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</o:shapelayout></xml><![endif]--></head><body bgcolor=white lang=EN-US link=blue vlink=purple><div class=WordSection1><p class=MsoNormal><span style='color:#1F497D'>Thanks for the explanation. I appreciate it.<o:p></o:p></span></p><p class=MsoNormal><span style='color:#1F497D'>Titus<o:p></o:p></span></p><p class=MsoNormal><span style='color:#1F497D'><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 lang=DE style='font-size:10.0pt;font-family:"Tahoma","sans-serif";color:windowtext'>From:</span></b><span lang=DE style='font-size:10.0pt;font-family:"Tahoma","sans-serif";color:windowtext'> Walter Stafford [mailto:stafford@bbri.org] <br><b>Sent:</b> Friday, December 10, 2010 1:03 PM<br><b>To:</b> Titus M. Franzmann<br><b>Cc:</b> rasmb@server1.bbri.org<br><b>Subject:</b> Re: [RASMB] radial scans 6 sector cells<o:p></o:p></span></p></div></div><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal>Hi Titus,<br><br>It matters if, for example, you have a single channel of data that is run at several different speeds and you tell your fitter that the loading concentration is the same for all data sets (because it really is). If you don't force that restriction, it doesn't matter but if the Rm and Rb positions are not accurately specified, the effective loading concentration will be different at each speed (i.e. each data set). If you can specify that the loading concentration is the same for each data set, then you can remove N-1 parameters, i.e. you fit for just one loading concentration. So it helps if you can locate the bottom. But John Philo has raised some important points, qv. Also if you have any pelleting or gel fomation at the bottom, all bets are off and you will have to float all loading concentrations anyway, even if you know the exact positions of Rm and Rb.<br><br>So as always - it depends ...<br><br>Walter<br><br>Titus M. Franzmann wrote: <o:p></o:p></p><p class=MsoNormal><span style='color:#1F497D'>Hello everyone, </span><o:p></o:p></p><p class=MsoNormal><span style='color:#1F497D'>I am having trouble following this discussion. At what particular point do these proper positions really become a matter? Is this a matter of “how close to the bottom can I reliably analyze an Eq-dataset”? Would the bottom position matter, if the absorbance close to the bottom is beyond Abs=1, and only you analyze the radial range that covers the signal below Abs=1?</span><o:p></o:p></p><p class=MsoNormal><span style='color:#1F497D'>Best</span><o:p></o:p></p><p class=MsoNormal><span style='color:#1F497D'>Titus</span><o:p></o:p></p><p class=MsoNormal><span style='color:#1F497D'> </span><o:p></o:p></p><p class=MsoNormal><span style='color:#1F497D'> </span><o:p></o:p></p><div><div style='border:none;border-top:solid windowtext 1.0pt;padding:3.0pt 0in 0in 0in;border-color:-moz-use-text-color -moz-use-text-color'><p class=MsoNormal><b><span style='font-size:10.0pt;font-family:"Tahoma","sans-serif";color:windowtext'>From:</span></b><span style='font-size:10.0pt;font-family:"Tahoma","sans-serif";color:windowtext'> <a href="mailto:rasmb-bounces@server1.bbri.org">rasmb-bounces@server1.bbri.org</a> [<a href="mailto:rasmb-bounces@server1.bbri.org">mailto:rasmb-bounces@server1.bbri.org</a>] <b>On Behalf Of </b>John Philo<br><b>Sent:</b> Friday, December 10, 2010 11:53 AM<br><b>To:</b> 'Walter Stafford'; 'Stephen Harding'; <a href="mailto:rasmb@server1.bbri.org">rasmb@server1.bbri.org</a><br><b>Subject:</b> Re: [RASMB] radial scans 6 sector cells</span><o:p></o:p></p></div></div><p class=MsoNormal> <o:p></o:p></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:blue'>Steve & Walter, </span><o:p></o:p></p><p class=MsoNormal> <o:p></o:p></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:blue'>Well I don't like to be belabor a point, but since I'm getting beat up a bit here... </span><o:p></o:p></p><p class=MsoNormal> <o:p></o:p></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:blue'>Yes, the conservation of mass calculations do need to know the meniscus and bottom positions, but that doesn't mean those positions have to be present in the final equilibrium scan you are actually fitting. (Clearly they don't since I have been using this data acquisition approach for many years, including when using my 'soft' conservation of mass constraints). From an experimental point of view it is also not very clear how to accurately define those locations from scans that include them (e.g. the infamous "where is the meniscus, really?" controversy).