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<TITLE>Re: [RASMB] fringe deviation</TITLE>
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<BLOCKQUOTE>Jack -<BR>
<BR>
I agree very much with your argument that one should always do velocity first. It is a great way of spotting what species are there, <I>and</I> you can get a Kd value out. So you may then be in a good position to design your equilibrium work (as you say, if not too much junk is present!). <BR>
<BR>
Just one little extra point. The hydrodynamic non-ideality is not usually a limiting problem. In 100 mM salt the monomer s-c behaviour is predictable, whilst under conditions (e.g. very low salt) where simple theory will not work, then you can still get on pretty well with an empirical s-c curve for the monomer - to which one can normally get access. But strangely enough not only do you not <I>have</I> to work above Kd, it is actually better in practice if you work <I>below</I> Kd. Even a log or two below. This derives from the fact that one normally knows how the monomer species behaves, and if one is nearer to monomer than dimer then the fitting algorithm is rather insensitive to the exact values which you assume for s(0)-dimer and ks-dimer.<BR>
<BR>
Regards to all<BR>
<BR>
Arthur<BR>
<TT><BR>
<BR>
This burst of emails on fringe deviations has touched on a comparison of<BR>
SE vs SV, and the ease of including or avoiding hydrodynamic and/or<BR>
thermodynamic nonideality. An essential part of this discussion is<BR>
selection of models. <BR>
<BR>
For example, knowing you have a monomer dimer system often stems from<BR>
having a coiled-coil domain that must induce dimerization. The problem<BR>
is you don't know 1) if it actually works in the context of your<BR>
construct, 2) you don't know if its also prone to aggregation, 3) you<BR>
don't know if it actually makes a trimer or tetramer instead or worse<BR>
yet a mixture of all of them. Yes I agree sed equilibrium analysis has<BR>
fewer parameters and is thus preferable on many levels. But sed<BR>
velocity as a function of concentration gives you a picture of the full<BR>
process including concentration dependence of zones and the presence of<BR>
Junk. If you have appreciable Junk then sed equilibrium will be harder<BR>
to interpret & often the velocity pattern offers selective quantitative<BR>
analysis of regions of the pattern in a way that sed equil will not. <BR>
Many of my systems are not stable over the time of an SE experiment and<BR>
thus SV is preferred.<BR>
<BR>
Thus my opinion is methods should be combined to establish a starting<BR>
point for analysis, a model, a hypothesis. It is also my experience<BR>
that for well behaved systems (I wish I had more of them) you will<BR>
necessarily get the same answer, the same Kd or energetics, from direct<BR>
fitting of SV and/or SE data (the caveat being the extent of<BR>
hydrodynamic nonideality).<BR>
<BR>
One additional point - Bo mentioned doing experiments above the Kd,<BR>
which of course requires knowing the answer in advance, or in fact doing<BR>
experiments over a wider and wider range of concentrations to achieve<BR>
that goal. 3 mm centerpieces have become common tools in my lab for<BR>
this reason. The problem often encountered is that you don't go high<BR>
enough to populate additional species and you stop at dimer in the<BR>
fitting when in fact tetramer or octamer may also be there if you push. <BR>
It is instructive to make species plots where the weight fraction of<BR>
species is plotted vs the total concentration where you can see how well<BR>
you span the Kd. <BR>
<BR>
-------------------------------------------------------------------<BR>
Dr. John J. "Jack" Correia<BR>
Department of Biochemistry<BR>
University of Mississippi Medical Center<BR>
2500 North State Street<BR>
Jackson, MS 39216<BR>
(601) 984-1522 <BR>
fax (601) 984-1501 <BR>
email address: jcorreia@biochem.umsmed.edu <BR>
homepage location: http://biochemistry.umc.edu/correia.html<BR>
dept homepage location: http://biochemistry.umc.edu/<BR>
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