Recombinant Human Coagulation Factor XI 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, pH 7.5 (TCN)
• Assay Buffer: 50 mM Tris, 250 mM NaCl, 1 mM EDTA, pH 7.5
• Recombinant Human Coagulation Factor XI (rhFactor XI) (Catalog # 2460-SE)
• Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
• EDTA, pH 8.0 (Sigma, Catalog # E-4884), 0.5 M stock in diH2O
• Substrate: t-Butyloxycarbonyl-Ile-Glu-Gly-Arg-7-amido-4-methylcoumarin (Bachem, Catalog # I-1100), 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 rhFactor XI at 100 µg/mL with 10 µg/mL Thermolysin in Activation Buffer.
2. Incubate at 37 °C for 1 hour.
3. Stop reaction with EDTA at a final concentration of 40 mM in Activation Buffer.
4. Dilute incubated rhFactor XI to 4 ng/µL in Assay Buffer.
5. Dilute Substrate to 200 µM in Assay Buffer.
6. In a plate, load 50 µL of 4 ng/µL rhFactor XI, and start the reaction by adding 50 µL of 200 µM Substrate.
7. Include a Substrate blank containing 50 µL of Assay Buffer and 50 µL of 200 µM Substrate.
8. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively in kinetic mode for 5 minutes.
9. Calculate specific activity:

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

• rhFactor XI:  0.2 µg
• Thermolysin: 0.02 µg
• Substrate: 100 µM