Recombinant Human Chymotrypsin C/CTRC Protein, CF

• Activation Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, pH 7.5 (TCN)
• Assay Buffer: 25 mM Tris, 0.5 mM CaCl₂, pH 8.0
• Recombinant Human Chymotrypsin C/CTRC (rhCTRC) (Catalog # 6907-SE)
• Trypsin (Sigma, Catalog # T1426)
• Recombinant Human Serpin F2/ alpha 2‑Antiplasmin (rhSerpin F2) (Catalog # 1470-PI) (optional)
• Substrate: Suc-AAPF-AMC (Bachem, Catalog # I-1465), 10 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 rhCTRC to 200 µg/mL in Activation Buffer.
2. Dilute Trypsin to 0.4 µg/mL in Activation Buffer.
3. Combine equal volumes of 200 µg/mL rhCTRC and 0.4 µg/mL Trypsin.
4. Incubate at 37 °C for 1 hour.
5. Optional: Stop Trypsin activity by adding rhSerpin F2 to a final concentration of 4 µg/mL.
6. Dilute rhCTRC to 1 ng/µL in Assay Buffer.
7. Dilute Substrate to 200 µM in Assay Buffer.
8. Load into a black well plate 50 µL of the 1 ng/µL rhCTRC and start the reaction by adding 50 µL of 200 µM Substrate. For a Substrate Blank, load Assay Buffer in place of the rhCTRC.
9. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
10. Calculate specific activity:

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

Per Well:

• rhCTRC: 0.050 µg
• Substrate: 100 µM