<html><body><div style="color:#000; background-color:#fff; font-family:garamond, new york, times, serif;font-size:14px"><div><span></span></div><div id="yui_3_16_0_1_1421892529622_2826">Hi John,</div><div id="yui_3_16_0_1_1421892529622_2825"><br></div><div id="yui_3_16_0_1_1421892529622_2816" dir="ltr">The opposite of non-ideality is reality. All real molecules take up space, so all real molecules sediment with some excluded volume non-ideality. Charge is another source of non-ideality: the ideal model of a molecule, besides being a geometric point with no volume, also has no attractive or repulsive forces.</div><div id="yui_3_16_0_1_1421892529622_2815" dir="ltr">The type of non-ideality that is not avoidable is excluded volume. So it is not exactly mg/ml that determines whether or not the non-ideality is significant, but the excluded volume. For most proteins it is reasonable to see little effect from concentration alone up to 1-2 mg/ml. But, put a molecule in a salt free buffer where charge is not screened (Actin) or take a molecule that has a really big carbohydrate attached (Pegasys) and nonideality can be evident at 0.5 mg/ml and below.</div><div id="yui_3_16_0_1_1421892529622_2827" dir="ltr"><br></div><div id="yui_3_16_0_1_1421892529622_2828" dir="ltr">In sedimentation velocity, the other non-ideality is the back flow. When a real molecule moves down the cell, real buffer has to move up the cell to fill in the otherwise to be empty spot where the molecule was. This hydrodynamic non-ideality is also always there, but usually negligible below 1-2 mg/ml.</div><div id="yui_3_16_0_1_1421892529622_2829" dir="ltr"><br></div><div id="yui_3_16_0_1_1421892529622_2830" dir="ltr">Did you do a concentration series of your antibody? If so, it is expected that around 5 mg/ml; you would have measurable concentration dependence of the sedimentation coefficient. If your peak moves the same S at 0.5 and 5 mg/ml I would suspect self association is adding S and offsetting the concentration dependence of S. Static light scattering is helpful to use as an orthogonal method when self association and non-ideality balance out in AUC-SV.</div><div id="yui_3_16_0_1_1421892529622_2843" dir="ltr"><br></div><div id="yui_3_16_0_1_1421892529622_2844" dir="ltr">David</div><div id="yui_3_16_0_1_1421892529622_2814" dir="ltr"><br></div><div id="yui_3_16_0_1_1421892529622_2813"><br> </div><div id="yui_3_16_0_1_1421892529622_2763" style="font-family: garamond, new york, times, serif; font-size: 14px;"> <div id="yui_3_16_0_1_1421892529622_2762" style="font-family: HelveticaNeue, Helvetica Neue, Helvetica, Arial, Lucida Grande, sans-serif; font-size: 16px;"> <div id="yui_3_16_0_1_1421892529622_2761" dir="ltr"> <hr size="1" id="yui_3_16_0_1_1421892529622_2781"> <font id="yui_3_16_0_1_1421892529622_2760" face="Arial" size="2"> <b><span style="font-weight: bold;">From:</span></b> John Sumida <jpsumida@u.washington.edu><br> <b><span style="font-weight: bold;">To:</span></b> rasmb@rasmb.org <br> <b><span style="font-weight: bold;">Sent:</span></b> Tuesday, January 20, 2015 9:00 PM<br> <b><span style="font-weight: bold;">Subject:</span></b> [RASMB] concentration range for non-ideal behavior<br> </font> </div> <div class="y_msg_container" id="yui_3_16_0_1_1421892529622_2806"><br><div id="yiv6573107090"><style><!--
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--></style><div id="yui_3_16_0_1_1421892529622_2809"><div class="yiv6573107090WordSection1" id="yui_3_16_0_1_1421892529622_2808"><div class="yiv6573107090MsoNormal" id="yui_3_16_0_1_1421892529622_2807"><span style='font-family: "Courier New"; font-size: 12pt;'>Dear RASMB,</span></div><div class="yiv6573107090MsoNormal" id="yui_3_16_0_1_1421892529622_2810"><span style='font-family: "Courier New"; font-size: 12pt;'> </span></div><div class="yiv6573107090MsoNormal" id="yui_3_16_0_1_1421892529622_2812"><span id="yui_3_16_0_1_1421892529622_2811" style='font-family: "Courier New"; font-size: 12pt;'>My recollection is that concentrations in excess of 1.0 mg/ml should be avoided due to the possibility of non-ideal interactions that would complicate the analysis of SV and SE measurements. However in two recent cases (in one case a mAb was being studied, and in another a globular viral capsid protein), we exceeded this value by a factor of five and failed to observe any evidence of non-ideal behavior. In both cases, it was possible to fit both SV and SE data without assuming any non-ideal interactions when the experiment was performed in PBS or HEPES.</span></div><div class="yiv6573107090MsoNormal" id="yui_3_16_0_1_1421892529622_2831"><span style='font-family: "Courier New"; font-size: 12pt;'> </span></div><div class="yiv6573107090MsoNormal" id="yui_3_16_0_1_1421892529622_2833"><span id="yui_3_16_0_1_1421892529622_2832" style='font-family: "Courier New"; font-size: 12pt;'>I would be curious to hear if it is the experience of the community that non-ideality is commonly to be expected at concentrations >1.0mg/ml or whether the concentration cutoff for non-ideality is less of an actual cutoff being more a function of the particular properties of the molecule being studied.</span></div><div class="yiv6573107090MsoNormal" id="yui_3_16_0_1_1421892529622_2834"><span style='font-family: "Courier New"; font-size: 12pt;'> </span></div><div class="yiv6573107090MsoNormal" id="yui_3_16_0_1_1421892529622_2835"><span style='font-family: "Courier New"; font-size: 12pt;'>Best regards</span></div><div class="yiv6573107090MsoNormal"><span style='font-family: "Courier New"; font-size: 12pt;'>John Sumida</span></div><div class="yiv6573107090MsoNormal"><span style='font-family: "Courier New"; font-size: 12pt;'>University of Washington</span></div></div></div></div><br>_______________________________________________<br>RASMB mailing list<br><a href="mailto:RASMB@list.rasmb.org" ymailto="mailto:RASMB@list.rasmb.org">RASMB@list.rasmb.org</a><br><a href="http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org" target="_blank">http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org</a><br><br><br></div> </div> </div> </div></body></html>