Recombinant Mouse Serpin A1a Protein, CF

• Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% Brij-35 (w/v), pH 7.5 (TCNB)
• Trypsin Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, pH 7.5 (TCN)
• Recombinant Mouse Serpin A1a (rmSerpin A1a) (Catalog # 7690-PI)
• Trypsin, from bovine pancreas (Sigma, Catalog # T1426)
• Substrate: MCA-RPKPVE-NVal-WRK(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. Prepare a curve of rmSerpin A1a (MW 44,000 Da). Make the following serial dilutions in Assay Buffer: 3600, 1800, 900, 450, 225, 112.5, 56.25, 28.13, and 14.1 nM.
2. Dilute Trypsin to 5.0 µg/mL in Trypsin Buffer.
3. Combine equal volumes of dilute rmSerpin A1a and Trypsin. Include a control containing equal volumes of Assay Buffer and Trypsin.
4. Incubate reactions at room temperature for 30 minutes.
5. Dilute incubated reaction mixtures 25-fold in Assay Buffer.
6. Dilute Substrate to 20 µM in Assay Buffer.
7. Load 50 µL of the incubated reactions into the wells of a black well 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% inhibiting concentration of rmSerpin A1a (IC₅₀) 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).

Per Well:

• Trypsin: 0.005 µg (2 nM)
• rmSerpin A1a: 36, 18, 9, 4.5, 2.25, 1.125, 0.563, 0.281, and 0.141 nM
• ES002 : 10 µM