Recombinant Mouse HAI-1 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 Mouse HAI-1 (rmHAI-1) (Catalog # 1141-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 with Assay Buffer.
2. Prepare a curve of rmHAI-1 (MW: 47792 Da) in Assay Buffer. Make the following serial dilutions: 100, 40, 20, 12, 8, 4, 2 and 1 nM.
3. Mix equal volumes of the rmHAI-1 curve dilutions and the diluted Trypsin. Include a Trypsin control (in duplicate) containing Assay Buffer and the diluted Trypsin without any rmHAI-1.
4. Incubate reactions for 60 minutes at 37 °C.
5. After incubation, dilute the mixtures five-fold in Assay Buffer.
6. Dilute Substrate to 20 µM in Assay Buffer.
7. Load into plate 50 µL of the diluted incubated mixtures, 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. Calculate specific activity for each point 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
• rmHAI-1: 5 nM, 2 nM, 1 nM, 0.6 nM, 0.4 nM, 0.2 nM, 0.1 nM, 0.05 nM
• Substrate: 10 µM