Recombinant Human TIMP-2 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, pH 7.5 (TCNB)
• Recombinant Human TIMP-2 (rhTIMP-2) (Catalog # 971-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. Dilute rhMMP-2 to 100 µg/mL in Assay Buffer with 1 mM of APMA.
2. Incubate at 37 °C for 1 hour.
3. Prepare a curve of rhTIMP-2 (MW: 21,500 Da). Make the following serial dilutions in Assay Buffer:  2000, 1000, 500, 300, 200, 150, 100, 50, 25, and 2.5 nM.
4. Dilute rhMMP-2 to 12.8 µg/mL in Assay Buffer.
5. For each inhibition point mix 25 µL of 12.8 µg/mL rhMMP-2, 16 µL of the rhTIMP-2 curve dilution, and 119 µL of Assay Buffer. Include two enzyme controls of 25 µL of 12.8 µg/mL rhMMP-2 and 135 µL Assay Buffer.
6. Incubate reaction mixtures at 37 °C for 2 hours.
7. Dilute incubated reaction mixtures by 5 fold in Assay Buffer.
8. Dilute Substrate to 10 µM in Assay Buffer.
9. In a plate load 50 µL of the 5-fold diluted incubated reaction mixtures to wells.
10. Start the reaction by adding 50 µL of 10 µM Substrate to all wells.
    Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively in kinetic mode for 5 minutes.
11. Derive the 50% inhibiting concentration of rhTIMP-2 (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
• rhTIMP-2 curve:  20, 10, 5, 3, 2, 1.5, 1.0, 0.5, 0.25, 0.025, and 0 nM
• Substrate: 5 µM