Recombinant Human Carbonic Anhydrase IV 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: 12.5 mM Tris, 75 mM NaCl, pH 7.5
• Absorbance Buffer: 12.5 mM Tris, pH 8.0
• Recombinant Human Carbonic Carbonic Anhydrase IV/CA4 (rhCA4) (Catalog # 2186-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 rhCA4 to 40 ng/µL in Assay Buffer.
2. Dilute Substrate to 2.0 mM in Assay Buffer.
3. Load 50 µL of 40 ng/µL rhCA4 into plate and start the reaction by adding 50 µL of 2.0 mM Substrate. As a Substrate Blank combine 50 µL Assay Buffer with 50 µL Substrate.
4. Cover and incubate at room temperature for 60 minutes in the dark.
5. After incubation add 100 µL Absorbance Buffer and read plate immediately.
6. Read (top read) absorbance in endpoint mode at 410 nm.
7. Calculate specific activity:

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

• rhCA4: 2 μg
• Substrate: 0.5 mM