheets

705-C8/CF (carrier free)

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

705-C8/CF

Formulation	Supplied as a 0.2 μm filtered solution in HEPES, NaCl, DTT and Sucrose.
Shipping	The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage:	Use a manual defrost freezer and avoid repeated freeze-thaw cycles. 6 months from date of receipt, -70 °C as supplied. 3 months, -70 °C under sterile conditions after opening.

Assay Procedure

Materials

Assay Buffer: 25 mM HEPES, 0.1% (w/v) CHAPS, 10 mM dithiothreitol (DTT), pH 7.5
Recombinant Human Caspase‑8 (rhCaspase‑8) (Catalog # 705-C8)
Substrate: Ac-Ile-Glu-Thr-Asp-AFC (MP Biomedicals, Catalog # AFC-140) (10 mM stock in DMSO)
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

Dilute rhCaspase-8 to 0.3 ng/µL in Assay Buffer.
Dilute Substrate to 100 µM in Assay Buffer.
Load 50 µL of 0.30 ng/µL rhCaspase-8 into a plate, and start the reaction by adding 50 µL of 100 µM Substrate. Include a Substrate Blank containing 50 µL of 100 µM Substrate and 50 µL Assay Buffer.
Read at excitation and emission wavelengths of 400 nm and 505 nm (top read), respectively, in kinetic mode for 5 minutes.
Calculate specific activity:

Specific Activity (pmol/min/µg) =	Adjusted V_max* (RFU/min) x Conversion Factor** (pmol/RFU)
	amount of enzyme (µg)

*Adjusted for Substrate Blank
**Derived using calibration standard 7-amino, 4-(trifluoromethyl)coumarin (Calbiochem, Catalog #164580).

Per Well:

rhCaspase-8: 0.015 µg
Substrate: 50 µM

Reconstitution Calculator

Background: Caspase-8

Caspase-8 (Cysteine-aspartic acid protease 8/Casp8a; also named MCH5, FLICA and MACH alpha 1) is a 28 kDa member of the peptidase C14A family of enzymes (1, 2, 3). It is widely expressed and is considered an initiating caspase for the apoptotic cascade (4). Caspase-8 acts on a wide variety of substrates, including procaspases‑3, 4, 6, 7, 9 and 10, c‑FLIP_L and procaspase-8 itself (1, 5 6). Human procaspase‑8a is a 54‑56 kDa, 479 amino acid (aa) protein (4, 7, 8, 9). It contains two N‑terminal death domains (aa 1‑177), followed by a catalytic site that utilizes His317Gly318 plus Cys360. Normally, it is an inactive, cytosolic monomer (1, 10, 11). But following death‑domain (DD) containing receptor oligomerization, Caspase‑8 is recruited to the death-inducing signaling complex (DISC) that forms around the death domains of the oligomerized receptor (12). FADD/CAP-1 is recruited first, followed by procaspase‑8/CAP‑4 and, possibly, c‑FLIP_L and procaspase‑10 (12). The recruitment, or concentration, of procaspase-8 induces homodimerization. This act alone is sufficient for activation. However, the activity level is modest at best, and appears to be directed towards either itself, or c‑FLIP_L, which is known to form a functional heterodimer with procaspase‑8 (5, 11). When directed towards itself, autocleavage occurs first between Asp374Ser375, generating a 43 kDa (p43) N‑terminal (aa 1‑374) and an 11 kDa C‑terminal (aa 375‑479) fragment. The C‑terminus is further cleaved between Asp384Leu385 to generate a mature p10 subunit (aa 385‑479). The p43 subunit is next cleaved twice, once between Asp216Ser217, and again between Asp210Ser211 to generate a 26 kDa DD‑containing prodomain (aa 1‑210) with an additional 18 kDa mature p18 subunit (aa 217‑374) (12). p18 and p10 noncovalently associate to form a 28 kDa heterodimer, which subsequently associates with another p18:p10 heterodimer to form an active, mature caspase‑8 molecule. This leaves the DISC to act on downstream apoptotic procaspases. In the event procaspase‑8 comes to the DISC complexed with c-FLIP_L, c-FLIP_L will be cleaved by procaspase‑8, generating a p43 fragment that is analogous to the Caspase‑8 p43 subunit. This fragment, however, appears not to be an intermediate in a proteolytic cascade. Rather, it serves as a functional subunit, interacting with TRAF2 and activating NF kappa B. This may account for many of the nonapoptotic activities associated with Caspase‑8 (5, 6, 13). Mature human and mouse Caspase‑8a heterodimers are 73% aa identical (14).

References

Chowdhury, I. et al. (2008) Comp. Biochem. Physiol. B 151:10.
Boatright, K.M. & G.S. Salvesen (2003) Curr. Opin. Cell Biol. 15:725.
Launay, S. et al. (2005) Oncogene 24:5137.
Srinivasula, S.M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:14486.
Hughes, M.A. et al. (2009) Mol. Cell 35:265.
Lamkanfi, M. et al. (2007) Cell Death Differ. 14:44.
Fernandes-Alnemri, T. et al. (1996) Proc. Natl. Acad. Sci. USA 93:7464.
Boldin, M.P. et al. (1996) Cell 85:803.
Muzio, M. et al. (1996) Cell 85:817.
Donepudi, M. et al. (2003) Mol. Cell 11:543.
Boatright, K.M. et al. (2003) Mol. Cell 11:529.
Golks, A. et al. (2006) Cell Death Differ. 13:489.
Scaffidi, C. et al. (1997) J. Biol. Chem. 272:26953.
Sakamaki, K. et al. (1998) Eur. J. Biochem. 253:399.

Entrez Gene IDs

841 (Human); 12370 (Mouse)

Alternate Names

AIS; androgen receptor; CASP8; Caspase8; Caspase-8; DHTRTFM; Dihydrotestosterone receptorHYSP1; HUMARA; Mch5; NR3C4KD; Nuclear receptor subfamily 3 group C member 4; SMAX1SBMA; spinal and bulbar muscular atrophy

Related Research Areas

Citations for Recombinant Human Caspase-8 Protein, CF

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

2 Citations: Showing 1 - 2
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Anamorsin, a novel caspase-3 substrate in neurodegeneration.
Authors: Yun N, Lee Y, Kim C, Shibayama H, Tanimura A, Hamanaka Y, Kanakura Y, Park I, Jo A, Shin J, Ju C, Kim W, Oh Y
J Biol Chem, 2014-06-27;289(32):22183-95.
Species: Mouse
Sample Types: Recombinant Protein
Applications: Enzyme Assay
Fine-tuning nucleophosmin in macrophage differentiation and activation.
Authors: Guery L, Benikhlef N, Gautier T, Paul C, Jego G, Dufour E, Jacquel A, Cally R, Manoury B, Vanden Berghe T, Vandenabeele P, Droin N, Solary E
Blood, 2011-08-29;118(17):4694-704.
Species: Human
Sample Types: Recombinant Protein
Applications: Enzyme Assay