[RASMB] AUC question

[John J. Correia]

The problem with extracting MW from a nonideal solution is the presence of both thermodynamic and hydrodynamic contributions to nonideality.  BM1c contributes to D, and Ksc contributes to both s and D.  When you have a system like this at high conc the boundary slows and sharpens dramatically, often giving a wide scattering artifact at the meniscus in the first few scans. To extract MW you must thus determine the c dependence of B and Ks and perform experiments over a wide conc range.  Both Ks and B are due to excluded volume and charge effects.  Salt dependence can thus possibly diminish the nonideality but not necessarily all of it. To reduce the complexity of fitting it helps to do sed equilibrium which will only have thermodynamic effects.  It is not a simple problem due to cross correlation effects, so fixing parameters is a good place to start.  Sedanal allows fitting to both BM1 and Ks. I presume often fitting packages do as well.

I am not sure if it really pertains but its a nice idea to remove ideology from AUC analysis at the same time.


________________________________
From: rasmb-bounces at rasmb.bbri.org [rasmb-bounces at rasmb.bbri.org] On Behalf Of Titus M. Franzmann [tmfr at umich.edu]
Sent: Friday, November 19, 2010 8:58 AM
To: 'Leslie T Alessandri'; rasmb at server1.bbri.org
Subject: Re: [RASMB] AUC question

                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

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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