Hello , After I have affinity (6x his pet24) purified protein from inclusion body using 50mM tris ph8 , 300mM Nacl , 250mM imidazole and 8M urea I have subjected it to dialysis in 50mM tris pH 7.6 and 150mM Nacl. Initially after 1 to 2 hours of dialysis at 4 degree C I saw some whitish precipitate in the bag. I took samples at 0hr ,1hr and 2hr post dialysis shown in the picture added below , there appears to be loss of or dilution of protein as in the picture added below. Protein is around 16.5kda
What can be the reason also what can I do to stop the same from happening? Kindly help
The protein in 8M urea is most likely denatured. When the urea is removed quickly, the protein may aggregate and precipitate. This may be why your see a loss of protein.
The usual way to renature urea-denatured protein by dialysis is by a more gradual process, dialyzing against decreasing concentrations of urea in several steps. It is also helpful to keep the protein concentration as low as feasible to reduce aggregation.
It is unlikely that all of the protein will be recovered in soluble form even with the more gradual process, and some of the soluble protein that is recovered will probably be aggregated. The refolded, reconcentrated protein should be subjected to gel filtration chromatography or sucrose gradient centrifugation to purify the monomer (or physiologically relevant oligomer) from the aggregate.
Dear Rajarshi Roy
i'm agree with Adam
Unfortunatelly not all the proteins can be succesfully refolded. it depends a lor from protein to protein, and the probability of success decrease on the basis of protein complexity (eg multifdomain proteins, proteins rich of cisteines, proteins that need cofactors for reach the correct folding and of course membrane proteins which require detergents to stay in solution)
Did you have done any trials (eg reducing expression temperature, change e.coli strain, change domain, add fusion tags) to improve protein solubility and avoid redolding?
best regards
Manuele
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