Hey guys, I'm working on protein that usually expresses well. Previously I've been getting it to express completely soluble and in milligram quantities, however lately I've been having problems getting soluble protein. I use His-tag purification first and then size exclusion and up until now I've had no problems. However the last few weeks I've been getting extremely low yields or the protein seems to be being expressed in insoluble form.
When I elute at the his tag stage of the purification I see a nice defined peak, but when I put the fractions through size exclusion the protein elutes as one continuous mass or a giant peak in the void volume. Dynamic light scattering indicated that these fractions were aggregates.
At first I thought the column might be the problem as it previously went over pressure and this could have affected the bedding, but I see no gap in the column, nor does there appear to be any air bubbles present. I've cleaned the column with 0.5 M NaOH and there appeared to be considerable amount of protein coming off.
Could there be any problems with the column I'm not thinking about? One other user has tried to put down a similar size protein and has had problems.
Best wishes
Harry
You've convinced me that the problem is with the column. Try running a standard protein mixture over it to see if it is working properly. If it isn't, it may have to be repacked or replaced.
HI Adam, thanks for your answer. Is it likely there is a gap in the column even if the pressure is normal and the Akta trace looks smooth? We will be trying another protein tomorrow.
Try with a known protein or mixture as Adam suggests, if as you say you noticed protein coming out with the washing it is likely that you need to clean the column further.
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