Recombinant Human HAI-1 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 HAI-1 (rhHAI-1) (Catalog # 1048-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 rhHAI-1 (MW: 47489 Da) in Assay Buffer. Make the following serial dilutions: 500, 200, 50, 25, 10, 5, 2, 0.5, and 0.05 nM.
3. Mix equal volumes of the rhHAI-1 curve dilutions and the diluted Trypsin. Include a control (in duplicate) containing Assay Buffer and the diluted Trypsin.
4. Incubate reactions for 1 hour at 37 °C.
   After incubation, dilute the mixtures 5 fold in Assay Buffer.
5. Dilute Substrate to 20 µM in Assay Buffer.
6. Load 50 µL of the diluted incubated mixtures into a plate and start the reaction by adding 50 µL of 20 µM Substrate.
7. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
8. Derive the 50% inhibiting concentration (IC₅₀) of rhHAI-1 by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
9. 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)

Per Well:

• Trypsin: 0.00125 µg
• rhHAI-1 curve:  25, 10, 2.5, 1.25, 0.5, 0.25, 0.1, 0.025, 0.0025 nM
• Substrate: 10 µM