[RASMB] AUC question

[Leslie T Alessandri]

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
100 Research Drive
Worcester, MA 01605
Office 508-688-3478
FAX 508-793-4885
leslie.alessandri at abbott.com



 
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