[RASMB] AUC question

[HGSR (Holger Martin Strauss)]

Hi Leslie and all,

just one more thought: as Patrick has said, the reliable outcome of a sedimentation velocity experiment is the sedimentation coefficient. Assigning molar masses to sedimentation coefficients can't be straigthforward, because of the known ambiguities of hydrodynamics and using f/f0 probably is a poor choice because its primary function is to deconvolute the boundary spread due to diffusion from that due to polydispersity. The reason why f/f0 should be floated is to get a better description of the data (rmsd) and sharper peaks, NOT to get accurate molar masses. If you float f/f0, it will be an average parameter, anyways, and hence the molar mass will be an average as well. Moreover, because a floated f/f0 is sensitive to the shape of the boundary, any distortion of the boundary (convection, bad centrepiece, non-ideality, self-association, etc.) will have an impact on f/f0 - but not so much on the sedimentation coefficient.

If you need to determine the molar mass of your main component(s), you might be better off to switch to the diffusion coefficient domain using a two-dimensional deconvolution in conjunction with multiple running speeds or a split discrete/c(s)-model or to use partial boundary modelling with Sedfit or dc/dt-kind of methods. This will "only" leave you with using a correct vbar for the Svedberg-equation, but this way at least the determined parameters themselves will be independent of assumptions that go into the c(s)-model, not the least being that all sedimenting components have the same vbar.

Best, Holger


From: Leslie T Alessandri [leslie.alessandri at abbott.com]
Sent: Friday, November 19, 2010 6:09 AM
To: rasmb at rasmb.bbri.org
Subject: [RASMB] AUC question

Hi all,

I am running sedimentation velocity experiments on an IgG that is ~201kDa in size.

Previous analysis was performed in triplicate in 15 mM histidine pH 5.2 and using Sedfit the MWs were calculated to be ~183kDa. Much lower than expected. The thought was that possibly solution non-ideology was in play and that if the samples were in 1X PBS the MW calculations would improve. Frictional coefficients were ~1.54 and were floated in Sedfit.

This week I have performed the experiment again in triplicate in 1X PBS and I get the same values for the MW ~183 kDa. Playing around with the data, I found that if I set the f/f0 to ~1.63 or so and do not float it, my MW values come up to a more expected result of around ~200 kDa.

Is it good practice to NOT float the f/f0 and set the value? Would there be good scientific rational for that? I was taught that the f/f0 should always be floated.

Best regards,
Leslie
________________________________

Leslie T Alessandri
Scientist
Process Sciences, Protein Analytics     Abbott Bioresearch Center<http://www.abbott.com/>
100 Research Drive
Worcester, MA 01605     Office 508-688-3478
FAX 508-793-4885
leslie.alessandri at abbott.com<mailto:leslie.alessandri at abbott.com>
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