I'm currently writing my bachelor thesis and I have a problem with the reaction between TMB (3-3´-5-5´-Tetramethylbenzidine), H2O2 and acetylthiocholine. At this stage I'm not yet adding the enzyme. According to various papers, a blue coloration should already occur after 7 minutes, but in my case it takes several hours. I use a phosphate buffer (pH 7.4) and a citrate-phosphate buffer (pH 5). After adding acetylthiocholine iodide in different concentrations and the phosphate buffer, the plate is incubated at 37°C for 15 minutes. After incubation, the TMB-H2O2 solution is added and measured on the ClarioStar for 60 min.
Does anyone have experience with this assay and has some answers for me?
Thank you in advance.
If the concentration of any of the ingredients is lower than it is supposed to be, the reaction will take longer than expected. The most likely problem would be with the hydrogen peroxide, which is unstable. If it is an old batch, then you might get better results with a fresh batch.
Hi.
The blue colour occurs due to oxidation of TMB by H2O2 in presence of ATCI. In your case, the oxidation is not fast enough meaning either the quality or quality of H2O2 is inadequate. You can try increasing the amount of freshly prepared H2O2.
Again, from personal experience, Ellmann assay is the best and easiest to study enzyme kinetics of AChE.
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