Recombinant Rat TIMP-1 Protein, CF

• Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05 % (w/v) Brij-35, pH 7.5 (TCNB)
• Recombinant Rat TIMP-1 (rrTIMP-1) (Catalog # 580-RT)
• 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 APMA in Assay Buffer.
2. Incubate at 37 ºC for 1 hour to activate rhMMP-2.
3. Dilute activated rhMMP-2 to 12.5 µg/mL in Assay Buffer.
4. Prepare a curve of rrTIMP-1 (MW: 21,500 Da) in Assay Buffer. Make the following serial dilutions: 5000, 2000, 1000, 500, 300, 200, 150, 100, 20, and 2 nM.
5. Mix 25.6 µL of 12.5 µg/mL rhMMP-2, 16 µL of rrTIMP-1 serial curve dilutions, and 118.4 µL of Assay Buffer in microtubes. Include two enzyme controls of 25.6 µL of 12.5 µg/mL rhMMP-2 and 134.4 µL Assay Buffer in microtubes.
6. Incubate reaction mixtures at 37 °C for 2 hours.
7. Dilute incubated reaction mixtures 5 fold in Assay Buffer.
8. Dilute Substrate to 10 µM in Assay Buffer.
9. In a plate load 50 µL of the diluted incubated reaction mixtures to wells, and start the reaction by adding 50 µL of 10 µM Substrate.
10. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
11. Determine the 50% inhibition concentration (IC₅₀) for rrTIMP-1 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).

Per Well:

• rhMMP-2: 0.02 µg
• rrTIMP-1 curve: 50, 20, 10, 5, 3, 2, 1.5, 1, 0.2, 0.02, and 0 nM
• Substrate: 5 µM