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 <r...@brandeis.edu> 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 Tel.: +41-(0)56-310-2332 Email: florian.brueck...@psi.ch