Recombinant Mouse Coagulation Factor XI Protein, CF

• 6 months from date of receipt, -70 °C as supplied.
• 3 months, -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, 1 mM EDTA, pH 7.5
• Recombinant Mouse Coagulation Factor XI (rmFactor XI) (Catalog # 4556-SE)
• Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
• EDTA, pH 8.0 (Sigma, Catalog # E-4884), 0.5 M stock in deionized water
• Substrate: BOC-Ile-Glu-Gly-Arg-AMC (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. Dilute rmFactor XI to 100 µg/mL in Activation Buffer containing 10 µg/mL of Thermolysin.
2. Incubate at 37 °C for 1 hour.
3. Stop reaction with EDTA at a final concentration of 40 mM.
4. Dilute incubated rmFactor XI to 2 ng/µL in Assay Buffer.
5. Dilute Substrate to 200 µM in Assay Buffer.
6. Load 50 µL of 2 ng/µL rmFactor XI into a plate, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 200 µM Substrate.
7. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
8. Calculate specific activity:

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

• rmFactor XI:  0.100 µg
• Thermolysin: 0.010 µg
• Substrate: 100 µM