</span><o:p></o:p></p><p class=MsoNormal> <o:p></o:p></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:blue'>Further, regarding the bottom position, what does the 'true' position actually mean? The bottom of the standard Beckman 6-channel cells is flat, not cut on a radius, so (without FC43) the length of the solution column varies as you move across the width of the cell. Even when you use FC43 there is a radial region at the bottom where the absorbance optics are detecting part sample solution and part FC43, so you really can't possibly get good data for the entire solution column (this issue should pretty much go away when you use interference slits). Often mass conservation approaches are not actually considering the rectangular rather than sector shape of the channel at all.</span><o:p></o:p></p><p class=MsoNormal> <o:p></o:p></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:blue'>These issues of not knowing precisely where the column ends are located, non-sector geometry, and not being able to get good data over the full column height, are exactly why true accurate mass conservation usually cannot be achieved and precisely why I implemented the 'soft' mass conservation constraints about 16 years ago [Philo, J. S. (2000). Improving sedimentation equilibrium analysis of mixed associations using numerical constraints to impose mass or signal conservation. <i>Methods Enzymol. </i>321, 100-120].</span><o:p></o:p></p><p class=MsoNormal> <o:p></o:p></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:blue'>John</span><o:p></o:p></p><div class=MsoNormal align=center style='text-align:center'><hr size=2 width="100%" align=center></div><p class=MsoNormal style='margin-bottom:12.0pt'><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"'> <a href="mailto:rasmb-bounces@rasmb.bbri.org">rasmb-bounces@rasmb.bbri.org</a> [<a href="mailto:rasmb-bounces@rasmb.bbri.org">mailto:rasmb-bounces@rasmb.bbri.org</a>] <b>On Behalf Of </b>Walter Stafford<br><b>Sent:</b> Thursday, December 09, 2010 8:21 PM<br><b>To:</b> Stephen Harding; <a href="mailto:rasmb@rasmb.bbri.org">rasmb@rasmb.bbri.org</a><br><b>Subject:</b> Re: [RASMB] radial scans 6 sector cells</span><o:p></o:p></p><p class=MsoNormal>So I agree with Steve:<br><br>Those data may not be recordable, especially near the bottom at higher speeds, but one must know those positions accurately to do the curve fitting properly (for two more components) because the best fit must match the observable part of the data (i.e. that range over which the data are being compared to the model for computation of the rms residuals), and the proper best fit (i.e. the model) is computed assuming those positions are known. For a self-associating system (which is a single component, albeit multi-species) it is not so important since the only error in that case would be the loading concentration - which we're not especially interested in. But for a hetero-associating system, say A+B=AB, the relative loading concentrations of the two components do matter in the fitting and in getting accurate values of the equilibrium constants. <br><br>Since I do most of my runs using interference optics and always do a blank run both before and after the main run in that case, at the speed of the run, I can use those scans to get the positions of the bases of the channels. And SEDANAL will do an interpolated blank subtraction.<br><br>Walter<br><br>Stephen Harding wrote: <o:p></o:p></p><p class=MsoNormal><span style='color:#1F497D'>I also disagree with John and agree almost 100% with Walter, i.e. meniscus and base positions have to be identifiable (and not just for SEDANAL) but I don’t think its possible to record (reliable) concentration data at the solution boundaries, particularly for heterogeneous systems.