[Rasmb] Tween 80 effect on c(s)

[John Philo]

Qin,
 
I agree with Peter's response, but I wanted to point out some additional
issues. It is quite difficult to get good cancellation of the micelle
signals by having Tween in the reference buffer, particularly for absorbance
studies. Most, if not all, of the absorbance of Tween is due to impurities,
and the absorbance varies markedly depending on the manufacturer and even
lot-to-lot. Unless your reference buffer is made from the very same bottle
of Tween, it is likely it won't match the Tween in the sample. Further,
Tween also makes the solutions very good at picking up UV-absorbing
extractables from plastic tubes, syringes, etc. which creates another source
of sample/reference differences. Even if everything does match perfectly,
you won't get perfect cancellation of the micelle signals unless you match
the meniscus positions in sample and reference too.
 
This has important consequences on the software side, because it is all too
easy to end up with NEGATIVE signals from the micelles (more detergent, or
at least more absorbance, on the reference side). The c(s) algorithms cannot
handle negative signals (all concentrations are forced to be positive). Thus
if you do have any negative signal (which you always will if the meniscus
positions are not matched) then the fitting algorithm cannot possibly
accurately represent the data and you will very likely get some sort of
false peak. Because of these issues I often use a buffer without the Tween
as the reference sample, which ensures the micelle signals are positive. 
 
That 2 S value for the micelles you cited is in fact experimental, not
calculated, but as Peter said the actual micelle size varies with solvent
conditions in addition to the usual density/viscosity effects. Definitely
you should run your buffer versus water to establish the exact micelle
sedimentation coefficient for your conditions. 
 
Overall though when your major protein component has a sedimentation
coefficient close to that of the micelles you likely to always have
significant interference from them.
 
John
 
 
-----Original Message-----
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 7:22 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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