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?
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#2
mdfenko
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Posted 18 November 2009 - 12:53 PM
you could try adding some 2-mercaptoethanol to break up disulfide bonds.
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#3
dxm
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Posted 01 December 2009 - 02:34 AM
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.
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.
#4
braincow
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Posted 03 December 2009 - 08:00 AM
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.
