Incomplete denaturing with 8M urea? - (Nov/13/2009 )

Does anyone have any experience with proteins that will not denature in 8M urea? It appears I have several ORFs that are expressed but when I lyse with 8M urea they stay with the pellet. I don't need native structure. I purify on a Ni-NTA column. Any suggestions on coaxing them out?

you could try adding some 2-mercaptoethanol to break up disulfide bonds.

1. beta-mercap
2. + SDS
3. +SDS+beta-mercap+boil for 10min (like what u do in SDS-PAGE)

I think boiling will destroy the native structure, if it do not totally destroy the protein.

If you're going to boil it, don't do it in urea lysis buffer unless you don't care about carbamylation. You could try using a urea-thiourea lysis buffer if your ORF is fairly hydrophobic.