I have tried to make reaction of Butyrylcholinesterase with it's substrate Butyrylcholine Chloride in 96 well-plate. I have added 150µl of 100mM sodium phosphate buffer (pH 8.0) with 30µl of enzyme (at various concentration: .3.5 to 8 U/mL) and 10µl of DTNB (0.5 mM). After thorough mixing, I started the reaction by adding 10µl of substrate (at various concentration: 0.2 to 20mM) and then incubated at 37°C (as suggestion of Sigma) and another one at 25°C (as follow some paper) for 30 min. But The reaction did not take place by monitoring the formation

Of the yellow 5-thio-2-nitrobenzoate anion as a result.

That condition have been done successfuly with Acetylcholinestarase.

Do you have any comment or suggestion for me?

Thank you very much!