Recombinant Human MMP-9 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-9 (rhMMP-9) (Catalog # 911-MP)
• p-aminophenylmercuric acetate (APMA), (Sigma, Catalog # A-9563), prepare a 100 mM stock in DMSO
• Substrate: 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-9 to 100 µg/mL in Assay Buffer.
2. Activate rhMMP-9 by adding APMA to a final concentration of 1 mM.
3. Incubate at 37 °C for 24 hours.
4. Dilute activated rhMMP-9 to 0.4 ng/µL in Assay Buffer.
5. Dilute Substrate to 20 µM in Assay Buffer.
6. Load 50 µL of the 0.4 ng/µL rhMMP-9 into a plate and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL of 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:

     *Adjusted for Substrate Blank

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

• rhMMP-9: 0.020 µg
• Substrate: 10 µM