On Mon, Aug 13, 2012 at 10:04 AM, Sangeetha Vedula <[log in to unmask]> wrote:

Hello all,

I am working with a protein that is probably a hexamer based on homology with other proteins but when I ran it on an analytical size gel filtration column, I see multiple peaks. I would like to determine the exact oligomerization state of the mixture and have considered blue native gel electrophoresis and gradient centrifugation. The monomer is about 54 kDa. All of the peaks are active enzymatically. I wish I could test higher concentrations to see if the equilibrium will shift towards a hexamer (or whatever the naturally highest state is) but the protein crashes out.

The protein was expressed in baculovirus and the multitude of states could be an artifact but I'd like to know what I am working with. Also, down the line, I'll need to pick one of the states for crystallization trials.

Has anyone encountered this problem? How did you determine the oligomerization state(s)?

I have basic biorad gel apparatus available and no gradient mixer.

Thanks for any input.

Best regards,

Sangeetha.