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<TITLE>Re: [RASMB] standard for sv</TITLE>
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Hi Marcello -<BR>
<BR>
The question you ask is a good one - although not easily answered. I have a paper currently submitted on this general area (absolute s values, what questions can be answered by modelling, and so on). I will try and summarise the points most relevant to your question:<BR>
<BR>
(1) there is no real point including a 'standard' in your experiments, <U>except</U> as noted below, point (3). All you will do is compound the errors (your experiment plus the 'standardisation' experiment). PSL spheres are tempting as standard particles, but their density is so close to that of a typical aqueous solvent that errors in their vbar become very serious. <U>Also</U>, the SE of the estimate of their mean diameter may look encouraging - but not all that encouraging when you think how critical this parameter is when you are trying to predict absolute friction (which is what is at stake), not to mention their absolute mass (oh dear - errors in radius x 3!)<BR>
<BR>
(2) If you are attempting to determine 'absolute' s values, then the biggest errors come in the temperature uncertainty. There is good stuff in the RASMB archives about this. At the end of the day, the accuracy of the XL-I/A temperature system is ±0.5 degrees only - not as good as one would like. But the <I>precision</I> is better than this, and one <U>can</U> up the accuracy by using the Liu & Stafford (1995) procedure.<BR>
<BR>
(3) HYDRO will only predict an absolute s value <U>if</U> one knows the 'hydration' term (aka the 3rd term in the scaled particle theory equation). One never knows this very well - but there is a decent way round this. <U>Provided</U> one has a reference conformer - i.e. a particle of the same (or extremely similar) nature to one's test system (a protein-DNA complex in your case), then one can assume that the 'hydration' term cancels <U>if one measures all one's s values relative to the reference conformer*</U>. That means running (reference conformer) and test species) in the same rotor. It's even better if one does 'difference sedimentation' (see RASMB archives again). Relative s values - for these we are talking down to 0.01% precision, enabling one to test a range of possible models rather critically.<BR>
<BR>
*having pushed one's reference conformer's details through HYDRO, of course<BR>
<BR>
Hope these thoughts are of some help<BR>
<BR>
Arthur<BR>
<BR>
<B>Reference<BR>
</B><BR>
Liu, S, Stafford, W F (1995) An Optical Thermometer for Direct Measurement of Cell Temperature in the Beckman Instruments XL-A Analytical Ultracentrifuge Analyt Biochem 224: 199-202.<BR>
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Arthur J Rowe<BR>
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<BLOCKQUOTE><B>From: </B>Marcelo Nollmann <marcnol@chem.gla.ac.uk><BR>
<B>Organization: </B>University of Glasgow<BR>
<B>Reply-To: </B>marcnol@chem.gla.ac.uk<BR>
<B>Date: </B>Thu, 17 Oct 2002 15:26:13 +0100<BR>
<B>Cc: </B>"'rasmb@rasmb-email.bbri.org'" <rasmb@rasmb-email.bbri.org><BR>
<B>Subject: </B>[RASMB] standard for sv<BR>
<BR>
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Hi everyone,<BR>
I am doing sv experiments on a dna-protein complex (complemented with <BR>
se), and then trying to identify the species by using hydro to model the <BR>
s-value that the possible architectures of the complex would have. I am <BR>
mainly analysing the sv data with sedfit (using c(s) analysis). I was <BR>
wondering if it was possible to<BR>
use a non-reactive standard either in a different cell or mixed with the <BR>
sample that would let me have more certainty on the s-values coming out <BR>
of the analysis. I thought of obvious things like lysozyme or bsa, or <BR>
maybe latex beads. Does anybody have any experience on this? I would <BR>
really appreciate any help or reference to previous work. (I am sorry if <BR>
the question is too naive)<BR>
Thanks a lot in advance,<BR>
Marcelo Nollmann<BR>
Glasgow University<BR>
<BR>
<BR>
-- <BR>
"He decidido enfrentar la realidad, asi que apenas se ponga<BR>
linda me avisan"<BR>
Felipe<BR>
("I've decided to face reality. Can anyone let me know as soon<BR>
as it gets nicer?")<BR>
<BR>
<BR>
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