Recombinant Human Serpin B3/SSCA1 Protein, CF

• Assay Buffer: 50 mM MES, 5 mM DTT, pH 6.0
• Recombinant Human Serpin B3/SCCA1  (rhSerpin B3) (Catalog # 6528-PI)
• Recombinant Human Cathepsin L (rhCathepsin L) (Catalog # 952-CY)
• Substrate: Z-Leu-Arg-AMC (Catalog # ES008)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: Gemini EM by Molecular Devices) or equivalent

1. Dilute rhCathepsin L to 40 µg/mL in Assay Buffer.
2. Incubate 40 µg/mL rhCathepsin L for 15 minutes on ice to activate.
3. Dilute activated rhCathepsin L to 0.2 µg/mL in Assay Buffer.
4. Prepare a curve of rhSerpin B3 (MW = 45386 Da) in Assay Buffer. Make the following serial dilutions: 1000, 200, 50, 35, 25, 15, 5, 2 and 1 nM.
5. Combine 25 µL of each dilution with 25 µL of the 0.2 µg/mL rhCathepsin L. Include an enzyme control containing Assay Buffer in place of rhSerpin B3.
6. Incubate reaction mixtures at 37 °C for 15 minutes.
7. Dilute reactions by adding 200 µL Assay Buffer to each.
8. Dilute Substrate to 20 µM in Assay Buffer.
9. Load into a black well plate 50 µL of the diluted incubated mixtures and start the reaction by adding 50 µL substrate.
10. Read at excitation and emission wavelengths of 380 nm and 460 nM (top read), respectively, in kinetic mode for 5 minutes.
11. Derive the 50% inhibition concentration (IC₅₀) value for rhSerpin B3 by plotting RFU/min vs. concentration with 4-PL fitting.
12. Calculate the specific activity for rhCathepsin L at each point using the following formula (if needed):

     *Adjusted for Substrate Blank
     **Derived using calibration standard 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).

• rhCathepsin L: 0.001 µg
• rhSerpin B3: 50, 10, 2.5, 1.75, 1.25, 0.75, 0.25, 0.1, and 0.05 nM
• Substrate: 10 μM