Recombinant Mouse MMP-8 Protein, CF

• 6 months from date of receipt, -20 to -70 °C as supplied.
• 3 months, -20 to -70 °C under sterile conditions after opening.

• Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
• Recombinant Mouse MMP-8 (rmMMP-8) (Catalog # 2904-MP)
• p-aminophenylmercuric acetate (APMA) (Sigma, Catalog # A-9563), 100 mM stock in DMSO
• Substrate MCA-Lys-Pro-Leu-Gly-Leu-DAP(DNP)-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 rmMMP-8 to 20 µg/mL in Assay Buffer.
2. Activate rmMMP-8 by adding APMA to a final concentration of 1 mM.
3. Incubate at room temperature for 2 hours.
4. Dilute activated rmMMP-8 to 0.5 ng/µL in Assay Buffer.
5. Dilute Substrate to 80 µM in Assay Buffer.
6. Load into a black well plate 50 µL of 0.5 ng/µL rmMMP-8, and start the reaction by adding 50 µL of 80 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 80 µM Substrate without any rmMMP-8.
7. Read at excitation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes.
8. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

• rmMMP-8: 0.025 μg
• Substrate: 40 µM