Dear Maayam!

Try acetylcholine chloride

Article Assessment of Acetylcholinesterase Activity Using Indoxylace...

4.2. Ellman’s Method for AChE

Ellman’s method was done with slight modifications of the reference [26,27]. The chemical principle is depicted in Figure 5. A disposable cuvette was consequently filled with 0.4 mL of 0.4 mg/mL DTNB, 25 μL of AChE solution (0.5 μkat in 1 mM acetylthiocholine), 425 μL of PBS, 50 μL of paraoxon in isopropanol or isopropanol alone. The reaction was started by adding 100 μL of acetylthiocholine chloride in a given concentration for assessment of Km and Vmax or 1 mM for toxicological and pharmacological investigations. Absorbance at 412 nm was measured immediately and after one minute. Enzyme activity was calculated estimating extinction coefficient ɛ = 14,150 M−1cm−1. The oxime drugs were tested in a similar protocol. 425 μL of PBS was reduced to 325 μL of PBS. Paraoxon was added in concentration providing 95% inhibition of AChE. Incubation time was set to 10 minutes. After that, 100 μL of oxime reactivator suspended in PBS was injected into the cuvette and kept for another 10 minutes. The reaction was started again by addition of acetylthiocholine.

Ellman's reagent detects thiol groups. To use this reagent to detect the activity of acetylcholinesterase, you must use acetylthiocholine as the substrate, not acetylcholine.

Article Anticholinesterase Efficacy of Bacopa monnieri against the B...

Please read above mentioned paper of ours for details.

If you are using Ellman method for measurement of AChE activity,

First dilute Homogenates with 2.6 ml 0.1 M Sodium phosphate buffer (pH 7.4) to which add 100 mM DTNB and 75 mM Acetylethiocholine Iodide (ATChI, substrate) Then measure the rate of color production at 412 nm in UV-VIS spectrophotometer.

Also you need to standardize the protocol.

Adam B Shapiro Thank you for your answer. when you dissolve the acetylthiocholine do you prepare a stock solution?

Yes. You can dissolve it at 10 mM in the assay buffer.