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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-family:"Courier New";color:blue'>Dear RASMB,<o:p></o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'>David and all who have responded, thank you for your comments. I think in the aggregate of comments received I have my answer and more questions.<o:p></o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'>In the range of concentrations measured 0.2 – 4.9mgs/ml the slope of the experimental s with concentration is negative but only slightly so (slope=-0.033 s/c, where c is the mass concentration g/cc). The extrapolated s at 0mgs/ml is 6.88, given the information received in these posts I think that at 4.2 mgs/ml I ought to expect a ~4.4% decrease in s due solely to excluded volume effects. If so the expected s-exp at 4.9mgs/ml ought to be 6.58. What is actually observed is a value of 6.72s. So I think what David and others have suggested is probably accurate, namely that self-association is offsetting the non-ideality here. <o:p></o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'>This raises some questions perhaps that would be good to test. I can’t shut off self-association but perhaps one could slow it down by reducing the temperature from 20C to 4C? I think excluded volume is only weakly dependent on temperature and self-association more strongly dependent on temperature? <o:p></o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'>Interestingly, the sw vs conc relationship changes as a function of buffer, whereas the slope of relationship is only weakly negative in HEPES (0.01M HEPES, 150mM NaCl, pH7.5) in PBS (0.137M NaCl) it is strongly positive.<o:p></o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'>Once again thank you all for your thoughts and suggestions.<o:p></o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'>Best regards,<o:p></o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'>John Sumida<o:p></o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'>University of Washington<o:p></o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'><o:p> </o:p></span></p><div><div style='border:none;border-top:solid #B5C4DF 1.0pt;padding:3.0pt 0in 0in 0in'><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>David Hayes<br><b>Sent:</b> Wednesday, January 21, 2015 6:25 PM<br><b>To:</b> RASMB<br><b>Subject:</b> Re: [RASMB] concentration range for non-ideal behavior<o:p></o:p></span></p></div></div><p class=MsoNormal><o:p> </o:p></p><div><div id="yui_3_16_0_1_1421892529622_2826"><p class=MsoNormal style='background:white'><span style='font-size:10.5pt;font-family:"Garamond","serif";color:black'>Hi John,<o:p></o:p></span></p></div><div id="yui_3_16_0_1_1421892529622_2825"><p class=MsoNormal style='background:white'><span style='font-size:10.5pt;font-family:"Garamond","serif";color:black'><o:p> </o:p></span></p></div><div id="yui_3_16_0_1_1421892529622_2816"><p class=MsoNormal style='background:white'><span style='font-size:10.5pt;font-family:"Garamond","serif";color:black'>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.<o:p></o:p></span></p></div><div id="yui_3_16_0_1_1421892529622_2815"><p class=MsoNormal style='background:white'><span style='font-size:10.5pt;font-family:"Garamond","serif";color:black'>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.<o:p></o:p></span></p></div><div id="yui_3_16_0_1_1421892529622_2827"><p class=MsoNormal style='background:white'><span style='font-size:10.5pt;font-family:"Garamond","serif";color:black'><o:p> </o:p></span></p></div><div id="yui_3_16_0_1_1421892529622_2828"><p class=MsoNormal style='background:white'><span style='font-size:10.5pt;font-family:"Garamond","serif";color:black'>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.<o:p></o:p></span></p></div><div id="yui_3_16_0_1_1421892529622_2829"><p class=MsoNormal style='background:white'><span style='font-size:10.5pt;font-family:"Garamond","serif";color:black'><o:p> </o:p></span></p></div><div id="yui_3_16_0_1_1421892529622_2830"><p class=MsoNormal style='background:white'><span style='font-size:10.5pt;font-family:"Garamond","serif";color:black'>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.