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</o:shapelayout></xml><![endif]--></head><body lang=EN-US link=blue vlink=purple><div class=WordSection1><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'> Hi Leslie, <o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'>I bet you will also hear back from more advanced AUC people.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'>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.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'>Best<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'>Titus<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:8.0pt;font-family:"Segoe UI","sans-serif";color:#1F497D'>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<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'><o:p> </o:p></span></p><div style='border:none;border-top:solid #B5C4DF 1.0pt;padding:3.0pt 0in 0in 0in'><p class=MsoNormal><b><span lang=DE style='font-size:10.0pt;font-family:"Tahoma","sans-serif"'>From:</span></b><span lang=DE style='font-size:10.0pt;font-family:"Tahoma","sans-serif"'> 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<o:p></o:p></span></p></div><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal><br><span style='font-size:10.0pt;font-family:"Arial","sans-serif"'>Hi all,</span> <br><br><span style='font-size:10.0pt;font-family:"Arial","sans-serif"'>I am running sedimentation velocity experiments on an IgG that is ~201kDa in size.</span> <br><br><span style='font-size:10.0pt;font-family:"Arial","sans-serif"'>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-size:10.0pt;font-family:"Arial","sans-serif"'>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-size:10.0pt;font-family:"Arial","sans-serif"'>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-size:10.0pt;font-family:"Arial","sans-serif"'>Best regards,<br>Leslie</span><o:p></o:p></p><table class=MsoNormalTable border=0 cellpadding=0><tr><td colspan=4 style='background:white;padding:.75pt .75pt .75pt .75pt'><div class=MsoNormal align=center style='text-align:center'><hr size=2 width="100%" noshade style='color:#A0A0A0' align=center></div></td></tr><tr><td style='background:white;padding:.75pt .75pt .75pt .75pt'><p class=MsoNormal><b><span style='font-size:7.5pt;font-family:"Arial","sans-serif"'>Leslie T Alessandri</span></b><span style='font-size:7.5pt;font-family:"Arial","sans-serif"'><br>Scientist<br>Process Sciences, Protein Analytics</span> <o:p></o:p></p></td><td valign=top style='background:white;padding:.75pt .75pt .75pt .75pt'><p class=MsoNormal><a href="http://www.abbott.com/"><span style='font-size:7.5pt;font-family:"Arial","sans-serif"'>Abbott Bioresearch Center</span></a><span style='font-size:7.5pt;font-family:"Arial","sans-serif"'><br>100 Research Drive<br>Worcester, MA 01605</span> <o:p></o:p></p></td><td valign=top style='background:white;padding:.75pt .75pt .75pt .75pt'><p class=MsoNormal><span style='font-size:7.5pt;font-family:"Arial","sans-serif"'>Office 508-688-3478<br>FAX 508-793-4885<u><span style='color:blue'><br></span></u></span><a href="mailto:leslie.alessandri@abbott.com"><span style='font-size:7.5pt;font-family:"Arial","sans-serif"'>leslie.alessandri@abbott.com</span></a> <o:p></o:p></p></td><td valign=top style='background:white;padding:.75pt .75pt .75pt .75pt'><p class=MsoNormal><span style='font-size:7.5pt;font-family:"Arial","sans-serif"'><br></span><img border=0 width=112 height=29 id="_x0000_i1026" src="cid:image001.gif@01CB87CE.E9FDACC0"><o:p></o:p></p></td></tr><tr><td colspan=4 style='padding:.75pt .75pt .75pt .75pt'><div class=MsoNormal align=center style='text-align:center'><hr size=2 width="100%" noshade style='color:#A0A0A0' align=center></div><p class=MsoNormal align=center style='text-align:center'><br><b> </b><o:p></o:p></p></td></tr><tr><td colspan=4 style='padding:.75pt .75pt .75pt .75pt'><p class=MsoNormal><span style='font-size:7.5pt;font-family:"Arial","sans-serif";color:gray'>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.</span><o:p></o:p></p></td></tr></table><p class=MsoNormal><br><br> <o:p></o:p></p></div></body></html>