Sanjiv Kumar wrote:
> I am working on a membrane protein. There was problem with the normal
> purification of the protein so I have tired to purify it under
> denaturing conditions (Using 8M Urea). Luckily I could purify the
> protein in large quantity. But now the problem is that I am not able
> to refold the protein by step wise removing 8M Urea by dialysis (8M,
> 6M, 4M, 2M, 1M, 0.5M and 0M urea). It is showing aggregation at very
> high urea concentration say 2M urea. Kindly suggest any alternate
> method that I can try for refolding of this protein. It is a
> prokaryotic protein, cloned in pET28a and expressed in BL21 DE3.
> Please Help.
>
> Sanjiv Kumar
Dear Sanjiv,
I guess that the protein is well expressed in the membranes if there is
only (if I could say) a problem with the purification, and that you are
also able to solubilize the membranes and extract the protein with
detergents.
Have you tried several detergents for the purification? Or several
buffers with or without salt?
David
--
David Cobessi
Institut de Biologie Structurale
41, Rue Jules Horowitz
38027 Grenoble Cedex-1, France
Tel:33(0)438789613
33(0)608164340
Fax:33(0)438785122
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