NAD+/NADH enzyme cycling assay in plants--HELP - enzyme assays (Sep/29/2006 )
I've been developing/modifying an assay to quantify NAD+/NADH levels in A. thaliana leaf tissue. I have the protocol attached, it's based on a variety of published acid/base extraction and detection protocols. The idea behind the assay is that you use alcohol dehydrogenase, which uses NAD+ as a cofactor; as NAD+ is converted to NADH, phenazine ethosulfate (an oxidizer and electron acceptor) uses NADH to oxidize a yellow tetrazolium compound into a blue formazan. the NADH is thus converted back to NAD+ and the enzyme cycle starts over, and thus the amount of NAD/NADH in the sample determines how much tetrazolium (yellow) is converted to formazan (blue).
My problems are mainly two-fold: 1) incredibly high variablity between separate samples from the same plant, 2) very high variability between experiments from day to day (in terms of NAD+/NADH ratios). Now, I do have very good reproducibility and low variability within each sample (ie, if I take an extract and divide it up and perform parallel reactions on each of them, the results will be the same), so I trust the assay/my lab hands. So what's the deal? is this just a very finicky assay and NAD+/NADH levels can just vary substantially from leaf to leaf? Has anyone else worked with this assay and have any advice/pointers? Thanks much!
Interference in this assay may come from any species that oxidises the tetrazolium. What controls are you using? I think you can cut out the tetrazolium stage and simply assay NAD/NADH changes directly. Use 340nm. How long does it take to prepare each leaf? In human cells there can be a problem with cell organelles lysing and releasing molecules which go on to falsley elevate outcome measures.
Thanks for the information. My leaf prep involves snap freezing them in eppi tubes in liquid N2, then grinding them with a plastic pestle, keeping it frozen on the N2. Once the tissue is ground I immediately add the acid or base for extraction. I'll give the 340nm idea a try.