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
