Recombinant Human Coagulation Factor XIV/Protein C, CF

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

• Activation Buffer: 50 mM Tris, 150 mM NaCl, 10 mM CaCl₂, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
• Assay Buffer: 50 mM Tris, 100 mM NaCl, 0.01% (w/v) Brij-35, pH 8.5
• Recombinant Human Coagulation Factor XIV/Protein C (rhPROC) (Catalog # 3349-SE)
• Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
• 1,10-Phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
• Fluorogenic Peptide Substrate: BOC-Val-Pro-Arg-AMC (Catalog # ES011)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhPROC to 100 µg/mL in Activation Buffer with 2.3 µg/mL Thermolysin.
2. Incubate for 30 minutes at 37 °C.
3. Stop Thermolysin activity by adding 1,10-Phenanthroline to 4 mM.
4. Incubate for 15 minutes at room temperature.
5. Dilute rhPROC to 4 ng/µL in Assay Buffer.
6. Dilute Substrate to 200 µM in Assay Buffer.
7. Load into a black well plate 50 µL of 4 ng/µL rhPROC, and start the reaction by adding 50 μL of 200 µM Substrate. 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 (AMC) (Sigma, Catalog # A-9891).

• rhPROC: 0.200 µg
• Substrate: 100 μM