Recombinant Human F13A1 His-tag Protein, CF

• Assay Buffer: 50 mM Tris, 150 mM NaCl, 10 mM CaCl₂, 0.05% Brij-35, pH 7.5 (TCNB)
• Recombinant Human F13A1 (rhF13A1) (Catalog # 10179-F1)
• Recombinant Human Coagulation Factor II/Thrombin (Catalog # 1473-SE)
• Substrate: Abz-NE(CAD-DNP)EQVSPLTLLK-OH (Zedira, Catalog # A101), 5 mM stock in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Florescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Activate rhF13A1 at 100 µg/mL with 40 µg/mL rhThrombin in Assay Buffer for 30 minutes at 37 °C.
2. Dilute activated rhF13A1 to 20 µg/mL in Assay Buffer.
3. Dilute Substrate to 100 µM in Assay Buffer.
4. Load in plate 50 µL of 20 µg/mL activated rhF13-A1, and start the reaction by adding 50 µL of 100 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 100 µM Substrate.
5. Read at excitation and emission wavelengths at 313 nm and 418 nm, respectively, in kinetic mode for 5 minutes.
6. Calculate specific activity:

 *Adjusted for Substrate Blank

**Derived using calibration standard Abz-Gly-OH (Bachem, Catalog # E-2920)

• rhF13A1: 1 µg
• Substrate: 50 µM