I want to see how my test compound affects ALDH activity. However, I am trying to do it without using any kit, such as Aldefluor or PicoProbe. Anyone willing to share their protocol? Thanks much!
The reaction of aldehyde dehydrogenase with an aldehyde results in reduction of NAD+ to NADH. This can be monitored as an increase in absorbance at 340 nm using a spectrophotomer or absorbance plate reader, or an increase in fluorescence (EX 340 nm, EM 460 nm) using a spectrofluorometer or fluorescence plate reader. You supply the aldehyde, NAD+, and ALDH in a suitable buffer. Check the literature on your enzyme to identify a suitable buffer and to find the substrate Kms.