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</o:shapelayout></xml><![endif]--></head><body lang=EN-US link="#0563C1" vlink="#954F72"><div class=WordSection1><p class=MsoNormal style='margin-bottom:6.0pt'><span style='font-size:10.0pt;font-family:"Arial",sans-serif;color:blue'>John, you should not have any problems running SV on this sample using absorbance detection. The tween 20 micelles sediment near 2 S (slow for their MW because the detergent density is less than for protein). <o:p></o:p></span></p><p class=MsoNormal style='margin-bottom:6.0pt'><span style='font-size:10.0pt;font-family:"Arial",sans-serif;color:blue'>For reasons too complex to go into here, it is quite difficult to match the Tween signals in the sample and reference channels. If the Tween is reasonably pure you probably won’t see more than a few hundredths of an OD from the UV-absorbing impurities in it (pure Tween does not absorb at 280 nm). Thus I would suggest simply leaving the Tween out of the reference channel, and then also running a control of Tween-containing buffer versus water so you know exactly how fast the micelles sediment.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial",sans-serif;color:blue'>John<o:p></o:p></span></p><div><div style='border:none;border-top:solid #E1E1E1 1.0pt;padding:3.0pt 0in 0in 0in'><p class=MsoNormal><b>From:</b> RASMB [mailto:rasmb-bounces@list.rasmb.org] <b>On Behalf Of </b>John Sumida<br><b>Sent:</b> Friday, August 05, 2016 1:00 PM<br><b>To:</b> RASMB <rasmb@list.rasmb.org><br><b>Subject:</b> [RASMB] sedimentation with P20<o:p></o:p></p></div></div><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal>Dear RASMB,<o:p></o:p></p><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal>I would like to design an experiment that would confirm (or not) the presence of a monomer with an expected mass of 60kDa. This protein is being prepped for an SPR experiment but has been shown to form significant aggregates in the absence of tween 20. We would like to confirm that in the presence of 0.05% Tween, the solution is primarily monomeric.<o:p></o:p></p><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal>DLS analysis of just the buffer containing 0.05%Tween shows a peak at 3nm. Analysis of the protein in the same buffer, likewise indicates a peak at 3nm.<o:p></o:p></p><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal>Calculating the stokes radius from the theoretical diffusion value (8.15 Ficks), the expected stokes radius of the protein is 2.4nm.<o:p></o:p></p><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal>Thus the problem is how to distinguish between protein and micelle which appear to have similar stokes radii.<o:p></o:p></p><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal>SEC is one approach that we are trying, but in thinking about getting to an answer with another method to bolster the SEC data, my understanding is that if there were no thermodynamic non-ideality present, an equilibrium measurement monitoring the absorbance of the protein may be another viable approach since one would not have to account for any hydrodynamic non-ideality that I expect would arise in a transport (Sed Vel) measurement.<o:p></o:p></p><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal>I would be interested in your thoughts and comments, and as always I thank you for your time and kind consideration.<o:p></o:p></p><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal>Best regards,<o:p></o:p></p><p class=MsoNormal>John Sumida Ph.D.<o:p></o:p></p><p class=MsoNormal>Bionalytical Core Facility Manager<o:p></o:p></p><p class=MsoNormal>University of Washington<o:p></o:p></p><p class=MsoNormal>Molecular Engineering & Sciences Rm G22<o:p></o:p></p><p class=MsoNormal><o:p> </o:p></p></div></body></html>