Yes, it could be the reason that protein concentration is high. The SDS-PAGE shows big blob of purified protein of probable size. I can try rapid dilution method.
Since it is in 8M Urea I have not estimated it yet by any protein estimation assay, but I am sure that protein concentration is high. I wish to know how to proceed after rapid dilution, say 8M Urea to 0.8M Urea in one step and after that? How do I go from 0.8M to 0M urea, here I have to go gradually? or stepwise? or again rapid dilution? This reduced the concentration of the protein. That is not very big problem as I have concentrators. Thanks a lot for help and suggestions. Sanjiv Kumar Lab. No. 411, Functional Genomics Unit, Institute of Genomics and Integrative Biology, New Delhi-110007 India On Tue, May 5, 2009 at 6:40 PM, Shao-Yang Ku <s...@embl-hamburg.de> wrote: > Your protein concentration may be too high, and the stepwise dilution or > dialysis may give your semi-folded protein plenty of chance to aggregate. I > had better luck with rapid dilution of unfolded protein in large quantity of > buffer in the presence of detergents ($$$). (Naturally you would try to use > the cheapest detergent first). After concentrating your protein, you may > recycle your refolding buffer if cost has become an issue. Many poly-ol > compounds in high concentration also suppress aggregation. However, the > absence of aggregates doesn't mean your protein is correctly folded. An > activity assay is essential. Also be aware that some anionic detergents will > precipitate with guanidinium chloride if you choose it as your denaturant. > It also shifts the standard curve of your colorimetric method that you may > use to estimate the protein concentration. > > Good Luck, > > SY > > > Quoting "Sanjiv Kumar" <sanjivi...@gmail.com>: > > 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 >> >> -- Sanjiv Kumar Lab. No. 411, Functional Genomics Unit, Institute of Genomics and Integrative Biology, New Delhi-110007 India
