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<TITLE>Re: [RASMB] upper concentration limit AUC</TITLE>
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<BLOCKQUOTE><FONT COLOR="#800000">Dear Joris and all RASMBers<BR>
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(1) There is a problem with using sedimentation velocity at protein concentrations in the 10-20 mg/ml level (or greater), in that the very sharp gradients which are formed deflect the beam out of the angular aperture of the optics. So you end up with a shadows ('schlieren') where the boundary information should be. Using shorter optical path length cells helps here - although you get increased problems with optical artifacts of the 2nd or 3rd order (see below) these cause much less problem than with SE work<BR>
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(2) But I guess that your colleague would be thinking about looking at Kd values via SE? In this case, no problem need arise using 12 mm optical path length cells <U>provided that you keep the sigma value down to below 1</U>. If you try to go for higher sigma values, then you will simply lose the trace at the higher radial values. Contrary to what is often supposed, provided the instrument is correctly focussed onto the 2/3rd plane of the cell (i.e. 8 mm beyond inner side of entry window) then there are no artifacts which arise from the 3rd order terms in the Svensson equation (which relates fringe shifts to RI increments). BUT - do not use shorter optical path length cells. The plane of focus now lies outside the actual fluid contents of the cell! Heaven knows what this does the the validity of your fringe increments.<BR>
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I describing the interpretation of high concentration SE runs in some details at the Newcastle AUC Meeting at the end of next week and in a MSS I am submitting therefrom, but some of the background is in my chapter in the 'White Book' - "Analytical Ultracentrifugation - Techniques & Methods" (eds Scott, Harding & Rowe RSC 2005) as well as in various publications (available on request).<BR>
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Kind regards<BR>
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Arthur<BR>
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<BLOCKQUOTE>Dear all, <BR>
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A colleague asked me whether analytical ultracentrifugation has an upper limit with regard to the concentration of the protein. They want to measure the protein at the same concentration as the NMR experiments (> 1 mM). I am not entirely sure but I thought this should be no problem. One could easily measure off-peak at another wavelength than 230nm, e.g. 235nm or 280nm, right?! Or does one run into non-ideality phenomena when doing sedimentation equilibrium at these high protein concentrations?! <BR>
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Thanks a lot in advance for any feedback. <BR>
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Best wishes, <BR>
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Joris Beld <BR>
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Hilvert Group <BR>
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ETH Zürich <BR>
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Switzerland <BR>
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