Coagulation factor VII/F7 Protein, Human (HEK293, His)

Materials
Assay buffer: 50 mM Tris, 2.5 mM CaCl₂, pH 8.5
Coagulation factor VII/F7 Protein, Human (406a.a, HEK293, His) (HY-P78763)
Coagulation factor III/F3 Protein, Human (HEK293, His) (Tissue Factor, HY-P7787)
Substrate: Boc-Val-Pro-Arg-AMC hydrochloride (HY-137784)
Standard: 7-amino, 4-Methyl Coumarin (HY-D0027)

Procedure
1. Dilute 7-amino, 4-Methyl Coumarin to 0, 9.765625, 19.53125, 39.0625, 78.125, 156.25, 312.5, 625, 1250, 2500, 5000, 10000 pmol with assay buffer respectively.
2. Add 100 μL of each concentration of standard to the wells.
3. Read in kinetic mode for 5 minutes with excitation and emission wavelengths of 380 nm and 460 nm (top read).
4. Plot a standard curve with the standard concentration as the abscissa and the measured RFU as the ordinate to obtain the standard curve.
5. Protein activity determination: First reconstitute Coagulation factor VII with sterile water to 100 μg/mL, then dilute Coagulation factor VII to 32 μg/mL with assay buffer.
6. Dilute Tissue Factor to 35.5 μg/mL with assay buffer.
7. Mix equal volumes of Coagulation factor VII and Tissue Factor, and incubate at 37°C for 5 minutes.
8. Dilute the substrate to 200 μM in assay buffer.
9. Transfer 50 μL of activated Coagulation factor VII to the wells of a black well plate.
10. Add 50 μL of 200 μM substrate to each well to start the reaction.
11. Read for 5 minutes in kinetic mode at excitation and emission wavelengths of 380 nm and 460 nm respectively.
12. Calculate the specific activity: