Recombinant Human Coagulation Factor III/Tissue Factor, CF

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

• Activation Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
• Assay Buffer: 50 mM Tris, 2.5 mM CaCl₂, pH 8.5
• Recombinant Human Coagulation Factor III/Tissue Factor (rhTF) (Catalog # 2339-PA)
• Recombinant Human Coagulation Factor VII (rhF7) (Catalog # 2338-SE)
• Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
• 1,10 Phenanthroline (Sigma, Catalog # 320056), 600 mM Stock in DMSO
• Substrate: BOC-Val-Pro-Arg-AMC (R&D Systems, Catalog # ES011), 100 mM in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhF7 to 100 µg/mL in Activation Buffer containing 6.25 µg/mL Thermolysin.
2. Incubate at 37 °C for 30 minutes to activate rhF7.
3. Stop reaction by adding 1,10 phenanthroline to a final concentration of 10 mM in Activation Buffer.
4. Incubate for 5 minutes at room temperature.
5. Prepare a dilution curve of rhTF in Assay Buffer. Make serial dilutions of: 71.04, 35.52, 17.76, 8.88, 4.44, 2.22, 1.11, 0.555, 0.278, and 0.139 µg/mL.
6. Dilute Thermolysin-activated rhF7 to 32 µg/mL in Assay Buffer.
7. Load 25 µL of each dilution from the dilution curve to a plate. Include two controls containing 25 µL Assay Buffer.
8. Add 25 µL of 32 µg/mL Thermolysin-activated rhF7 to all the wells.
9. Incubate at 37 °C for 5 minutes.
10. Dilute Substrate to 200 µM in Assay Buffer.
11. Add 50 µL of 200 μM Substrate to all wells.
12. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
13. Derive the 50% activating concentration (AC₅₀) of rhTF by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
14. The specific activity for rhF7 at each point may be determined using the following formula (if needed):

     *Adjusted for Substrate Blank

     **Derived using calibration standard 7-amino, 4-methyl coumarin (Sigma, Catalog # A-9891).

• rhF7: 0.800 µg
• rhTF: 17.76, 8.88, 4.44, 2.22, 1.11, 0.556, 0.278, 0.139, 0.0695, and 0.0348 µg/mL
• Substrate: 100 µM