Recombinant Human Serpin C1/Antithrombin-III Protein, CF

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

• Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
• Recombinant Human Serpin C1/Antithrombin-III (rhSerpin C1) (Catalog # 1267-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) , 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 Thrombin to 1 µg/mL with Heparin at 48.6 µg/mL in Assay Buffer.
2. Prepare a curve of rhSerpin C1 (MW: 50,378 Da) in Assay Buffer. Make the following serial dilutions: 1000, 500, 250, 125, 62.5, 41.7, 27.8, 13.9, 6.94, and 2.31 nM.
3. Mix equal volumes of rhSerpin C1 curve dilutions and Thrombin/Heparin mixture. Include a control (in duplicate) containing equal volumes of Assay Buffer and Thrombin/Heparin mixture.
4. Incubate reaction mixtures at room temperature for 30 minutes.
5. After incubation, dilute 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% inhibition concentration (IC₅₀) for rhSerpin C1 by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
10. Calculate specific activity for Thrombin at each point using the following formula (if needed):

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

• Thrombin: 0.005 µg (1.0 nM)
• rhSerpin C1:  50, 25, 12.5, 6.25, 3.13, 2.09, 1.39, 0.695, 0.347, and 0.116 nM
• Substrate: 100 µM