Good morning everybody.
I am purify MsrA from E. coli. I first run an anion exchange and now I am running a Phenyl sepharose (HIC).
the buffer I am using is 20mM MOPS pH7 1.2M Ammonium sulphate 5mM DTT for equilibration and 20mM MOPS pH7 5mM DTT for elution.
The running stopped last evening due to overpressure alarm, so the protein stayed on the column overnight.
Now I am eluting and the sample was passing through the column with an intense brown color.
Does someone know why this could be? I have looke online but explanations are only about reduced nickel column.
Thanks in advance and have a nice day!
Did you filter your sample on 0.22 µm cellulose membranes?
Big particles might be clogging the column and triggering over pressure alarm.
The overpressure was due to aggregates, this I know, since at first the sample aggregated due to too high ammonium sulphate concentration; I then re-resuspend the sample in lower amount of ammonium sulphate and the solution was clear, but probably part of it wasn't in solution.
Due you think the brown colour could be related to this?
Is it possible that MsrA is binding a metal? This could cause the coloration, or perhaps even aggregation. You could try adding EDTA or some other chelator.
I agree with Walter. Possibly you have a metal in the sample that causes DTT oxidation causing the brown color of your sample. EDTA should help.
Be cautious with EDTA, if the metal bound is important for maintaining conformation of your protein then you would potentially cause it to precipitate as it unfolds. Even if it is not important for conformation it could be important for the function of the protein.
What were your anion exchange buffers and how did you buffer exchange into your HIC buffers?
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