Recombinant Mouse Carboxypeptidase B1/CPB1 Protein, CF

• Activation Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
• Assay Buffer: 25 mM Tris, 0.1 M NaCl, pH 7.5
• Recombinant Mouse Carboxypeptidase B1/CPB1 (rmCPB1) (Catalog # 2898-ZN)
• Trypsin (Sigma, Catalog # T-1426)
• AEBSF (Catalog # EI001), 100 mM stock in DMSO
• Substrate: Hippuryl-Arg (Sigma, Catalog # H-2508), 25 mM in diH2O
• 96-well Clear UV Plate (Corning, Catalog # 3635)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Activate rmCPB1 at 100 µg/mL with 1 µg/mL Trypsin in Activation Buffer.
2. Incubate at room temperature for 30 minutes.
3. Add AEBSF at a final concentration of 1 mM to stop reaction.
4. Dilute activated rmCPB1 to 2 µg/mL in Assay Buffer.
5. Dilute Substrate to 2 mM in Assay Buffer.
6. In a plate, load 50 µL of 2 µg/mL rmCPB1, and include a Substrate Blank containing 50 µL Assay Buffer.
7. Start the reaction by adding 50 µL of 2 mM Substrate into wells.
8. Read in kinetic mode for 5 minutes at an absorbance of 254 nm.
9. Calculate specific activity using the following formula:

     *Adjusted for Substrate Blank
     **Derived using calibration standard Hippuric acid (Sigma Catalog # 112003).

Per Well:

• rmCPB1: 0.1 µg
• Substrate: 1 mM