Recombinant Human TIMP-4 (Sf 21-expressed) 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% (w/v) Brij-35, pH 7.5 (TCNB)
• Recombinant human TIMP-4 (rhTIMP-4) (Catalog # 974-TSF)
• Recombinant Human MMP‑2 (rhMMP‑2) (Catalog # 902-MP)
• 4-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 with 1 mM APMA in Assay Buffer.
2. Incubate at 37 ^ºC for 1 hour (to activate).
3. Prepare a curve of rhTIMP-4 (MW: 22,400 Da) in Assay Buffer.  Make serial dilutions of: 5000, 2000, 1000, 500, 300, 200, 150, 100, 20, and 2 nM.
4. After activation, dilute 100 µg/mL rhMMP-2 to 12.5 µg/mL in Assay Buffer.
5. Mix 16 µL of rhTIMP-4 curve dilutions, 25.6 µL of diluted rhMMP-2, and 118.4 µL of Assay Buffer.  Include a control (in duplicate) containing 134.4 µL of Assay Buffer and 25.6 µL of diluted rhMMP-2.
6. Incubate reactions for 2 hours at 37 °C.
7. After incubation, dilute the mixtures five fold in Assay Buffer.
8. Dilute Substrate to 10 µM in Assay Buffer.
9. Load 50 µL of the diluted incubated mixtures in a plate, 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. Derive the 50% inhibiting concentration (IC₅₀) for rhTIMP-4 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:  0.020 µg
• rhTIMP-4:  50, 20, 10, 5, 3, 2, 1.5, 1, 0.2, and 0.02 nM
• Substrate: 5 µM