Recombinant Human TIMP-1 Protein, CF

• Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% Brij-35 (w/v), pH 7.5 (TCNB)
• Recombinant Human TIMP-1 (rhTIMP-1) (Catalog # 970-TM)
• Recombinant Human MMP‑2 (rhMMP‑2) (Catalog # 902-MP)
• p-aminophenylmercuric acetate (APMA), (Sigma, Catalog # A-9563), 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. Activate rhMMP-2 at 100 µg/mL with 1 mM of APMA at final concentration respectively, in Assay Buffer.
2. Incubate activation of rhMMP-2 at 37°C for 1 hour.
3. Prepare a curve of rhTIMP-1 (MW: 20,695 Da). Make the following serial dilutions in Assay Buffer: 2000 nM, 1,000 nM, 500 nM, 300 nM, 200 nM, 150 nM, 100 nM, 20 nM, and 2 nM.
4. Dilute activated 100 µg/mL rhMMP-2 to 12.8 µg/mL in Assay Buffer.
5. Mix 25 µL of 12.8 µg/mL rhMMP-2, 16 µL of rhTIMP-1 serial curve dilutions, and 119 µL of Assay Buffer in micro-tubes.
6. Include two enzyme controls of 25 µL of 12.8 µg/mL rhMMP-2 and 135 mL Assay Buffer in micro-tubes.
7. Incubate reaction mixtures at 37 °C for 2 hours.
8. Dilute incubated reaction mixtures by a 5-fold dilution in Assay Buffer.
9. Dilute Substrate to 10 µM in Assay Buffer.
10. In a plate load 50 µL of 5-fold diluted incubated reaction mixtures to wells.
11. Start the reaction by adding 50 µL of 10 µM Substrate to wells.
12. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively in kinetic mode for 5 minutes.
13. Derive the IC₅₀ value of rhTIMP-1 from the curve.
14. Calculate specific activity for each point 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
• rhTIMP-1 curve: 20 nM, 10 nM, 5 nM, 3 nM, 2 nM, 1.5 nM, 1.0 nM, 0.2 nM, 0.02 nM, and 0 nM
• Substrate: 5 µM