Recombinant Mouse TIMP-2 Protein, CF

• Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
• Recombinant Mouse TIMP-2 (rmTIMP-2) (Catalog # 6304-TM)
• Recombinant Human MMP‑2 (rhMMP-2) (Catalog # 902-MP)
• 4-Aminophenylmercuric acetate (APMA) (Sigma, Catalog # A9563), 40 mM stock in DMSO
• Substrate: MCA-PLGL-DPA-AR-NH₂ (Catalog # ES001), 2 mM stock in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: Gemini EM by Molecular Devices) or equivalent

1. Dilute rhMMP-2 to 100 µg/mL in Assay Buffer.
2. Add APMA to the rhMMP-2 to a final concentration of 1 mM.
3. Incubate the 100 µg/mL rhMMP-2 at 37 °C for 1 hour to activate.
4. Prepare a curve of rmTIMP-2 (MW = 21,700 kDa) in Assay Buffer. Make the following serial dilutions: 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, and 1.56 nM.
5. Dilute the activated rhMMP-2 to 2 µg/mL in Assay Buffer.
6. Combine 20 µL of each dilution with 20 µL of the 2 µg/mL rhMMP-2. Include an enzyme control containing Assay Buffer in place of rmTIMP-2.
7. Incubate reaction mixtures at 37 °C for two hours.
8. Dilute reactions by adding 160 µL Assay Buffer to each.
9. Dilute Substrate to 20 µM in Assay Buffer.
10. Load into a black well plate 50 µL of the diluted incubated mixtures, and start the reaction by adding 50 µL substrate.
11. Read at excitation and emission wavelengths of 320 nm and 405 nM (top read), respectively, in kinetic mode for 5 minutes.
12. Derive the 50% inhibiting concentration (IC₅₀) for rmTIMP-2 by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
13. 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-P-L-OH (Bachem, Catalog # M-1975).

Per Well:

• rhMMP-2: 0.01 µg
• rmTIMP-2: 20 nM, 10 nM, 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, 0.313 nM, 0.156 nM, 0.078 nM
• Substrate: 10 µM