Recombinant Mouse Fetuin A/AHSG 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, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
• Recombinant Mouse Fetuin A/AHSG (rmFetuin A) (Catalog # 1563-PI)
• Recombinant Human Active Trypsin 3/PRSS3 (rhTrypsin 3) (Catalog # 3714-SE)
• Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(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. Dilute rhTrypsin 3 to 0.6 µg/mL in Assay Buffer.
2. Prepare a curve of rmFetuin A (MW: 36651 Da) in Assay Buffer. Make the following serial dilutions: 6500, 1952, 651, 195.4, 65.1, 19.6, and 6.5 nM.
3. Combine 20 µL of 0.6 µg/mL rhTrypsin 3, 100 µL of each dilution from the rmFetuin A curve, and 80 µL of Assay Buffer. Include a control (in duplicate) containing 180 µL of Assay Buffer and 20 µL of the diluted rhTrypsin 3.
4. Incubate mixtures at room temperature for 30 minutes.
5. Dilute Substrate to 20 µM in Assay Buffer.
6. Load 50 µL of the incubated mixtures into a plate, and start the reaction by adding 50 µL of 20 µM Substrate.
7. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
8. Derive the 50% inhibition concentration (IC₅₀) value for rmFetuin A by plotting RFU/min (or specific activity) vs. concentration with 4‑PL fitting.
9. The specific activity for rhTrypsin 3 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).

• rhTrypsin 3: 0.003 µg (1.17 nM)
• rmFetuin A curve: 1625, 488, 163, 48.9, 16.3, 4.9, and 1.625 nM
• Substrate: 10 µM