I have 5mM beta mercaptoethanol in my elution buffer used for protein purification. Will it interfere with an assay monitoring NADH to NAD+ oxidation spectrophotometrically? I am working with a dehydrogenase enzyme and measuring OD at 340 nm.
I dont think it should. rather it might be helpful. once I was working with a dehydrogenase with cysteine as nucleophile in the active site. I have to use a reducing agent to keep the protein active.
If you are worried about bME reducing NAD, then bME can nor reduce NAD.
BME oxidized quickly, when exposed to air; so, after a few days its reducing effect is gone and only its stink remains. BME's redox potential at pH 7 is -0.26V.
Be careful though with DTT; its is the far better reducing agent, with a redox potential of -0.33V @ pH 7 and it does not oxidize when exposed to air.
Hi Tom
Could you please explain the protocol you followed ?
I want to characterize my purified protein as an oxidoreductase using NADPH .
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