| Assay Method Information | |
| | Acetylcholinesterase Inhibition Assay |
| Description: | The assay was carried out according to the Ellman method [Ellman et al., Biochem. Pharmacol., 7:88-95], optimized for 96-well microplates using a Tecan Sunrise™ absorbance reader. The initial reaction velocities were measured at different concentrations of the tested compound solution and fixed ATCI concentrations (0.062-1.25 mM). The reaction mixture contained 50 μL of 0.26 U mL^−1 AChE, 25 μL of 7.6 mM DTNB, and 20 μL of the tested compound solution together with an aliquot volume of 6.24 mM ATCI solution in order to obtain the required concentration of ATCI in the final volume (250 μL) of reaction mixture, and 0.1 M phosphate buffer pH 8.0 in order to achieve the final volume of reaction mixture. For the blank, 50 μL of 0.26 U mL^−1 AChE was replaced by the same volume of 0.1 M phosphate buffer pH 8.0. In a typical assay, solutions of AChE, DTNB, and the compound to be tested, as well as the phosphate buffer, were mixed and incubated for 30 min at 30°C. Next, the reaction was started by the addition of ATCI solution. The increase in absorbance at 412 nm was measured every 11 s for 220 s. Reaction velocities were determined from the slopes of the linear portions of the graphs of time versus concentration of 5-thio-2-nitrobenzoate. All assays were done in triplicate. |
| Affinity data for this assay | |
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