I am trying to do an enzyme assay using Wild type enzyme
the protocol involves tris buffer pH 8, MgCl2, S-adenosyl methionine, Substrate, and I inititate the reaction with my enzyme, but after running HPLC, I cant see my product, only the substrate.
I tried to increase enzyme conc, increase incubation time ( From 1 hr to overnight) .. but nothing changes
N.B. I purify my protein using Ni affinity column using buffer containing imidazole .. and then used gel filtration chromatography to remove imidazole
can you suggest to me why the reaction cant happen
Assuming your enzyme is a methyltransferase made in E.Coli and purified by Ni-NTA, does your methyltransferase require it to be phosphorylated to be active? Have you checked whether your enzyme is folded properly? It sounds more likely the enzyme is the problem rather than the in vitro methyltransferase assay
Have you considered that the sample matrix is poisoning your enzyme. Try a positive control with the pure enzyme (no sample)!
Can you try trapping the product in a secondary reaction and then measure that product?
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