hi to all,
i have a stock solution of an ab amyloid peptide previously treated according to the standard protocols. this solution has been stocked at -20°C for a couple of years, and the peptide is no more in a monomeric state and is not properly folded. i was wandering if it is possible to restore the monomeric state and the correct folding with such treatment. unfortunately all the protocols i found are for the liophylized peptide, and not for a peptide solution.
thank you
Did you try treating the solution with denaturing agent viz, urea/Guanidinium hydrochloride or so and then refolding by treating with favorable buffer solution. I guess this might help you to get the correct folding state, however I am not too sure it will help you to get the monomeric state. In order to get the monomeric state, you might have to dilute your sample a lot and then do the refolding followed by HPLC/FPLC purification.
Hi Antonio, sorry, but if you were storing the samples at -20oC in a frost-free freezer then I'm afraid they are gone forever.
As a general rule for storing conformationally sensitive proteins. Store them at -80oC, store them lyophilized or don't store them at all.
The protein by this time is almost certainly in the aggregated state. You could try to recover it by:
The reverse phase HPLC step is important at this point since you could no longer assume the protein is chemically pure. Met35 is likely oxidized and it is possible the peptide has undergone other modifications during the long storage period, such as deamidation, which can have a strong impact on aggregation kinetics and structures. [1,2]
1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3410046/
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2373442/
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