Recombinant Human Serpin A1/alpha-1-Antitrypsin 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 Serpin A1/ alpha 1‑Antitrypsin (rhSerpin A1) (Catalog # 1268-PI)
• Trypsin (Sigma, Catalog # T1426)
• 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 rhSerpin A1 (MW: 45,667 Da) in Assay Buffer.  Make the following serial dilutions:
   200 nM, 100 nM, 50 nM, 33.3 nM, 22.2 nM, 14.8 nM, 9.88 nM, and 3.29 nM.
3. Combine 25 µL of 0.25 µg/mL Trypsin with 25 µL of rhSerpin A1 serial curve dilutions. Include two controls of 25 µL Assay Buffer with 25 µL of 0.25 µg/mL Trypsin.
4. Incubate at room temperature for 30 minutes.
5. After incubation, add 200 µL of Assay Buffer to each serial curve dilution.
6. Dilute Substrate to 20 µM in Assay Buffer.
7. In a plate, load 50 µL of the diluted rhSerpin A1 curve, and start the reaction by adding 50 µL of 20 µM Substrate to wells.
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 hSerpin A1 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
• rhSerpin A1: 10, 5, 2.5, 1.665, 1.11, 0.74, 0.494, 0.165 and 0 nM
• Substrate: 10 µM