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<TITLE>Re: [RASMB] A question about sample preparation: dilution (20 fold) vs dialysis</TITLE>
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<BLOCKQUOTE>Hi Yi-Ming<BR>
<BR>
I think you could have real problems at 230 nm, but at 280 nm I am pretty confident you will be OK. The baseline will not be all that large - indeed if I understand your proposed dilution schedule correctly then there will be only the slightest difference in Tween 80 concentration between the solution and reference channels. And since your velocity software (SEDFIT ?) will always float the baseline - that is routine - then everything should work.<BR>
<BR>
Even at 230 nm it is worth giving it a try.<BR>
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Regards<BR>
<BR>
Arthur<BR>
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*******************************************************<BR>
Arthur J Rowe<BR>
Professor of Biomolecular Technology<BR>
NCMH Business Centre<BR>
University of Nottingham<BR>
School of Biosciences<BR>
Sutton Bonington<BR>
Leicestershire LE12 5RD UK<BR>
<BR>
Tel: +44 (0)115 951 6156<BR>
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Web: www.nottingham.ac.uk/ncmh/business<BR>
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Hi<BR>
I have a question about sample preparation for analytical<BR>
ultracentrifugation. I am going to run a sedimentation velocity experiment<BR>
to examine the size distribution of the samples. Due to the limit in sample<BR>
amount, I will directly dilute the sample (20 folds) in running buffer<BR>
instead of dialyzing it against running buffer. This will make some<BR>
difference between the reference and the sample. I want to know whether<BR>
this is tolerable for sedimentation velocity experiment with absorbance<BR>
(280 nm and 230 nm) detection. The differences of sample buffer and running<BR>
buffer are in glycine, Tween 80 and sucrose concentrations as following:<BR>
<BR>
Sample buffer: 1.9% glycine, 0.5% sucrose, 0.01% Tween 80<BR>
<BR>
Running buffer: no glycine, 8% sucrose, 0.04% Tween 80<BR>
<BR>
In both buffers, Tween 80 concentrations are over CMC (critical micelle<BR>
concentration).<BR>
<BR>
Thanks!<BR>
<BR>
Yi-Ming<BR>
<BR>
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