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<div><font size="2" face="Tahoma">The problem with extracting MW from a nonideal<a></a><a></a> solution is the presence of both thermodynamic and hydrodynamic contributions<a></a> to nonideality<a></a><a></a>. BM1c<a></a><a></a> contributes to D, and Ksc<a></a><a></a>
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<a></a><a></a> range. Both Ks and B are due to excluded volume and charge effects. Salt dependence can thus possibly diminish<a></a> the nonideality<a></a><a></a> but not necessarily all of it. To reduce
the complexity of fitting it helps to do sed<a></a><a></a> 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<a></a> allows fitting to
both BM1<a></a> and Ks. I presume often fitting packages do as well.</font></div>
<div><font size="2" face="Tahoma"><font face="tahoma"></font></font> </div>
<div><font size="2" face="Tahoma"><font face="tahoma">I am not sure if it really pertains<a></a> but its a nice<a></a> idea to remove ideology from AUC analysis at the same time.</font></font></div>
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<font color="#000000" size="2" face="Tahoma"><b>From:</b> rasmb-bounces@rasmb.bbri.org [rasmb-bounces@rasmb.bbri.org] On Behalf Of Titus M. Franzmann [tmfr@umich.edu]<br>
<b>Sent:</b> Friday, November 19, 2010 8:58 AM<br>
<b>To:</b> 'Leslie T Alessandri'; rasmb@server1.bbri.org<br>
<b>Subject:</b> Re: [RASMB] AUC question<br>
</font><br>
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<p class="MsoNormal"><span style="FONT-FAMILY: 'Calibri','sans-serif'; COLOR: #1f497d; FONT-SIZE: 11pt"> Hi Leslie,
</span></p>
<p class="MsoNormal"><span style="FONT-FAMILY: 'Calibri','sans-serif'; COLOR: #1f497d; FONT-SIZE: 11pt">I bet you will also hear back from more advanced AUC people.</span></p>
<p class="MsoNormal"><span style="FONT-FAMILY: 'Calibri','sans-serif'; COLOR: #1f497d; FONT-SIZE: 11pt">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.</span></p>
<p class="MsoNormal"><span style="FONT-FAMILY: 'Calibri','sans-serif'; COLOR: #1f497d; FONT-SIZE: 11pt">Best</span></p>
<p class="MsoNormal"><span style="FONT-FAMILY: 'Calibri','sans-serif'; COLOR: #1f497d; FONT-SIZE: 11pt">Titus</span></p>
<p class="MsoNormal"><span style="FONT-FAMILY: 'Calibri','sans-serif'; COLOR: #1f497d; FONT-SIZE: 11pt"></span> </p>
<p class="MsoNormal"><span style="FONT-FAMILY: 'Segoe UI','sans-serif'; COLOR: #1f497d; FONT-SIZE: 8pt">Dr. Titus M. Franzmann<br>
S. G. Walter Lab<br>
Mol., Cell. and Develop. Biol. Dept. <br>
University of Michigan<br>
4140C Nat. Sci. Bldg<br>
830 N. University Ave.<br>
Ann Arbor, MI 48109-1048</span></p>
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<p class="MsoNormal"><span style="FONT-FAMILY: 'Calibri','sans-serif'; COLOR: #1f497d; FONT-SIZE: 11pt"></span> </p>
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<p class="MsoNormal"><b><span style="FONT-FAMILY: 'Tahoma','sans-serif'; FONT-SIZE: 10pt" lang="DE">From:</span></b><span style="FONT-FAMILY: 'Tahoma','sans-serif'; FONT-SIZE: 10pt" lang="DE"> rasmb-bounces@server1.bbri.org [mailto:rasmb-bounces@server1.bbri.org]
<b>On Behalf Of </b>Leslie T Alessandri<br>
<b>Sent:</b> Friday, November 19, 2010 9:10 AM<br>
<b>To:</b> rasmb@server1.bbri.org<br>
<b>Subject:</b> [RASMB] AUC question</span></p>
</div>
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<p class="MsoNormal"><br>
<span style="FONT-FAMILY: 'Arial','sans-serif'; FONT-SIZE: 10pt">Hi all,</span> <br>
<br>
<span style="FONT-FAMILY: 'Arial','sans-serif'; FONT-SIZE: 10pt">I am running sedimentation velocity experiments on an IgG that is ~201kDa in size.</span>
<br>
<br>
<span style="FONT-FAMILY: 'Arial','sans-serif'; FONT-SIZE: 10pt">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.</span>
<br>
<br>
<span style="FONT-FAMILY: 'Arial','sans-serif'; FONT-SIZE: 10pt">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.</span>
<br>
<br>
<span style="FONT-FAMILY: 'Arial','sans-serif'; FONT-SIZE: 10pt">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.</span>
<br>
<br>
<span style="FONT-FAMILY: 'Arial','sans-serif'; FONT-SIZE: 10pt">Best regards,<br>
Leslie</span></p>
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<p class="MsoNormal"><b><span style="FONT-FAMILY: 'Arial','sans-serif'; FONT-SIZE: 7.5pt">Leslie T Alessandri</span></b><span style="FONT-FAMILY: 'Arial','sans-serif'; FONT-SIZE: 7.5pt"><br>
Scientist<br>
Process Sciences, Protein Analytics</span> </p>
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