</span><o:p></o:p></p><p class=MsoNormal><span style='color:#1F497D'>Steve</span><o:p></o:p></p><div><div style='border:none;border-top:solid windowtext 1.0pt;padding:3.0pt 0in 0in 0in;border-color:-moz-use-text-color -moz-use-text-color'><p class=MsoNormal style='margin-left:.5in'><b><span style='font-size:10.0pt;font-family:"Tahoma","sans-serif";color:windowtext'>From:</span></b><span style='font-size:10.0pt;font-family:"Tahoma","sans-serif";color:windowtext'> <a href="mailto:rasmb-bounces@rasmb.bbri.org">rasmb-bounces@rasmb.bbri.org</a> [<a href="mailto:rasmb-bounces@rasmb.bbri.org">mailto:rasmb-bounces@rasmb.bbri.org</a>] <b>On Behalf Of </b>Walter Stafford<br><b>Sent:</b> 09 December 2010 23:14<br><b>To:</b> <a href="mailto:jphilo@mailway.com">jphilo@mailway.com</a><br><b>Cc:</b> <a href="mailto:rasmb@rasmb.bbri.org">rasmb@rasmb.bbri.org</a><br><b>Subject:</b> Re: [RASMB] radial scans 6 sector cells</span><o:p></o:p></p></div></div><p class=MsoNormal style='margin-left:.5in'>I disagree with John. <br><br>For example, my software (SEDANAL) needs to have both the menicus and the base positions identifiable in order to do consrevation of mass calculations for multi-compnent systems. For an equilibrium run - since time is not of the essentce here - is to collect enough data to include the solution boundaries.<br><br>actually, I usually collect all three channels of data in one scan and process it afterward - once for each channel. Less muss and less fuss.<br><br><br><br><o:p></o:p></p><pre style='margin-left:.5in'> <o:p></o:p></pre><pre style='margin-left:.5in'>===============================================<o:p></o:p></pre><pre style='margin-left:.5in'>Walter F. Stafford, Ph.D.<o:p></o:p></pre><pre style='margin-left:.5in'>Senior Scientist<o:p></o:p></pre><pre style='margin-left:.5in'>Boston Biomedical Research Institute<o:p></o:p></pre><pre style='margin-left:.5in'>64 Grove Street<o:p></o:p></pre><pre style='margin-left:.5in'>Watertown, MA 02472<o:p></o:p></pre><pre style='margin-left:.5in'>617-658-7808<o:p></o:p></pre><pre style='margin-left:.5in'>skype: w.stafford3<o:p></o:p></pre><pre style='margin-left:.5in'>----------------------<o:p></o:p></pre><pre style='margin-left:.5in'>"Be kind, for everyone you meet is fighting a hard battle." -Plato<o:p></o:p></pre><pre style='margin-left:.5in'>------------------------------------ <o:p></o:p></pre><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><div style='margin-left:.5in'><div class=MsoNormal align=center style='text-align:center'><span style='font-size:12.0pt;font-family:"Times New Roman","serif"'><hr size=2 width="100%" align=center></span></div></div><p class=MsoNormal style='mso-margin-top-alt:0in;margin-right:0in;margin-bottom:12.0pt;margin-left:.5in'><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"'> <a href="mailto:rasmb-bounces@rasmb.bbri.org">rasmb-bounces@rasmb.bbri.org</a> [<a href="mailto:rasmb-bounces@rasmb.bbri.org">mailto:rasmb-bounces@rasmb.bbri.org</a>] <b>On Behalf Of </b>Lake Paul<br><b>Sent:</b> Thursday, December 09, 2010 1:26 PM<br><b>To:</b> 'Roy Hantgan'; <a href="mailto:rasmb@rasmb.bbri.org">rasmb@rasmb.bbri.org</a><br><b>Subject:</b> Re: [RASMB] radial scans 6 sector cells</span><o:p></o:p></p><p class=MsoNormal style='margin-left:.5in'><span style='color:#1F497D'>Roy,</span><o:p></o:p></p><p class=MsoNormal style='margin-left:.5in'><span style='color:#1F497D'>Funny you should mention this, I did my first 6-sector experiment yesterday. The 5.7 default might not cover the inside sector, so you might have to adjust it to 5.65. I did this by trial and error and by getting help from Dr. John Burgner. The 5.65 worked well I get alittle of the top of the cell and both menisci. Hope this helps.</span><o:p></o:p></p><p class=MsoNormal style='margin-left:.5in'><span style='color:#1F497D'>Lake</span><o:p></o:p></p><div><div style='border:none;border-top:solid windowtext 1.0pt;padding:3.0pt 0in 0in 0in;border-color:-moz-use-text-color -moz-use-text-color'><p class=MsoNormal style='margin-left:.5in'><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"'> <a href="mailto:rasmb-bounces@rasmb.bbri.org">rasmb-bounces@rasmb.bbri.org</a> [<a href="mailto:rasmb-bounces@rasmb.bbri.org">mailto:rasmb-bounces@rasmb.bbri.org</a>] <b>On Behalf Of </b>Roy Hantgan<br><b>Sent:</b> Thursday, December 09, 2010 3:42 PM<br><b>To:</b> <a href="mailto:rasmb@rasmb.bbri.org">rasmb@rasmb.bbri.org</a><br><b>Subject:</b> [RASMB] radial scans 6 sector cells</span><o:p></o:p></p></div></div><p class=MsoNormal style='margin-left:.5in'>Dear colleagues:<o:p></o:p></p><p class=MsoNormal style='margin-left:.5in'>Can you please remind me how you set the radial distance for sed eq runs in 6-sector cells?