I'd like to perform a SEC step prior to on-column refolding of a protein I am expressing/purifying but am worried about the extended time the protein will be in the presence of urea (and subsequent protein carbamylation) in the current refolding workflow I have.
Is there any concern of protein modification with a 4-6 hour denatured protein purification workflow using 6M GndHCl?
It is suggested to perform the purification relatively cold environment to keep the protein intact and prevent any non-desired PTM...Extended periods or elevated heat can lead to carbamylation if the chaotrope is selected as urea. However, I guess, isocyanic acid is not derived from guanidin and the expected reaction is not occur...You need a reactive carbonyl moiety to do N-terminal protein modification, and guanidin cannot serve this.
There is considerably less danger with both GuHCl and GuSCN. Sometimes urea is required, in grad school I needed to do fully denatured AEX, so you want very fresh urea solutions and can remove the isocyanic acid prior to use. If salt isnt to be avoided in your urea, ammonium bicarb can minimize any associated carbamylation. Good luck!
- Badges
- Science topic
- Similar topics
- Comment