[Rasmb] Tween 80 effect on c(s)

[Qin "Chin" Zou]

Thank you all for your input. That did help clear out many of my thoughts.
First, to answer Tom's questions, I did not have chances to do the
experiments you suggested. And I try not to disturb my samples too much.
Peter and John suggested not including Tween in reference buffer. This may
be a good idea to make things more straightforward. Thanks. Also at 5mg/ml,
the fits were bad, which may indicate the non-ideality. 
As far as the Tween 80 absorbance, I did scan the buffer from 290-230nm in
UV spec and there are some absorbance between 285-260nm and it then really
takes off at < 260nm. This is at Tween concentration of 0.02%.
 
Now I would like to further ask if it is possible to just look at the ratio
such as 280nm/250nm since for Tween this ratio could be quite small while
there are still enough signals for the protein. I would think to do this at
protein concentration around 1-2mg/ml (this protein has an extinction
coefficient of 0.55). Thanks.
 
Chin
 
 
  _____  

From: rasmb-admin at server1.bbri.org [mailto:rasmb-admin at server1.bbri.org] On
Behalf Of Qin "Chin" Zou
Sent: Wednesday, September 21, 2005 9:22 PM
To: RASMB at server1.bbri.org
Subject: [Rasmb] Tween 80 effect on c(s)
 
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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