[RASMB] AUC question

[Brown, Patrick (NIH/NIBIB) [E]]

Leslie:
All one is really measuring in an SV experiment is the velocity that the protein moves relative to the gravitational field- the sedimentation coefficient.  In order to extract information on the mass of your protein, you'll need to take into account it's buoyancy (and shape). What value are you using for vbar, and buffer density?  Have you measured them?  Often SEDNTERP is used to calculate vbar based on the amino acid composition, but if you have any glycosylation, post-translational modifications, or other cofactors such as bound metals or porphyrins...and such, the calculated vbar can be wrong.  This can have a significant affect on your apparent mass.  In order to get an idea of what affect it can have, I would encourage you to try fitting your data with different vbar values, for example instead of the default setting (0.73), try 0.72, and 0.71  and you'll see that your apparent mass will increase.  There are several methods that one could use to establish a vbar, such as densimetry or density contrast which you could look up in the literature.
Finally, as you pointed out, it seems best to float the frictional ratio (and meniscus).
Hope this was helpful,
Patrick


________________________________________
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>
[cid:_1_07FF566007FF5260004DD0B8852577E0]
________________________________



This communication may contain information that is proprietary, confidential, or exempt from disclosure. If you are not the intended recipient, please note that any other dissemination, distribution, use or copying of this communication is strictly prohibited. Anyone who receives this message in error should notify the sender immediately by telephone or by return e-mail and delete it from his or her computer.