Hi there,

You should monitor absorbance spectrum in time in order to check if the increase of absorbance is due to a peak formation (NADH) or shift of the spectrum to higher absorbance values (aggregate formation).

Okay, Thanks Dominique!

Some thiol-based protein/peptide such as GSNO or other S-nitrosothiols have similar absorbance at 340 nm, which might contribute to the increase of 340 nm AB reading.

 Thanks Sun, for my case, it seems that something was bound to the protein during purification. The AB increase tends to diminish after 30min incubation with NAD, after that the AB increase again when I add my substrate.

Thanks Valesca,

I will follow your suggestions. Do you think imidazole could interfere with the 340 absorbance? I am worried that my protein sample indeed contain imidazole, have got similar experience?

same here. i experienced an increasing trend wherein i am expecting a decreasing trend with my enzyme assay of my purified his-tagged protein. I removed the imidazole by dialyzing my eluted protein with the same buffer except that it has no imidazole in it (elution buffer contains imidazole; dialyzing buffer has the same composition with elution buffer except imidazole).. I dialyzed my purified protein for 24hours, still there was no activity at all while it sometimes increase.. so i just tried the crude lysate that showed activity.

 @Rhudith   I don't know about your case, but with mine the background activity (without adding substrate) seems to be an enzymatic process. while I still have no idea why the abs increases, I do have experiences that enzyme loses activity gradually with dialysis. some people suggest dialyzing the protein immediately after elution for less than 12h at 4 degree, haven't got time to try it.