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| | Carbonic Anhydrase II Inhibition Assay |
| Description: | In this assay, 4-nitrophenyl acetate (4-NPA), a colorless compound, was hydrolyzed to 4-nitrophenol and CO2. The reaction was followed by measuring the formation of 4-nitrophenol, a yellow colored compound. The reaction was performed at 25 °C inbuffer containing HEPES and Tris-HCl at a total concentration of 20 mM and pH of 7.4 for each sample. The reaction mixture contained 140 μL of the HEPES-tris solution, 20 μL of freshly prepared aqueous solution of purified bovine erythrocyte CA-II (0.1 mg/mL of deionized water for 96-well), 20 μL of test compound in DMSO (10% final concentration), 20 μL of substrate 4-NPA at a concentration of 0.7 mM diluted in ethanol. The reaction was initiated by addition of 4-NPA after 15 min incubation of test compound, and each compound was tested 3-times at different concentrations. In this assay, the reaction was performed using 96-well plates. The plate was placed in a spectrophotometer and the amount of product formed was monitored at a 1 min interval for 30 min at 400 nm [Arslan et al., Biochemistry, 66:982-983]. |
| Affinity data for this assay | |
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