<br>I’ve only run sed eq in double sector cells. <o:p></o:p></p><p class=MsoNormal style='margin-left:.5in'>Thanks for your advice,<o:p></o:p></p><p class=MsoNormal style='margin-left:.5in'>Roy <o:p></o:p></p><p class=MsoNormal style='margin-left:.5in'><span style='font-size:10.0pt;font-family:"Arial","sans-serif"'>Roy R. Hantgan, Ph.D.</span><o:p></o:p></p><p class=MsoNormal style='margin-left:.5in'><span style='font-size:10.0pt;font-family:"Arial","sans-serif"'>Associate Professor of Biochemistry / Associate in Molecular Medicine</span><o:p></o:p></p><p class=MsoNormal style='margin-left:.5in'><span style='font-size:10.0pt;font-family:"Arial","sans-serif"'>Director, Macromolecular Interactions Core Laboratory (MICL)</span><o:p></o:p></p><p class=MsoNormal style='margin-left:.5in'><span style='font-size:10.0pt;font-family:"Arial","sans-serif"'>Wake Forest University School of Medicine</span><o:p></o:p></p><p class=MsoNormal style='margin-left:.5in'><span style='font-size:10.0pt;font-family:"Arial","sans-serif"'>3058 Hanes Bldg / TEL 336-716-4675</span><o:p></o:p></p><pre style='margin-left:.5in'> <o:p></o:p></pre><pre style='margin-left:.5in'> <o:p></o:p></pre><pre style='margin-left:.5in'>_______________________________________________<o:p></o:p></pre><pre style='margin-left:.5in'>RASMB mailing list<o:p></o:p></pre><pre style='margin-left:.5in'><a href="mailto:RASMB@rasmb.bbri.org">RASMB@rasmb.bbri.org</a><o:p></o:p></pre><pre style='margin-left:.5in'><a href="http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb">http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb</a><o:p></o:p></pre><pre style='margin-left:.5in'> <o:p></o:p></pre></blockquote><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Times New Roman","serif"'> </span><o:p></o:p></p><p>This message and any attachment are intended solely for the addressee and may contain confidential information. If you have received this message in error, please send it back to me, and immediately delete it. Please do not use, copy or disclose the information contained in this message or in any attachment. Any views or opinions expressed by the author of this email do not necessarily reflect the views of the University of Nottingham. <o:p></o:p></p><p>This message has been checked for viruses but the contents of an attachment may still contain software viruses which could damage your computer system: you are advised to perform your own checks. Email communications with the University of Nottingham may be monitored as permitted by UK legislation. <o:p></o:p></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Times New Roman","serif"'><br><br><br></span><o:p></o:p></p><pre> <o:p></o:p></pre><pre>-- <o:p></o:p></pre><pre> <o:p></o:p></pre><pre>===============================================<o:p></o:p></pre><pre>Walter F. Stafford, Ph.D.<o:p></o:p></pre><pre>Senior Scientist<o:p></o:p></pre><pre>Boston Biomedical Research Institute<o:p></o:p></pre><pre>64 Grove Street<o:p></o:p></pre><pre>Watertown, MA 02472<o:p></o:p></pre><pre>617-658-7808<o:p></o:p></pre><pre>skype: w.stafford3<o:p></o:p></pre><pre>----------------------<o:p></o:p></pre><pre>"Be kind, for everyone you meet is fighting a hard battle." -Plato<o:p></o:p></pre><pre>------------------------------------ <o:p></o:p></pre><pre><o:p> </o:p></pre><pre><o:p> </o:p></pre><pre>_______________________________________________<o:p></o:p></pre><pre>RASMB mailing list<o:p></o:p></pre><pre><a href="mailto:RASMB@rasmb.bbri.org">RASMB@rasmb.bbri.org</a><o:p></o:p></pre><pre><a href="http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb">http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb</a><o:p></o:p></pre><pre> <o:p></o:p></pre><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Times New Roman","serif"'><br><br><o:p></o:p></span></p><pre>-- <o:p></o:p></pre><pre><o:p> </o:p></pre><pre>===============================================<o:p></o:p></pre><pre>Walter F. Stafford, Ph.D.<o:p></o:p></pre><pre>Senior Scientist<o:p></o:p></pre><pre>Boston Biomedical Research Institute<o:p></o:p></pre><pre>64 Grove Street<o:p></o:p></pre><pre>Watertown, MA 02472<o:p></o:p></pre><pre>617-658-7808 <o:p></o:p></pre><pre>skype: w.stafford3<o:p></o:p></pre><pre>----------------------<o:p></o:p></pre><pre>"Be kind, for everyone you meet is fighting a hard battle." -Plato<o:p></o:p></pre><pre>------------------------------------<o:p></o:p></pre></div></body></html>