Recombinant Human 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 Human MMP-3 (rhMMP-3) (Catalog # 513-MP)
• Chymotrypsin (Sigma, Catalog # C-3142), 1 mg/mL stock in 1 mM HCl
• Phenylmethyl Sulfonyl Fluoride (PMSF) (Sigma, Catalog # P-7626), 0.2 M stock in 2-Propanol
• 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. Activate rhMMP-3 at 20 µg/mL in Assay Buffer containing 5 µg/mL Chymotrypsin.
2. Incubate reaction at 37 °C for 30 minutes.
3. Stop activation with 2 mM PMSF. Pre-warm the PMSF to 37 °C prior to adding to sample.
4. Dilute activated rhMMP-3 to 2.5 ng/µL in Assay Buffer.
5. Dilute Substrate to 20 µM in Assay Buffer.
6. In a plate load 50 µL of 2.5 ng/µL rhMMP-3, and start the reaction by adding 50 µL of 20 µM Substrate to wells. 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-3: 0.125 µg
• Substrate: 10 µM