Recombinant Rat MMP-8 Protein, CF

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

Recombinant Rat MMP-8 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave a fluorogenic peptide substrate Mca-KPLGL-Dpa-AR-NH 2 (Catalog # ES010). The specific activity is >75 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived rat MMP-8 protein Leu21-Pro466, with a C-terminal 6-His tag
• Accession #: O88766
• N-terminal Sequence: Leu21
• Structure / Form: Pro form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Mmp8
• 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: 52 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: 64 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 degreesC as supplied. 3 months, -20 to -70 degreesC under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in MES, NaCl and CaCl 2 .
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (v/v) Brij-35, pH 7.5 (TCNB)
  • Recombinant Rat MMP‑8 (rrMMP-8) (Catalog # 3245-MP)
  • p-aminophenylmercuric acetate (APMA) (Sigma, Catalog # A-9563), 100 mM stock in DMSO
  • ZnCl₂, 1 M stock in deionized water
  • Substrate MCA-Lys-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH₂ (Catalog # ES010) , 2 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 rrMMP-8 to 100 µg/mL in Assay Buffer.
  2. Activate rrMMP-8 by adding APMA and ZnCl₂ to final concentrations of 1 mM and 25 µM respectively.
  3. Incubate at room temperature for 4 hours.
  4. Dilute activated rrMMP-8 to 2 ng/μL in Assay Buffer.
  5. Dilute Substrate to 20 µM in Assay Buffer.
  6. Load 50 µL of the 2 ng/µL rrMMP-8 into in plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 20 µM Substrate.
  7. Read at excitation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes.
  8. 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:
  • rrMMP-8: 0.10 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 Rat MMP-8 Protein, CF

• CLG1HNC
• Collagenase 2
• EC 3.4.24
• EC 3.4.24.34
• matrix metallopeptidase 8 (neutrophil collagenase)
• matrix metalloproteinase 8 (neutrophil collagenase)
• Matrix metalloproteinase-8
• MMP8
• MMP-8
• neutrophil collagenase
• PMNL collagenase
• PMNL-CL

Background

Matrix metalloproteinases (MMPs) are a family of zinc and calcium dependent endopeptidases with the combined ability to degrade all the components of the extracellular matrix. MMP-8 (neutrophil collagenase, collagenase 2) is expressed in neutrophils, where it is stored in specific granules. MMP-8 release from the neutrophils is stimulated by various factors such as interleukins 1 and 8, TNF-alpha and GM-CSF. MMP-8 is capable of cleaving types I, II and III triple-helical collagen, gelatin peptides, fibronectin, proteoglycans, aggrecan, serpins, beta -casein and peptides such as angiotensin and substance P. In addition to its function in phagocytosis, MMP-8 has a high capacity for infiltrating connective tissue, and is implicated in the breakdown of the extracellular matrix in diseases such as rheumatoid arthritis. Structurally, MMP-8 consists of several domains: a pro-domain that is cleaved upon activation, a catalytic domain containing the zinc-binding site, a short hinge region and a hemopexin-like domain (1).

1. H. Tschesche et al. (2004) Handbook of Proteolytic Enzymes (eds. A.J. Barrett et al.) p.480, Academic Press, San Diego.

Bioinformatics

• Gene Symbol: Mmp8
• Uniprot:
  • O88766()