[Rasmb] Tween 80 effect on c(s)

[Peter Schuck]

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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