Recombinant Mouse Mast Cell Protease-6/Mcpt6 Protein, CF

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

Recombinant Mouse Mast Cell Protease-6/Mcpt6 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH₂ (Catalog # ES002). The specific activity is >500 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse Mast Cell Protease-6/Mcpt6 protein
  Ala22-Ser276, with a C-terminal 10-His tag
• Accession #: P21845
• N-terminal Sequence: Ala22
• Structure / Form: Pro form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Tpsb2
• 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: 37 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Maturation Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
  • Assay Buffer: 0.05 M MES, 0.1 M NaCl, pH 6.5
  • Recombinant Mouse Mast Cell Protease‑6/Mcpt6 (rmMCP-6) (Catalog # 3736-SE)
  • Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH₂ (Catalog # ES002), 2 mM stock in DMSO
  • Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
  • Heparin (Sigma, Catalog # H3393), 20 mg/mL deionized water
  • 1,10-Phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rmMCP-6 to 200 µg/mL in Maturation Buffer.
  2. Dilute Thermolysin to 0.2 µg/mL in Maturation Buffer.
  3. Combine 25 µL of 200 µg/mL rmMCP-6 with 25 µL of 0.2 µg/mL Thermolysin.
  4. Incubate at room temperature for 15 minutes.
  5. Dilute 1,10-Phenanthroline to 10 mM in Assay Buffer.
  6. Stop maturation by adding 50 µL of 10 mM 1,10-phenanthroline to reactions.
  7. Dilute Heparin to 1 mg/mL in Assay Buffer.
  8. Add 50 µL of Heparin and 350 µL of Assay Buffer to the stopped reaction vials.
  9. Incubate for 30 minutes at room temperature.
  10. Dilute reaction vials to a final enzyme concentration of 2 ng/µL in Assay Buffer.
  11. Dilute Substrate to 20 µM in Assay Buffer.
  12. Load 50 µL of 2 ng/µL rmMCP-6 mixture into wells of a plate and start the reaction by adding 50 µL of Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of Substrate.
  13. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
  14. 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 MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

  Per Well:
  • rmMCP-6: 0.100 µg
  • Substrate: 10 µM

Notes

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

Alternate Names for Recombinant Mouse Mast Cell Protease-6/Mcpt6 Protein, CF

• Mast Cell Protease6
• Mast Cell Protease-6
• Mcpt6

Background

Tryptases are trypsin-like serine proteases, and beta tryptases appear to be the main isoenzymes expressed in mast cells (1). Mast Cell Protease-6 (MCP-6, Mcpt6, or Tpsb2) is one of beta tryptases produced from mouse mast cells. It is stored in secretory granules of mast cells, where it forms active tetramers with heparin proteoglycan. Because of the unique arrangement of the active sites in the tetramer, which are facing a narrow central pore, MCP-6 is resistant to macromolecular protease inhibitors (2). When mast cells are activated, MCP-6 is released along with other proteins in secretory granules, participating in provoking inflammatory conditions (3). MCP-6 has been implicated as a mediator in the pathogenesis of asthma and other allergic disorders in mouse models.

1. Caughey, G. H. (2004) in Handbook of Proteolytic Enzymes, Barrett, A.J. et al. eds. p. 1535 .
2. Sommerhoff, C.P. et al. (1999) Proc. Natl. Acad. Sci. USA. 96:10984.
3. Hallgren, J. and G. Pejler, (2006) FEBS J. 273:1871.

Publications for Mast Cell Protease-6/Mcpt6 (3736-SE)(2)

Publications using 3736-SE

• Hirai , Shizuka, Ohyane , Chie, Kim , Young-Il, Lin , Shan, Goto , Tsuyoshi, Takahashi , Nobuyuki, Kim , Chu-Sook, Kang , Jihey, Yu , Rina, Kawada , Teruo Involvement of mast cells in adipose tissue fibrosis. Am J Physiol Endocrinol Metab, 2013-12-10;306(3):E247-55. 2013-12-10 [PMID: 24326418] (Bioassay, Mouse)
• de Souza DA, Toso VD, Campos MR, Lara VS, Oliver C, Jamur MC Expression of Mast Cell Proteases Correlates with Mast Cell Maturation and Angiogenesis during Tumor Progression. PLoS ONE, 2012-07-18;7(7):e40790. 2012-07-18 [PMID: 22815822] (Bioassay, Mouse)

Bioinformatics

• Gene Symbol: Tpsb2
• Uniprot:
  • P21845()