Recombinant Mouse MMP-3 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 (rmMMP-3) (Catalog # 548-MM)
• p-aminophenylmercuric acetate (APMA) (Sigma, Catalog # A-9563), 100 mM stock in DMSO
• Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys-(DNP)-NH₂ (Catalog # ES002), 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-3 to 100 µg/mL with 1 mM APMA in Assay Buffer.
2. To activate, incubate reaction at 37 °C for 1 hour.
3. Dilute activated rmMMP-3 to 1 ng/µL in Assay Buffer.
4. Dilute Substrate to 20 µM in Assay Buffer.
5. In a plate load 50 µL of 1 ng/µL rmMMP-3 to wells, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank of 50 µL Assay Buffer and 50 µL of 20 µM Substrate.
6. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
7. Calculate specific activity:

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

• mMMP-3: 0.050 µg
• Substrate: 10 µM