Recombinant Human Carbonic Anhydrase III 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: 12.5 mM Tris, 75 mM NaCl, pH 7.5
• Recombinant Human Carbonic Anhydrase III/CA3 (rhCA3) (Catalog # 2185-CA)
• Substrate: 4-Nitrophenyl Acetate (4-NPA) (Sigma, Catalog # N8130), 100 mM stock in acetone
• 96-well Clear Plate (Costar, Catalog # 92592)
• Plate reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute rhCA3 to 400 ng/µL in Assay Buffer.
2. Dilute Substrate to 2.0 mM in Assay Buffer.
3. In a plate load 50 µL of 400 ng/µL rhCA3, and include a Substrate Blank containing 50 µL Assay Buffer.
4. Start the reaction by adding 50 µL of 2.0 mM Substrate to wells containing rhCA3 and Assay Buffer.
5. Read at a wavelength of 400 nm (bottom read) in kinetic mode for 5 minutes.
6. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard 4-Nitrophenol (Sigma, Catalog # 241326).

• rhCA3: 20 µg
• Substrate: 1.0 mM