Recombinant Mouse Coagulation Factor XIV/Protein C, CF

• 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, 10 mM CaCl₂, 150 mM NaCl, 0.01% (w/v) Brij-35, pH 8.5
• Recombinant Mouse Coagulation Factor XIV/Protein C (rmPROC) (Catalog # 4885-SE)
• Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
• 1,10 phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
• Substrate: BOC-beta -benzyl-Asp-Pro-Arg-AMC (Bachem, Catalog # I-1560), 10 mM in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rmPROC to 200 µg/mL in Activation Buffer.
2. Dilute Thermolysin to 10 µg/mL in Activation Buffer.
3. Combine 25 µL of the diluted rmPROC and 25 µL of the diluted Thermolysin.
4. Incubate at 37 °C for 2 hours.
5. Dilute 1,10 phenanthroline to 5 mM in Assay Buffer.
6. Add 200 µL of 5 mM 1,10 phenanthroline to the reaction mixtures to stop the Thermolysin activation.
7. Dilute rmPROC to 2 µg/mL in Assay Buffer.
8. Dilute Substrate to 200 µM in Assay Buffer.
9. Load 50 µL of 2 µg/mL rmPROC into a plate, and start the reaction by adding 50 µL of 200 µM Substrate.
10. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 200 µM Substrate.
11. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
12. Calculate specific activity:

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

Per Well:

• rmPROC: 0.100 µg
• Substrate: 100 µM