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| | Ellman Assay |
| Description: | The modified Ellman assay for inhibition of acetylcholinesterase (AChE) and the mouse ear vesication assay (MEVA) have been described in detail by us (see S. C. Young et al, J Appl Tox, 2012, 32: 135-141). AChE (Type V-S from electrophorus electricus), acetylthiocholine iodide (ATChI), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and tacrine from EMD Chemicals (Gibbstown, N.J.). Cholinesterase inhibition was assayed spectrophotometrically at 412 nm according to Ellman's method. Assays were performed in polystyrene 96-well plates (Corning 96-well flat transparent) and a conventional micro-plate reader was employed for kinetic readings (Tecan Infinite 200 multimode). The following reagents were added to the wells: 200 μL of 0.5 mM DTNB in sodium phosphate buffer (100 mM, pH 8), 30 μL of inhibitor stock solution in methanol, 20 μL of 1.25 units/mL of AChE in sodium phosphate buffer (20 mM, pH 7), and 50 μL of 3 mM ATCh in buffer (100 mM, pH 8). Immediately aft |
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