Recombinant Mouse TIMP-4 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, pH 7.5 (TCNB)
• Recombinant Mouse TIMP-4 (rmTIMP-4) (Catalog # 7667-TM)
• Recombinant Human MMP‑2 (rhMMP-2) (Catalog # 902-MP)
• p-aminophenylmercuric acetate (APMA) (Sigma, Catalog # A9563), 100 mM stock in DMSO
• Substrate: MCA-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂,(Catalog # ES001), 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 rhMMP-2 to 100 µg/mL in Assay Buffer with 1 mM APMA.
2. Incubate at 37 °C for 1 hour.
3. Prepare a curve of rmTIMP-4 (MW: 22,600 Da). Make the following serial dilutions in Assay Buffer: 4000, 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6, and 7.8 nM.
4. Dilute activated rhMMP-2 to 12.5 µg/mL in Assay Buffer.
5. For each inhibition point, mix 25.6 µL of 12.5 µg/mL rhMMP-2, 16 µL of the rmTIMP-4 curve dilution, and 118.4 µL of Assay Buffer. Include two enzyme controls of 25.6 µL of 12.5 µg/mL rhMMP-2 and 134.4 µL Assay Buffer.
6. Incubate reaction mixtures at 37 °C for 2 hours.
7. Dilute incubated reaction mixtures 5-fold in Assay Buffer.
8. Dilute Substrate to 20 µM in Assay Buffer.
9. Load 50 µL of the incubated reactions into the wells of a black well plate. Start the reaction by adding 50 µL of 20 µM Substrate.
10. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read) in kinetic mode for 5 minutes.
11. Derive the 50% inhibiting concentration of rmTIMP-4 (IC₅₀) by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
12. The specific activity for rhMMP-2 at each point may be determined using the following formula (if needed):

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

• rhMMP-2: 0.02 µg (2.82 nM)
• rmTIMP-4: 40, 20, 10, 5, 2.5, 1.25, 0.63, 0.31, 0.16, and 0.078 nM
• Substrate: 10 µM