Recombinant Mouse TIMP-1 Protein, CF

• 6 months from date of receipt, -20 to -70 °C as supplied.
• 3 months, -20 to -70 °C under sterile conditions after reconstitution.

• Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% Brij-35 (w/v), pH 7.5 (TCNB)
• Recombinant Mouse TIMP-1 (rmTIMP-1) (Catalog # 980-MT)
• 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. Dilute rhMMP-2 to 100 µg/mL in Assay Buffer containing 1 mM APMA.
2. Incubate at 37 °C for 1 hour (activation).
3. Prepare a curve of rmTIMP-1 (MW: 20,200 Da). Make the following serial dilutions in Assay Buffer:  5000, 2000, 1000, 500, 300, 200, 150, 100, 20, and 2 nM.
4. Dilute activated rhMMP-2 to 12.5 µg/mL in Assay Buffer.
5. Mix 25.6 µL of diluted rhMMP-2, 16 µL of rmTIMP-1 serial curve dilutions, and 118.4 µL of Assay Buffer.
6. Include two enzyme controls of 25.6 µL of diluted rhMMP-2 and 134.4 μL Assay Buffer.
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 the 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 50% inhibiting concentration (IC₅₀) for rmTIMP-1 by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
14. 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
• rmTIMP-1:  50, 20, 10, 5, 3, 2, 1.5, 1, 0.2, and 0.02 nM
• Substrate: 5 µM