Recombinant Human Serpin B2/PAI-2 Protein, CF

• 6 months from date of receipt, -70 °C as supplied.
• 3 months, -70 °C under sterile conditions after opening.

• Assay Buffer: 50 mM Tris, 0.01% Tween® 20, pH 8.5
• Recombinant Human Serpin B2 (rhSerpin B2) (Catalog # 9206-PI)
• Recombinant Human u-Plasminogen Activator (uPA)/Urokinase (rhuPA) (Catalog # 1310-SE)
• Substrate: Z-Gly-Gly-Arg-AMC (Bachem, Catalog # I-1140), 10 mM stock in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: Gemini EM by Molecular Devices) or equivalent

1. Prepare a curve of rhSerpin B2 (MW: 43.8 kDa) in Assay Buffer. Make the following serial dilutions: 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, and 0.3125 nM. 
2. Dilute rhuPA to 2 μg/mL in Assay Buffer.
3. Combine equal volumes of each point of the rhSerpin B2 curve with 2 µg/mL rhuPA. Include an enzyme control containing equal volumes of Assay Buffer and rhuPA.
4. Incubate reaction mixtures at room temperature for 15 minutes.
5. Dilute Substrate to 200 µM in Assay Buffer.
6. Load 50 µL of the diluted incubated mixtures into empty wells of a plate, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 200 µM Substrate.
7. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
8. Derive the 50% inhibition concentration (IC₅₀) value for rhSerpin-B2 by plotting RFU/min (or specific activity) versus concentration with 4-PL fitting.
9. Calculate specific activity for each point using the following formula (if needed):

     *Adjusted for Substrate Blank
     **Derived using calibration standard 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A9891)

• rhSerpin-B2: 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, and 0.078 nM
• rhuPA: 0.050 µg
• Substrate: 100 µM