[RASMB] AUC question

Fri Nov 19 06:58:03 PST 2010

                Hi Leslie, 

I bet you will also hear back from more advanced AUC people.

One thing I noticed during my years of working on mass determination of
proteins using AUC, is that the experimental run conditions are critical to
get very accurate masses. The shape domain (ff0) is critical. When you spin
too fast, the shape domain is poorly described. Spinning too slow will gain
information on the shape domain, but you compromise your resolution on the
S-domain. Depending on how important this question off mass determination
is, I found that an approach that worked for me is spinning the samples at
multiple speeds, improving resolution on both domains and fitting them
globally.

Best

Titus

Dr. Titus M. Franzmann
S. G. Walter Lab
Mol., Cell. and Develop. Biol. Dept. 
University of Michigan
4140C Nat. Sci. Bldg
830 N. University Ave.
Ann Arbor, MI 48109-1048

From: rasmb-bounces at server1.bbri.org [mailto:rasmb-bounces at server1.bbri.org]
On Behalf Of Leslie T Alessandri
Sent: Friday, November 19, 2010 9:10 AM
To: rasmb at server1.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 

 <http://www.abbott.com/> Abbott Bioresearch Center
100 Research Drive
Worcester, MA 01605 

Office 508-688-3478
FAX 508-793-4885
 <mailto:leslie.alessandri at abbott.com> leslie.alessandri at abbott.com 

  _____  

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