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Recombinant Human Caspase-8 Protein

Catalog #: 705-C8
2 Citations Datasheet / COA / SDS
Newer Version Available: 705-C8/CF
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Discontinued Product

705-C8 has been discontinued and is replaced by 705-C8/CF.

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Recombinant Human Caspase-8 Protein 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

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705-C8 (with carrier)

Product Datasheet
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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

Formulation Supplied as a 0.2 μm filtered solution in HEPES, NaCl, DTT and Sucrose with BSA as a carrier protein.
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.

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
  1. Dilute rhCaspase-8 to 0.3 ng/µL in Assay Buffer.
  2. Dilute Substrate to 100 µM in Assay Buffer.
  3. 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.
  4. Read at excitation and emission wavelengths of 400 nm and 505 nm (top read), respectively, in kinetic mode for 5 minutes.
  5. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

 Adjusted Vmax* (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

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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

Citations for Recombinant Human Caspase-8 Protein

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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  1. 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
  2. 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

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