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Hi Jenny, I think your problem is caused by a high protein concentration. Since your protein is soluble before the His column (which concentrates the protein). Try to find a stabilization buffer, add glycerol or change pH. Another crucial parameter could be the redox environement. Which will be difficult to test because you use a his tag. Change the tag to GST, you can then use 10mM DTT in your buffer. I would not recommand the use of guanidine, it will unfold your protein... Good luck Alex Jenny <[EMAIL PROTECTED]> a écrit :
Hi All, I'm trying to purify a protein which is expressed in soluble form.It's a His-tag protein, so when I run His-column, it's really easy to clog.In the next step size exclusion purification, the protein come off at the void volume, which suggests that the protein is severely aggregated.Does any one have any suggestions that I can modify the purification condition so as to avoid aggregation problem?Or can I add GuHCl during His column purification so that it won't clog so easily?Thanks a lot. Jenny
