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
