I'm purifying a protein that has a functional cysteine residue at the active site. In the file below, on the left is the SDS of the protein post-IMAC purification. These fractions are then subjected to anionic exchange to further purify the sample.
If you look at the anionic exchange gel, there are four bands whereby two of the bands have secondary bands underneath. The band without the secondary band is the same sample but with addition of 1mM of B-mercaptoethanol.
I know from experience that with addition of reducing my agents, my protein lose functionality and precipitates. I think this secondary band is a fraction of the protein that is oxidised and still not fully denatured, thereby still retaining a partially folded conformation of the protein. Has anyone encountered the same issue with a protein before? I always heat my sample with 95C at 10 minutes prior running the SDS-PAGE.
Dear Liyana,
from the gel it seems to me more likely that the protein is degrading: the gel is denaturing your protein (it contains SDS), probably your loading buffer has SDS in it, additionally you could boil you samples and make sure it is all denatureted. However the best way to find out, is to bring your sample to mass spec. That will definitively tell you what is happening!
Good Luck, Dom
Hello Domenico,
Thank you for your suggestion. I have boiled my samples thoroughly and I will definitely consider doing mass-spec (its really expensive so I'll put that option last!)
Hello Liyana,
I also got two bands for one of the protein and proceeded with mass spec (as suggested earlier) and found that upper band was methionine oxidized. This could be a probable reason in your case as well.
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