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| | IC50-Activity Assay (AKR1B10) |
| Description: | The IC50-activity assays were carried out on the basis of the quantification of the NADPH consumption that takes place when the enzyme catalyzes the conversion of the aldehyde substrate into the alcohol product. The assays were performed at 25 °C in a 100 mM sodium phosphate buffer (pH 7.0), with the protein amount reaching the Vmax, and 0.2 mM NADPH. The final reaction volume was 600 μL per reaction. All compounds were dissolved in dimethyl sulfoxide (DMSO), and the corresponding solution was added to the cell to be assayed in a final concentration of 2% (v/v) DMSO and incubated for 5 min at 25 °C prior to the addition of the substrate. The reaction was initiated by the addition of 60 mM glyceraldehyde and 0.2 mM pyridine-3-aldehyde, and the decrease in opticaldensity at 340 nm was monitored for at least 3 min at 25 °C in a UV−vis spectrophotometer (Cary 400 Bio (Varian)). |
| Affinity data for this assay | |
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