Carbonic Anhydrase IX (CA9) also known as membrane antigen MN and renal cell carcinoma (RCC) associated antigen G250, is a transmembrane enzyme expressed primarily in carcinoma cells. It is one of the best markers for hypoxia and for RCC. rhCA9 corresponds to the extracellular portion of human CA9. It catalyzes the reversible reaction of CO2 H2O = HCO3- H , which is fundamental to many processes such as respiration, renal tubular acidification and bone resorption. The recombinant human CA9 activity was measured by its ability to hydrolyze 4-Nitrophenyl acetate (4-NPA) to 4-Nitrophenol. The reaction was performed in 12.5 mM Tris, 75 mM NaCl, pH 7.5 (Assay Buffer), initiated by addition 50 μL of various concentrations of CA9 (diluted by Assay Buffer) to 50 μL of 2 mM Substrate 4-NPA (100 mM stock in Acetone, diluted by deionized water). Incubated at 37℃ for 5min, then read at a wavelength of 400 nm.