Hi,

This could be due to various reasons such as temperature, concentration of NAD, enzyme and/or substrate. To begin with you could alter these parameters one at a time and check the absorbance.

Considering that the extinction coefficient change for the conversion of NAD(P) to NAD(P)H is 6.22/(mM-cm) and a minimal absorbance change for a good signal is around 0.1, you can calculate the optimal initial concentration of NAD.

Possible reasons why an enzyme activity assay's absorbance at 340 nm decreased are as follows;

a) Inaccurate sample preparation: Improper dilution, contamination, or sample degradation can cause the absorbance at 340 nm to decrease.

b) Instrument error: Problems with the spectrophotometer's technical components, such as damaged cuvettes, grimy lenses, or misconfigured settings, can affect the precision of absorbance readings.

c) Incomplete reaction: If the reaction is prevented from finishing, less product will be produced, resulting in a smaller decrease in absorbance.

d) Enzyme inhibition: When inhibitors or competitive inhibitors are present in the reaction mixture, the enzyme's activity is reduced, resulting in a decrease in absorbance at 340 nm.

e) Reduced absorbance at 340 nm can be caused by non-specific binding, which occurs when the substrate or product binds in an unintended way to other elements in the reaction mixture.

Finally, review each step of the assay, including sample preparation, instrumentation, reaction conditions, and reagents.

Dear Ethan Matthews ,

I think another thing you should consider is that not all plastic ware is working at low wavelength. You might want to test this. If applicable make a lamda scan with your plate reader and an empty plate.

You might want to use in this case Glas bottom plates or UV-Star plates.

good luck

Soenke