[RASMB] A question about sample preparation: dilution (20 fold) vs dialysis

[Arthur Rowe]

Hi Yi-Ming

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.

Even at 230 nm it is worth giving it a try.

Regards

Arthur


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Arthur J Rowe
Professor of Biomolecular Technology
NCMH Business Centre
University of Nottingham
School of Biosciences
Sutton Bonington
Leicestershire LE12 5RD   UK

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Hi
I have a question about sample preparation for analytical
ultracentrifugation. I am going to run a sedimentation velocity experiment
to examine the size distribution of the samples. Due to the limit in sample
amount, I will directly dilute the sample (20 folds) in running buffer
instead of dialyzing it against running buffer.  This will make some
difference between the reference and the sample. I want to know whether
this is tolerable for sedimentation velocity experiment with absorbance
(280 nm and 230 nm) detection. The differences of sample buffer and running
buffer are in glycine, Tween 80 and sucrose concentrations as following:

Sample buffer: 1.9% glycine, 0.5% sucrose, 0.01% Tween 80

Running buffer: no glycine, 8% sucrose, 0.04% Tween 80

In both buffers, Tween 80 concentrations are over CMC (critical micelle
concentration).

Thanks!

Yi-Ming








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