I have a protein forming a very stable dimer in solution, but still running as monomer in SDS page. I have tryed to disrupt the dimer with Beta-ME and high salt, but it is still running as dimmer in analytical gel filtration. Do you known other chemicals that I could use??
regards and thanks in advance.
Dear Juan Pablo,
DTT is also a reducing agent that, like B-ME, reduces the disulfide bridges. Additionally, you could use urea in your buffer and boil the solution 5 minutes prior to loading it onto your SDS-PAGE.
Works well with collagen triple helices.
Good luck!
Hi Jeroen, thanks for your help. The problem is not in the gel, in the gel i have the monomer, but i would like to get the monomer in solution, and that its run like a monomer in the FPLC. Do you now some additive to achieve this??
regards
Dear Juan Pablo.
Why do you need run it as monomer? I mean, Do you need that it retain it rolfind? Because in SDS-page you completelly destroid the protein folding. If you would like to do someting similar in solution you can use UREA6-8M + DTT and perfom the size exclusion in this buffer but you will completelly lost the protein folding.
On the contrary, If you want to maintain the folding but just destabilize the dimer you can certanilly try to change other parameter as pH for example or use weak detergents, but the result is not obviuous. You can also induce unfolding and precipitation. The structure of this protein is already known?
Because if the structure is known, you can try to desing mutation to reduce the protein protein interaction at the surface and obtain a stable monomeric protein form as was done for Cu.ZN sod1 (Article The crystal structure of the monomeric human SOD mutant F50E...
) but of course it is something that need time and efforts.
good luck
Manuele
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