Recombinant Mouse Coagulation Factor VII 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.

• 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, pH 9.0
• Recombinant Mouse Coagulation Factor VII (rmFactor VII) (Catalog # 3305-SE)
• Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
• 1,10-Phenanthroline (Sigma, Catalog # 320056)
• Recombinant Mouse Coagulation Factor III/Tissue Factor (rmTF) (Catalog # 3178-PA)
• Substrate: Boc-Val-Pro-Arg-AMC (Catalog # ES011), 10 mM stock in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Activate rmFactor VII at 100 µg/mL with 10 µg/mL Thermolysin in Activation Buffer.
2. Incubate at 37 °C for 30 minutes.
3. After incubation, stop reaction with 1,10-Phenanthroline at a final concentration of 10 mM in Activation Buffer.
4. Incubate reaction mixtures at 37 °C for 5 minutes.  The enzyme concentration is now at 75 µg/mL.
5. Dilute rmTF to 15.3 µg/mL in Assay Buffer.
6. In a plate, load 13.3 µL of 75 µg/mL activated rmFactor VII followed by adding 36.7 µL of 15.3 µg/mL rmTF.
7. Incubate plate at 37 °C for 5 minutes.
8. Dilute Substrate to 200 µM in Assay Buffer.
9. After plate incubation, start the reaction by adding 50 µL of 200 µM of Substrate to wells.
10. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively in kinetic mode for 5 minutes.
11. Calculate specific activity:

     *Adjusted for Substrate Blank

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

• rmFactor VII: 1 µg
• rmTF: 0.56 µg
• Substrate: 100 µM