Recombinant Human MMP-15/MT2-MMP Protein, CF

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

• Activation Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 5 µM ZnCl₂, 0.05% (w/v) Brij-35, pH 7.5
• Assay Buffer: 50 mM Tris, 500 mM NaCl, 5 mM CaCl₂, 1 µM ZnCl₂, 0.02% (w/v) Brij-35, pH 8.0
• Recombinant Human MMP‑15/MT2‑MMP (rhMMP‑15) (Catalog # 916-MP)
• Recombinant Human Active Trypsin 3/PRSS3 (rhTrypsin 3) (Catalog # 3714-SE) )
• 4-(2-Aminoethyl-benzensulfonyl fluoride hydrochloride) (AEBSF) (Tocris, Catalog # 5175), 100 mM stock in deionized water
• Substrate: MCA-Lys-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH₂ (Catalog # ES010)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Activate rhMMP-15 at 100 µg/mL with 0.1 µg/mL rhTrypsin 3 in Activation Buffer.
2. Incubate at 37 °C for 2 hours.
3. Add AEBSF for a final concentration of 1 mM and incubate at room temperature for 15 minutes to stop the reaction.
4. Dilute activated rhMMP-15 to 1 ng/µL in Assay Buffer.
5. Dilute Substrate to 20 µM in Assay Buffer.
6. Load 50 µL of the 1 ng/µL rhMMP-15 in a black well 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 without any rhMMP-15.
7. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), 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).

• rhMMP-15: 0.05 µg
• Substrate: 10 µM