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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">Hi-<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">AUC doesn’t lie… If the complexes are near or in the void volume, SEC won’t provide much resolution. Likewise, DLS is fitting the correlation function the best
it can, and it will fit it with a single decay if that suffices to describe the data.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">There is insufficient force across a complex for it to be torn apart by the gravitational field. The force available will be the gradient of the gravitational
potential energy, with the diameter of the complex being the distance, which is very short. On the other hand, if you suspect pressure effects in the AUC, you have some choices for diagnosis.<o:p></o:p></span></p>
<p class="MsoListParagraph" style="text-indent:-.25in;mso-list:l0 level1 lfo1"><![if !supportLists]><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><span style="mso-list:Ignore">1.<span style="font:7.0pt "Times New Roman"">
</span></span></span><![endif]><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">The pressure effect depends on the number of units affected (the stoichiometry of the complex), which is moderately high for a MDa complex of 60 KDa
subunits. Pressure usually leads to dissociation of complexes. The pressure is non-uniform across the cell, and depends on the rotor speed. You should get horrible fits to the data since dissociation will be greater towards the base of the cell. Also, the
extent of dissociation will be rotor speed dependent (by the square of the rotor speed). See if the distribution shifts towards larger complexes at lower rotor speeds. If the distributions are insensitive to rotor speed, you have a good indication that the
distribution of sizes that is pressure independent.<o:p></o:p></span></p>
<p class="MsoListParagraph" style="text-indent:-.25in;mso-list:l0 level1 lfo1"><![if !supportLists]><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><span style="mso-list:Ignore">2.<span style="font:7.0pt "Times New Roman"">
</span></span></span><![endif]><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">Layer a bit of mineral oil over the sample. This will add static pressure to the sample. If the distribution is pressure dependent, you will see greater
dissociation when you analyze the data.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">Best wishes,<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">Tom<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>
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<p class="MsoNormal"><b><span style="font-size:10.0pt;font-family:"Tahoma","sans-serif"">From:</span></b><span style="font-size:10.0pt;font-family:"Tahoma","sans-serif""> RASMB [mailto:rasmb-bounces@list.rasmb.org]
<b>On Behalf Of </b>Toth IV, Ronald T<br>
<b>Sent:</b> Monday, December 14, 2015 5:15 PM<br>
<b>To:</b> rasmb@rasmb.org<br>
<b>Cc:</b> Kumru, Ozan S<br>
<b>Subject:</b> [RASMB] Possible for a complex to break apart from force of sedimentation?<o:p></o:p></span></p>
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<p class="MsoNormal"><o:p> </o:p></p>
<p><span style="font-family:"Calibri","sans-serif";color:black">Hi all! We’re looking at a ~60 kDa protein that forms a ~ 1 MDa oligomeric disulfide linked complex. By SEC and DLS the sample is very monodisperse at about what we would expect the MDa complex
to look like. However by AUC we see a very heterogeneous distribution between 5s and 30s. I was wondering if anyone else had observed a system where a complex had broken apart from the force of sedimentation?<o:p></o:p></span></p>
<p><span style="font-family:"Calibri","sans-serif";color:black"><o:p> </o:p></span></p>
<p><span style="font-family:"Calibri","sans-serif";color:black">Thanks in advance! <o:p></o:p></span></p>
<p><span style="font-family:"Calibri","sans-serif";color:black"><o:p> </o:p></span></p>
<p><span style="font-family:"Calibri","sans-serif";color:black"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Tahoma","sans-serif";color:black">- Ronald Toth</span><span style="color:black"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Tahoma","sans-serif";color:black"> </span><span style="color:black"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Tahoma","sans-serif";color:black">Research Associate</span><span style="color:black"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Tahoma","sans-serif";color:black">Macromolecule and Vaccine Stabilization Center</span><span style="color:black"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Tahoma","sans-serif";color:black">University of Kansas</span><span style="color:black"><o:p></o:p></span></p>
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