[Rasmb] Tween 80 effect on c(s)

Peter Schuck pschuck at helix.nih.gov
Tue Sep 27 13:41:00 PDT 2005


John,

just a small note to add to your comment - it is actually possible to fit 
negative signals in the newest version of sedphat (4.02) in the hybrid 
discrete/continuous model, if you check the box  entitled "neg." in the 
field of the discrete species #1.  In this case, this particular Lamm 
equation solution will be multiplied with minus 1.  This option was 
introduced precisely to deal better with situations you describe, where we 
have excess buffer components in the reference.  We encountered this 
problem with samples that were not dialyzed properly (or somebody picked 
the wrong buffer).

I think you're absolutely correct that this can create false peaks, if not 
accounted for.

You can currently download this version from the sedphat site.  Originally, 
I wanted to wait with the official announcement a few days for the 
concurrent update of the website, and for the new release of a slight 
further upgrade to version 4.04, which has a few more bugs fixed and 
slightly revised interface for ITC data analysis.  This will be out in a 
matter of days.

Peter




At 07:22 PM 9/26/2005, you wrote:
>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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