Recombinant Human TFPI-2 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 TFPI‑2 (rhTFPI-2) (Catalog # 2545-PI)
• Trypsin (Sigma, Catalog # T-1426)
• Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH₂ (Catalog # ES002)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute Trypsin to 0.25 µg/mL in Assay Buffer.
2. Prepare a curve of rhTFPI-2 (MW: 23,197 Da) in Assay Buffer. Make the following serial dilutions: 4000, 800, 400, 200, 100, 50, 25, 12.5, 6.25, and 1.25 nM.
3. Combine equal volumes of diluted Trypsin and rhTFPI-2 at each concentration of the curve. Include two controls containing equal volumes of Assay Buffer and diluted Trypsin without any rhTFPI-2.
4. Incubate mixtures at 37 °C for 15 minutes.
5. Dilute reaction mixtures five fold in Assay Buffer.
6. Dilute Substrate to 20 µM in Assay Buffer.
7. Load 50 µL of the incubated mixtures in a plate, and start the reaction by adding 50 µL of 20 µM Substrate.
8. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively in kinetic mode for 5 minutes.
9. Derive the 50% inhibiting concentration (IC₅₀) for rhTFPI-2 by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
10. The specific activity for trypsin at each point may be determined using the following formula (if needed):

     *Adjusted for Substrate Blank

     **Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

• Trypsin: 0.00125 µg
• rhTFPI-2: 200, 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.0625 nM.
• Substrate: 10 µM