My protein mainly has alpha helices and very few beta barrel as predicted by PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred/psiform.html). There are no literature available on this protein so I can not be very sure. I have not tried purifying this protein either with detergent or with *Guanidine Hydrochloride. I can try with both *Guanidine HCl and detergent. Since I got good purification with the 8M urea, I was thinking if this protein could be refolded and used further. Isn't removal of detergent more difficult then Urea?
Sanjiv Kumar Lab. No. 411, Functional Genomics Unit, Institute of Genomics and Integrative Biology, New Delhi-110007 India On Tue, May 5, 2009 at 5:27 PM, vinothkumar <[email protected]>wrote: > Is it a beta barrel or alpha helical protein? If it is a beta barrel then > there is lot of information available, I hope that when you were removing > urea you used some detergent and at some concentration higher than CMC > (generally for refolding you should use more 3-4 times CMC) that is because > in urea (depending on conc.) the CMC differs. For alpha helical protein it > is bit more difficult because of their hydrophobic nature, they may require > specific lipids for assembly. There aren't very many examples of alpha > helical protein (with many TM helices) refolded and structure solved. Hope > this helps. > > > Vinoth > > On 5 May 2009, at 12:27, 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 >> > > Vinothkumar K.R. > Structural Studies Division > MRC-Laboratory of Molecular Biology > Hills Road, Cambridge. > CB2 0QH > > Tel: 44-1223-402405 > > > > > >
