I wonder whether the stability of acetoacetyl-CoA might be improved by using pH 7.0 instead of 8.0.

Good afternoon dear, acetoacetyl-CoA was very important for brain, so it's must be with a special conditions such as temperature 37, Ph 7.

I tested different ph for my assay and figured basic ph worked better 8.0 or 8.4. The enzyme activity is important too. I had low detergent for solubility too. I think it was 0.1% triton.

I wonder whether you are noticing basic issues and not very specific ones.  I will list them in no particular order and I hope the list will help

- why NADH instead of NADPH?

- Buffer (Tris) is the right one according to literature ?

- AcetoacetylCoa makes anything strange if solved in tris and with NADPH? I Assume that you buy it from a "good" vendor and nothing happens in Tris plus NADPH, i.e. no unstability should be noticed when you mix these two compounds in the appropriate inert buffer the absence of any cell extract.

- If everything above goes well  you should be careful with the extract you use from Polyphosphate-accumulating organisms. First of all you must remove all small compounds from the extract (use a PD10 dessalting column) and be sure you use the fraction in which the reductase should be in these organisms (not familiar to me)

- If still you notice strange things please tell details and with these new details I may have more suggestions .

Thanks everyone for your suggestions! I tried the enzymatic assay at pH of 7.0 and 8.0. However, 8.0 showed less degradation than 7.0. Measuring at 30°C seemed to have improved the AcAcCoA stability.