Recombinant Human MMP-13 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% Brij-35 (w/v), pH 7.5 (TCNB)
• Recombinant Human MMP-13 (rhMMP-13) (Catalog # 511-MM)
• p-Aminophenylmercuric acetate (APMA), (Sigma, Catalog # A-9563) 100 mM stock in DMSO
• Fluorogenic Peptide Substrate I: MCA-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂ (Catalog # ES001)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhMMP-13 to 100 µg/mL in Assay Buffer.
2. Add APMA to a final concentration of 1 mM.
3. Incubate at 37 °C for 2 hours to activate.
4. Dilute activated rhMMP-13 to 0.2 ng/µL in Assay Buffer.
5. Dilute Substrate to 20 µM in Assay Buffer.
6. Load 50 µL of the 0.2 ng/µL rhMMP-13 into 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.
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-13: 0.010 µg
• Substrate: 10 µM