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

[JA KORNBLATT]

hello yi-ming from jack kornblatt
i am a novice like yourself so you should take my views with a grain of
salt. most tween 80 solutions are coloured and the degree of absorption
varies with the producer. a 1% tween 80 solution usually has an od of
0.5 to 1 at 280. it is even less friendly at lower wavelengths. if your
baseline determination is off by 0.04 A is it really a catastrophe?
in some cases it would be.
the problem is further complicated by the fact that tween 80 does not
usually dialyze out of or into the dialysis sac. the result is that you are
courting trouble.

you might want to ask dr neal robinson in texas for his views.
he has done a great deal over the years with membrane proteins and
different detergents.

the sugar concentration is less of a problem vis a vis dialysis but most
nondestructive determinations do not give the sort of precision you might
like to have.

good luck
jack

On Tue, 2 Mar 2004 Yi-Ming_Li at hgsi.com wrote:

> Date: Tue, 02 Mar 2004 12:23:22 -0500
> From: Yi-Ming_Li at hgsi.com
> To: rasmb at rasmb-email.bbri.org
> Subject: [RASMB] A question about sample preparation: dilution (20 fold)
>     vs dialysis
>
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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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