Recombinant Human Serpin E1/PAI-1 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, 0.01% (v/v) Tween® 20, pH 8.5
• Recombinant Human Serpin E1/PAI-1 (rhSerpin E1/PAI-1) (Catalog # 1786-PI)
• Recombinant Human u‑Plasminogen Activator (uPA)/Urokinase (rhuPA) (Catalog # 1310-SE)
• Substrate: Z-Gly-Gly-Arg-AMC (Bachem, Catalog # I-1140)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhuPA to 2 µg/mL in Assay Buffer.
2. Prepare a rhSerpin E1/PAI-1 (MW: 44146 Da) dilution curve in Assay Buffer. Make the following serial dilutions:  1500, 750, 375, 187.5, 125, 75, 50, 25, and 12.5 nM.
3. Mix equal volumes of the rhSerpin E1/PAI-1 curve and the diluted rhuPA at 2 µg/mL. Include 2 controls containing rhuPA and Assay Buffer only.
4. Incubate mixtures at room temperature for 15 minutes.
5. After incubation, dilute the mixtures 5 fold in Assay Buffer.
6. Dilute the Substrate to 200 µM in Assay Buffer.
7. Load 50 µL of the incubated reactions into a plate, and start the reaction by adding 50 µL of 200 µM Substrate to each well.
8. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
9. Determine the 50% inhibiting concentration (IC₅₀) for rhSerpin E1 by plotting RFU/min (or specific activity) vs. concentration with 4‑PL fitting.
10. The specific activity for rhuPA at each point may be determined using the following formula (if necessary):

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

• rhuPA: 0.010 µg
• rhSerpin E1/PAI-1 curve: 75, 37.5, 18.75, 9.375, 6.25, 3.75, 2.5, 1.25, and 0.625 nM
• Substrate: 100 µM