Recombinant Human SLPI 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% Brij-35 (v/v), pH 7.5 (TCNB)
• Recombinant Human SLPI (rhSLPI) (Catalog # 1274-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 rhSLPI (MW: 11,726 Da) in Assay Buffer. Make the following serial dilutions: 5000, 500, 250, 125, 83.8, 55.6, 37, 24.7, 8.23, and 2.06 nM.
3. Mix 25 µL of 0.25 µg/mL Trypsin and 25 µL of the rhSLPI dilutions. Include an enzyme control (in duplicate) of 25 µL Assay Buffer and 25 µL of 0.25 µg/mL Trypsin.
4. Incubate reaction mixtures at 37 °C for 60 minutes.
5. Dilute Substrate to 20 µM in Assay Buffer.
6. After incubation dilute the enzyme inhibitor mixtures five fold in Assay Buffer.
7. In a plate load 50 µL of the diluted reaction mixtures to wells.
8. Start the reaction by adding 50 µL of 20 µM Substrate to wells.
9. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively in kinetic mode for 5 minutes.
10. Derive the IC₅₀ value for rhSLPI from the curve.
11. 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
• rhSLPI curve: 250, 25, 12.5, 6.25, 4.19, 2.78, 1.85, 1.235, 0.415, 0.103 and 0 nM
• Substrate: 10 µM