Recombinant Human Serpin A5 Protein, CF

• Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
• Recombinant Human Serpin A5/Protein C Inhibitor (rhSerpin A5) (Catalog # 1266-PI)
• Recombinant Human Coagulation Factor II/Thrombin (Catalog # 1473-SE)
• Heparin (Sigma, Catalog # H3393), 20 mg/mL stock in deionized water
• 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 Thrombin to 0.4 µg/mL with 48.6 µg/mL Heparin in Assay Buffer.
2. Prepare a curve of rhSerpin A5 (MW: 45,003 Da) in Assay Buffer. Make the following serial dilutions:  200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.781, and 0.391 nM.
3. Mix equal volumes of rhSerpin A5 curve dilutions and Thrombin/Heparin mixture. Include a control (in duplicate) containing equal volumes of Assay Buffer and diluted Thrombin/Heparin mixture.
4. Incubate reaction mixtures at room temperature for 30 minutes.
5. After incubation, dilute the reaction mixtures by 1/5 in Assay Buffer.
6. Dilute Substrate to 200 µM in Assay Buffer.
7. In a plate load 50 µL of the diluted reaction mixtures to wells, and start the reaction by adding 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. Derive the 50% inhibiting concentration (IC₅₀) value by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
10. The specific activity for Thrombin at each point may be determined using the following formula (if needed):

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

Per Well:

• Thrombin: 0.002 µg
• rhSerpin A5: 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.0781, 0.0391 and 0.0195 nM
• Substrate: 100 µM