Recombinant Human Caspase-10 (Mch 4) Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Caspase-10 (Mch 4) Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate N-acetyl-Leu-Glu-His-Asp-7-amino-4-trifluoromethyl coumarin (Ac-LEHD-AFC). The specific activity is > 200 pmol/min/µg, as measured under the described conditions.
• Source: E. coli-derived human Caspase-10 protein
  Val220-Ile479, with a C-terminal 6-His tag
• Accession #: AAC50644
• N-terminal Sequence: Val220 & Ala373
• Protein/Peptide Type: Recombinant Enzymes
• Gene: CASP10
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 30 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 17 kDa and 10 kDa, reducing conditions

Packaging, Storage & Formulations

• 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.
• Buffer: Supplied as a 0.2 μm filtered solution in HEPES, NaCl and Sucrose.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Assay Procedure:
  • Assay Buffer: 25 mM HEPES, 0.1% CHAPS (w/v), 10 mM DTT, pH 7.5
  • Recombinant Human Caspase‑10 (Mch4) (rhCaspase-10) (Catalog # 834-CP)
  • Substrate: Ac-Leu-Glu-His-Asp-AFC (Ac-LEHD-AFC), 10 mM stock in DMSO
  • Black 96-well Plate
  • Fluorescent Plate Reader
  1. Dilute rhCaspase-10 to 20 ng/µL in Assay Buffer.
  2. Dilute Substrate to 100 µM in Assay Buffer.
  3. Load into a plate 50 µL of 20 ng/µL rhCaspase-10 and start the reaction by adding 50 µL of 100 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 100 µM Substrate.
  4. Read at excitation and emission wavelengths of 400 nm and 505 nm (top read), respectively, in kinetic mode for 10 minutes.
  5. Calculate specific activity using data from minutes 5 - 10 only:

  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.

  Per Well:
  • rhCaspase-10: 1 µg
  • Substrate: 50 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Caspase-10 (Mch 4) Protein, CF

• Apoptotic protease Mch-4
• CASP10
• CASP-10
• caspase 10, apoptosis-related cysteine peptidase
• caspase 10, apoptosis-related cysteine protease
• Caspase10
• Caspase-10
• EC 3.4.22.63
• FADD-like ICE2
• FAS-associated death domain protein interleukin-1B-converting enzyme 2
• FLICE2
• ICE-like apoptotic protease 4
• interleukin-1B-converting enzyme 2
• Mch4
• MCH4ALPS2

Background

Caspase-10 (also known as Mch4 and FLICE2) is a 28‑29 kDa member of the peptidase C14A family of enzymes (1‑4). It is widely expressed, being found in dendritic cells, T and B cells, neurons, keratinocytes, and intestinal epithelium (5‑8). Caspase-10 appears to be an initiator caspase, and is known to act on a select number of substrates, including Bid, ABCF1, AKAP1, PARP, HSP90 beta and procaspases-3, 4, 6, 7, and 9 (1, 2, 9‑12). There are several alternatively spliced forms of human Caspase‑10, and all forms contains the same pro region (aa 1‑219) with two death effector domains (DEDs) (SwissProt # Q92851). Recombinant Human Caspase-10 (Mch4) corresponds to the mature region of isoform B and is based on the sequence described by Fernandes‑Alnemri et al. (4). Normally, procaspase‑10 is an inactive, cytosolic monomer (2, 9). But following DED containing receptor trimerization, procaspase‑10 is recruited to a complex termed DISC that forms around the death domains of the oligomerized receptor (1, 2, 9, 13). FADD is recruited first, followed by both procaspase‑10 and 8. The recruitment, or concentration, of both procaspase‑8 and ‑10 induces their homodimerization and activation. The actual activation of Caspase‑10 can occur either through homodimerization and self‑cleavage, or caspase‑8‑induced cleavage (9, 11, 12). Cleavage initially generates a 43 kDa (p43) N‑terminal and an 12 kDa (p12) C‑terminal fragment. The p43 subunit is next cleaved at Asp219‑Val220 to generate a 25 kDa (p25) DED‑containing prodomain and a 17 kDa mature p17 subunit (14). p17 and p12 noncovalently associate to form a 29 kDa heterodimer, which subsequently associates with another p17/p12 heterodimer to form an active, mature Caspase-10 molecule. This leaves the DISC to act on downstream substrates. There does not appear to be a mouse gene equivalent to the human CASP10 gene (15).

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. Valmiki, M.G. & J.W. Ramos (2009) Cell. Mol. Life Sci. 66:814.
4. Fernandes-Alnemri, T. et al. (1996) Proc. Natl. Acad. Sci. USA 93:7464.
5. Wang, J. et al. (2001) Proc. Natl. Acad. Sci. USA 98:13884.
6. Grossmann, J. et al. (1998) Am. J. Physiol. 274:G1117.
7. Marconi, A. et al. (2004) J. Cell Sci. 117:5815.
8. Nunnari, G. et al. (2005) J. Neurovirol. 11:319.
9. Chen, M. & J. Wang (2002) Apoptosis 7:313.
10. Bae, S. et al. (2008) Int. J. Mol. Med. 21:381.
11. Milhas, D. et al. (2005) J. Biol. Chem. 280:19836.
12. Chen, H. et al. (2009) Mol. Cell. Biol. 29:3657.
13. Tibbetts, M.D. et al. (2003) Nat. Immunol. 4:404.
14. Sprick, M.R. et al. (2002) EMBO J. 21:4520.
15. Reed, J.C. et al. (2003) Genome Res. 13:1376.

Publications for Caspase-10/FLICE2 (834-CP)(1)

Publication using 834-CP

• 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 Fine-tuning nucleophosmin in macrophage differentiation and activation. Blood, 2011-08-29;118(17):4694-704. 2011-08-29 [PMID: 21876121] (Enzyme Assay, Human)

Bioinformatics

• Gene Symbol: CASP10
• Uniprot:
  • AAC50644()