Recombinant Human TFPI 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 TFPI1 (rhTFPI1) (Catalog # 2974-PI)
• Trypsin (Sigma, Catalog # T-1426)
• Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH₂ (Catalog # ES002), 2 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 Trypsin to 0.25 µg/mL in Assay Buffer.
2. Prepare a curve of rhTFPI1 (MW: 30,525 Da) in Assay Buffer. Make the following serial dilutions: 500, 100, 50, 30, 20, 10, 5, 2.5, and 0.833 nM.
3. Combine equal volumes of 0.25 µg/mL Trypsin and rhTFPI1 serial curve dilutions. Include two controls containing equal volumes of Assay Buffer and 0.25 µg/mL Trypsin.
4. Incubate reaction mixtures at 37 °C for 15 minutes.
5. Dilute incubated reaction mixtures 5-fold in Assay Buffer.
6. Dilute Substrate to 20 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 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 rhTFPI1 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
• rhTFPI1 curve: 25, 5, 2.5, 1.5, 1.0, 0.5, 0.25, 0.125, and 0.0417 nM
• Substrate: 10 µM