Recombinant Human Coagulation Factor IX Protein, CF

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

• Assay Buffer: 100 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 8.0
• Recombinant Human Coagulation Factor IX (rhFactor IX) (Catalog # 9260-SE)
• Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
• Substrate: Fluorogenic Peptide Substrate II, MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)NH₂ (Catalog # ES002)
• 1,10-Phenanthroline (Sigma, Catalog # 320056)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Activate rhFactor IX at 100 µg/mL wih 10 µg/mL Thermolysin in Assay Buffer.
2. Incubate at 37 °C for 90 minutes.
3. Stop the reaction by adding 1,10-Phenanthroline to the reaction tube at a final concentration of 10 mM.
4. Dilute activated rhFactor IX to 4 ng/µL in Assay Buffer.
5. Dilute Substrate to 20 µM in Assay Buffer.
6. Load 50 µL of the 4 ng/µL rhFactor IX into a black well plate, and start the reaction by adding 50 µL of 20 µM Substrate.  Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL 20 µM Substrate.
7. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
8. Calculate specific activity:

      *Adjusted for Substrate Blank
      **Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975)

• rhFactor IX: 0.2 µg
• Substrate: 10 µM