Doing fluorescence analysis, I've identified a peak related to a NADH-dehydrogenase complex, but I don't know which type of dehydrogenase is. Is there any kit I can use for this? Or any other protocol?
Do you fractionate as a part of your protocol? If so can you see the protein(s) on a gel? Is there more than one? My immediate inclination is to MS/MS the fraction, identify proteins and look at their functional annotation to see if there is any NADH dependent anzyme present, or BLAST search with the MS/MS sequence to find an annotated homologue from another organism
Do you fractionate as a part of your protocol? If so can you see the protein(s) on a gel? Is there more than one? My immediate inclination is to MS/MS the fraction, identify proteins and look at their functional annotation to see if there is any NADH dependent anzyme present, or BLAST search with the MS/MS sequence to find an annotated homologue from another organism
The sample is from a purification process, and there are more proteins from the same microorganism. As I've found in literature, the peak I see with fluorescence analysis is related to a NADH-dehydrogenase complex. I'll run a gel for gathering more information. Thank you.
It would be best if you could purify the protein to some extent by following the fluorescent peak. Depending on the organism, it could be something like alcohol dehydrogenase, or some other dehydrogenase that is involved in making food for the organism. So I would focus on what type of organism, and what was its food supply. There are so many enzymes that use NADH, it could be just about anything, but the most highly expressed ones, that you may actually be able to visualize right away with coomasie staining are probably housekeeping enzymes, like those involved in glycolysis, etc. and where the activity resides can maybe help as well. Is it in mitochondria, or is it in membranes., etc etc.
If the sample is pure, you can set up maybe a 96 well plate, and add different substrates for dehydrogenase such as alcohol, pyruvate, citrate, etc as many as you can think of, and then look for a fluorescence change, or add NADH and follow A340 changes. It the enzyme consumes the substrate, the fluorescence or absorbance should change.
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