My protein is stable in 150mM NaCl and 20mM Tris. I tried refolding it by dialysis using this buffer but it didn't work.
Is your protein expected to have disulphide bonds? If so, you should include some redox agents such as a mixture of reduced/oxidized glutathione. This will help the formation of s-s bonds. However the ratio of the two components needs to be optimized since it depends on the redox potential of the particular disulphide bond(s)
Farkas has rightly suggested you... However commercially a lot of kits are available such as Protein Folding Kit from Novagen or Foldit from Hampton Research. However it is suggested that Urea should be partially or completely removed from the system by either dialysis or diluting and then concentrating your protein sample via 30KD membrane which will remove most of the denaturant in permeate whereas retaining your 60KD protein in the retentate.
Hi,
I would recommend browsing through a large collection of protocols for refolding hundreds of proteins here:
http://refold.med.monash.edu.au/
Unfortunately the site was hacked some time ago, so you can only access it with limited functionality. At least you can get the protocol details for all the proteins in the database.
Btw, how do you know that you're protein is stable in this buffer? Do you have native protein as reference? Is there an alternative expression system that you could try? If yes, I would suggest trying a eukaryotic system.
This paper from Yves Muller's group reports a nice method to screen for refolding conditions; basically, they employed limited proteolysis to identify which conditions were suitable to refold proteins:
http://peds.oxfordjournals.org/content/14/3/183.full
The question suggests there is a simple answer around, but it isn't. I agree Heide and Deepti who are both right. I suggest a refolding screen but save the money and prepare it by your own: http://www.ncbi.nlm.nih.gov/pubmed/20851186.
You can screen for aggregates or activity as you like.
for a first shot try this (rapid dilution instead of dialysis):
1) reduce disulfides with 5-10fold excess TCEP
2) exchange buffer to remove urea (100 mM phosphate and 6M guanidium chloride, pH 6)
3) concentrate up to 5-10 mg/ml
4) pulsed rapid dilution: final protein concentration 50 µg/ml (add protein solution slowly dropwise in 5-10 steps to the refolding buffer under gently stirring); refolding buffer (100-200fold volume of protein concentrate) is the Tris buffer you mentioned. If your protein contains disulfide bridges, add a redox system. caution: the drops must disrupt intermediately. take your time. All solution must be particle-free.
If it doesn't work screen for a refolding buffer and apply it to the procedure above.
Good Luck!
You can try using a stepwise dialysis (reducing the urea concentration gradually, instead of all at once). Also, do you know the pI of the protein? you could adjust the pH to allow more or less charge on your protein (depending if the aggregation you're seeing is an electrostatic effect or hydrophobic collapse).
Alternatively you could either co-express with chaperone proteins (or lower the temperature) or add a known binding protein during dialysis to induce proper folding.
Dear Valentina
Your comment helped me very much to refold my denatured protein following dialysis
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