<o:p></o:p></span></p></div><div id="yui_3_16_0_1_1421892529622_2843"><p class=MsoNormal style='background:white'><span style='font-size:10.5pt;font-family:"Garamond","serif";color:black'><o:p> </o:p></span></p></div><div id="yui_3_16_0_1_1421892529622_2844"><p class=MsoNormal style='background:white'><span style='font-size:10.5pt;font-family:"Garamond","serif";color:black'>David<o:p></o:p></span></p></div><div id="yui_3_16_0_1_1421892529622_2814"><p class=MsoNormal style='background:white'><span style='font-size:10.5pt;font-family:"Garamond","serif";color:black'><o:p> </o:p></span></p></div><div id="yui_3_16_0_1_1421892529622_2813"><p class=MsoNormal style='background:white'><span style='font-size:10.5pt;font-family:"Garamond","serif";color:black'><br> <o:p></o:p></span></p></div><div id="yui_3_16_0_1_1421892529622_2763"><div id="yui_3_16_0_1_1421892529622_2762"><div id="yui_3_16_0_1_1421892529622_2761"><div class=MsoNormal align=center style='text-align:center;background:white'><span style='font-family:"Helvetica","sans-serif";color:black'><hr size=1 width="100%" align=center></span></div><p class=MsoNormal style='background:white'><b><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:black'>From:</span></b><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:black'> John Sumida <<a href="mailto:jpsumida@u.washington.edu">jpsumida@u.washington.edu</a>><br><b>To:</b> <a href="mailto:rasmb@rasmb.org">rasmb@rasmb.org</a> <br><b>Sent:</b> Tuesday, January 20, 2015 9:00 PM<br><b>Subject:</b> [RASMB] concentration range for non-ideal behavior</span><span style='font-family:"Helvetica","sans-serif";color:black'><o:p></o:p></span></p></div><div id="yui_3_16_0_1_1421892529622_2806"><p class=MsoNormal style='background:white'><span style='font-family:"Helvetica","sans-serif";color:black'><o:p> </o:p></span></p><div id=yiv6573107090><div id="yui_3_16_0_1_1421892529622_2809"><div id="yui_3_16_0_1_1421892529622_2808"><div id="yui_3_16_0_1_1421892529622_2807"><p class=MsoNormal style='background:white'><span style='font-family:"Courier New";color:black'>Dear RASMB,</span><span style='font-family:"Helvetica","sans-serif";color:black'><o:p></o:p></span></p></div><div id="yui_3_16_0_1_1421892529622_2810"><p class=MsoNormal style='background:white'><span style='font-family:"Courier New";color:black'> </span><span style='font-family:"Helvetica","sans-serif";color:black'><o:p></o:p></span></p></div><div id="yui_3_16_0_1_1421892529622_2812"><p class=MsoNormal style='background:white'><span style='font-family:"Courier New";color:black'>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><span style='font-family:"Helvetica","sans-serif";color:black'><o:p></o:p></span></p></div><div id="yui_3_16_0_1_1421892529622_2831"><p class=MsoNormal style='background:white'><span style='font-family:"Courier New";color:black'> </span><span style='font-family:"Helvetica","sans-serif";color:black'><o:p></o:p></span></p></div><div id="yui_3_16_0_1_1421892529622_2833"><p class=MsoNormal style='background:white'><span style='font-family:"Courier New";color:black'>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><span style='font-family:"Helvetica","sans-serif";color:black'><o:p></o:p></span></p></div><div id="yui_3_16_0_1_1421892529622_2834"><p class=MsoNormal style='background:white'><span style='font-family:"Courier New";color:black'> </span><span style='font-family:"Helvetica","sans-serif";color:black'><o:p></o:p></span></p></div><div id="yui_3_16_0_1_1421892529622_2835"><p class=MsoNormal style='background:white'><span style='font-family:"Courier New";color:black'>Best regards</span><span style='font-family:"Helvetica","sans-serif";color:black'><o:p></o:p></span></p></div><div><p class=MsoNormal style='background:white'><span style='font-family:"Courier New";color:black'>John Sumida</span><span style='font-family:"Helvetica","sans-serif";color:black'><o:p></o:p></span></p></div><div><p class=MsoNormal style='background:white'><span style='font-family:"Courier New";color:black'>University of Washington</span><span style='font-family:"Helvetica","sans-serif";color:black'><o:p></o:p></span></p></div></div></div></div><p class=MsoNormal style='margin-bottom:12.0pt;background:white'><span style='font-family:"Helvetica","sans-serif";color:black'><br>_______________________________________________<br>RASMB mailing list<br><a href="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><o:p></o:p></span></p></div></div></div></div></div></body></html>