Recombinant Human Serpin A3 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 A3/ alpha 1-Antichymotrypsin (rhSerpin A3) (Catalog # 1295-PI)
• Chymotrypsin (Sigma, Catalog # 3142), 40-80 units/mg, prepare a 1 mg/mL stock in 1 mM HCl
• 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 Chymotrypsin to 0.6 µg/mL in Assay Buffer.
2. Prepare a curve of rhSerpin A3 (MW: 46,373 kDa) in Assay Buffer. Make the following serial dilutions including the most concentrated dilution possible: (most concentrated), 1000, 500, 250, 175, 120, 80, 40, 20, and 5 nM.
3. Mix equal volumes of rhSerpin A3 curve dilutions and 0.6 µg/mL Chymotrypsin in microtubes. Include two enzyme controls of equal volumes of Assay Buffer and 0.6 µg/mL Chymotrypsin.
4. Incubate reaction mixtures at 37 °C for 30 minutes.
5. Dilute incubated reaction mixtures by five fold in Assay Buffer.
6. Dilute Substrate to 20 µM in Assay Buffer.
7. In a plate load 50 µL of the diluted reaction mixtures to wells, 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% inhibiting concentration (IC₅₀) for rhSerpin A3 by ploting RFU/min (or specific activity) vs concentration with 4-PL fitting.
10. The specific activity for Chymotrypsin 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).

• Chymotrypsin: 0.003 µg
• rhSerpin A3: (most concentrated), 50, 25, 12.5, 8.75, 6, 4, 2, 1 and 0.25 nM
• Substrate: 10 µM