Recombinant Mouse HAI-2B 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-2B (rmHAI-2B) (Catalog # 1107-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 rmHAI-2B (MW: 14,281 Da) in Assay Buffer. Make the following serial dilutions: 1000, 200, 50, 25, 12.5, 6.25, 3.13, 1.56, and 0.391 nM.
3. Mix equal volumes of the rmHAI-2A curve dilutions and 0.25 µg/mL Typsin. Include a control (in duplicate) containing Assay Buffer and 0.25 µg/mL Typsin.
4. Incubate reactions for 30 minutes at room temperature.
5. After incubation, dilute the mixtures 5 fold in Assay Buffer.
6. Dilute Substrate to 20 µM in Assay Buffer.
7. Load 50 µL of the diluted incubated mixtures into 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% inhibition concentration (IC₅₀) value for rmHAI-2B by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
10. The specific activity of 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
• rmHAI-2B curve: 50, 10, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078 and 0.0195 nM
• Substrate: 10 µM