Dear Raji
the detergent micelle increases the hydrodynamic radius of a membrane protein and therefore affects the behavior on SEC. The following papers show examples how the molecular mass and therefore oligomeric state of membrane proteins can be determined by SEC: Slotboom, D. J., Duurkens, R. H., Olieman, K., and Erkens, G. B. (2008). Static light scattering to characterize membrane proteins in detergent solution. Methods 46, 73–82. Kunji, E. R. S., Harding, M., Butler, P. J. G., and Akamine, P. (2008). Determination of the molecular mass and dimensions of membrane proteins by size exclusion chromatography. Methods 46, 62–72.
Regards
Florian
Date: Thu, 21 Jun 2012 11:50:09 -0400
From: Raji Edayathumangalam <[log in to unmask]>
Subject: Detergent and protein oligomerization
Hi Everyone,
Sorry for the non-CCP4 post.
I have a very basic question about detergents, critical micelle
concentration and behavior on gel filtration.
A 33kDa membrane protein was purified by gel filtration in a buffer
containing 0.4%(w/v) beta-NG (CMC: 6.5mM) and 0.046%(w/v)LDAO (CMC:
0.14mM). So the concentrations of beta-NG and LDAO in the gel-filtration
buffer are ~2X and ~14X that of the CMCs of the respective detergents. The
elution volume of the protein peak (plus detergent) on Superdex200
corresponds to a molecular mass of 100kDa.
I think that the 100kDa mass above includes contributions from both the
protein as well as the detergent micelles. If this is correct, is it then
accurate to try to glean the oligomerization state of the protein (and
conclude that it is a trimer or tetramer) without taking into account
detergent micellar mass and its influence on elution volume?
How should one interpret the 100kDa mass estimate from the gel filtration?
Thanks.
Raji
--
Raji Edayathumangalam
Instructor in Neurology, Harvard Medical School
Research Associate, Brigham and Women's Hospital
Visiting Research Scholar, Brandeis University
-------------------------------------------------------------
Dr. Florian Brückner
Biomolecular Research Laboratory
OFLG/102
Paul Scherrer Institut
CH-5232 Villigen PSI
Switzerland
