Human alpha 2-Macroglobulin 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)
• Human alpha ₂-Macroglobulin (h alpha ₂-Macroglobulin) (Catalog # 1938-PI)
• Recombinant Human Serpin F2 (rhSerpin F2) (Catalog # 1470-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)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhTrypsin 3 to 1.28 µg/mL with Assay Buffer.
2. Prepare a curve of h alpha ₂-Macroglobulin with Assay Buffer. Dilute h alpha ₂-Macroglobulin (MW: 720000 Da) to the following concentrations: 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 0.3125 nM.
3. Combine 30 µL of h alpha ₂-Macroglobulin curve with 30 µL of 1.28 µg/mL rhTrypsin 3. Include a control containing 30 µL of Assay Buffer with 30 µL of rhTrypsin 3 in duplicate. Also include a h alpha ₂-Macroglobulin control in duplicate for each sample tested containing 30 µL of 200 nM h alpha ₂-Macroglobulin and 30 µL Assay Buffer.
4. Incubate at 37 °C for 1 hour.
5. Dilute rhSerpin F2 to 40 µg/mL with Assay Buffer.
6. Add 60 µL of 40 µg/mL rhSerpin F2 to each reaction.
7. Incubate at 37 °C for 15 minutes.
8. Dilute Substrate to 20 µM in Assay Buffer.
9. Load into plate 50 µL of h alpha ₂-Macroglobulin curve containing rhTrypsin 3 and rhSerpin F2, and start the reaction by adding 50 µL of 20 µM Substrate to each well.
10. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
11. Derive the 50% inhibiting concentration (IC₅₀) of h alpha ₂-Macroglobulin, by its trapping of rhTrypsin 3, toward rhSerpin F2 activity by plotting RFU/min (or specific activity) vs. concentration (h alpha ₂-Macroglobulin) with 4-PL fitting.
12. The specific activity for rhTrypsin 3 at each point may be determined using the following formula (if needed):

     *Adjusted for h alpha ₂-Macroglobulin control

     **Derived using calibration MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

• h alpha ₂-Macroglobulin: 25, 12.5, 6.25, 3.125, 1.563, 0.781, 0.391, and 0.0391 nM
• rhTrypsin 3: 0.016 µg
• rhSerpin F2: 1 µg
• Substrate: 10 µM