Recombinant Mouse Serpin F2/alpha 2-Antiplasmin Protein, CF

• 6 months from date of receipt, -20 to -70 °C as supplied.
• 3 months, -20 to -70 °C under sterile conditions after opening.

• Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB
• Recombinant Mouse Serpin F2/ alpha ₂‑Antiplasmin (rmSerpin F2) (Catalog # 1239-PI)
• Trypsin (Sigma, Catalog # T-1426)
• Substrate: Mca-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH₂ (Catalog # ES002
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute Trypsin to 0.25 µg/mL in Assay Buffer and KEEP ON ICE.
2. Prepare a curve of rmSerpin F2 (MW: 53,503 Da) in Assay Buffer. Make the following serial dilutions: 100, 20, 15, 10, 7, 4, 2, and 1 nM.
3. Combine equal volumes of diluted Trypsin and rmSerpin F2 curve dilutions. Include two blank controls containing equal volumes of Assay Buffer and diluted Trypsin without any rmSerpin F2.
4. Incubate mixtures at 37 °C for 15 minutes.
5. Dilute the reaction mixtures 1/5 with Assay Buffer.
6. Dilute Substrate to 20 µM with Assay Buffer.
7. Load in a black well plate 50 µL of the incubated mixtures, 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) of rmSerping F2 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).

• Trypsin:  0.00125 µg
• rmSerpin F2 curve: 5, 1, 0.75, 0.5, 0.35, 0.2, 0.1, and 0.05 nM
• Substrate: 10 µM