[Rasmb] Tween 80 effect on c(s)

[JA KORNBLATT]

hello chin
peter suggests omitting tween from the reference buffer.
we've little experience with tween and the auc but a lot of experience
with tween itself.
it absorbs strongly at 276 nm where you are working. a 1% solution may
give an abs of >> 1 relative to water depending on the tween sample.
unfortunately, every sample of tween is different from every other sample.
best regards
jack


On Thu, 22 Sep 2005, Peter Schuck wrote:

> Date: Thu, 22 Sep 2005 22:53:59 -0400
> From: Peter Schuck <pschuck at helix.nih.gov>
> To: Qin "Chin" Zou <heat-capacity at indy.rr.com>, RASMB at server1.bbri.org
> Subject: Re: [Rasmb] Tween 80 effect on c(s)
>
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> Hi Chin,
> I would probably try it without Tween in the reference buffer.  In
> general, the size of detergent micelles can depend on the solvent and
> experimental conditions, and even though you didn't say what you're
> buffer is, some deviations from the 2S you quote would not be
> surprising. I assume you're referring to s20w values, otherwise it will
> depend even more on the experimental conditions.  Have you looked at
> what happens with the Tween without the protein?  Also, at 5 mg/ml
> protein we usually cannot be so stringent about the quality of fit,
> since we should expect significant non-ideality (I assume since you're
> talking about peaks, you look at a s-value distribution, for which
> there's to my knowledge no correction for the non-ideality). This effect
> would give you lower s-values, in addition to distorting the boundary
> shapes, and therefore limit the amount of detail we can currently
> extract from the sedimentation velocity experiment.
> Peter
>
>
> Qin "Chin" Zou wrote:
>
> > Hi all,
> >
> > I wonder if anyone has looked at the effect of Tween 80 on the s
> > distribution. I remember that John Philo once asked the similar
> > question and had calculated s for Tween 80 around 2s.
> >
> > I have an 18KD protein that runs at about 1.7s when there is no Tween
> > 80 in the buffer. However, in the presence of Tween 80, there were two
> > species 1.2 and 1.5s (without regularization. If F=.68, then two peaks
> > are overlapped).
> >
> > The experiment was run at 60K rpm, absorbance at 276nm. Also, the fit
> > was not good with rmsd above 0.01. In addition, I tried to scan at
> > 289nm, where the absorbance of Tween 80 is minimal, with higher
> > protein concentration (5mg/ml). Although the 1.2s peak is not there
> > anymore, the main peak is at 1.4s instead of the expected 1.7s. Also
> > the fit was quite bad with rmsd around 0.03.  For both experiments, I
> > had 0.02% Tween in the reference buffer.
> >
> > Is it possible that the 1.2s peak is due to the Tween absorbance at
> > the region between the meniscuses of reference and sample sectors? I
> > have not tried with the same column height in both sectors. It is hard
> > to match them exactly. Would it be better to run this kind of
> > experiment without including Tween 80 in reference buffer and note the
> > potential 2s peak as the Tween peak? For my protein of 1.7s, it could
> > be still difficult. Any input will be appreciated.
> >
> >
> >
> > Qin "Chin" Zou
> >
> > Eli Lilly and Co.
> >
> >
> >
> >
> >
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