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<BLOCKQUOTE><FONT COLOR="#800000">Greetings all<BR>
<BR>
Sorry to pick up this matter a bit late - have been away for a couple of weeks (holiday!).<BR>
<BR>
Just to say that whilst the ± <I>precision</I> for determination of s values is indeed around 0.5%, the value quoted by John, the <I>absolute accuracy</I> is several times worse that this. Which does affect M values if you combine s (AUC) with D (from DLS). Less of a problem in some ways if you do it all from AUC, as some of the errors self-cancel.<BR>
<BR>
This issue has been discussed in<BR>
<BR>
N Errington, A J Rowe (2003) "Probing conformation and conformational change in proteins is optimally undertaken in relative mode" European Biophysics Journal 32 (5) 511-517<BR>
<BR>
Regards to everyone<BR>
<BR>
Arthur<BR>
<BR>
</FONT></BLOCKQUOTE><FONT COLOR="#800000">-- <BR>
*************************<BR>
Arthur Rowe<BR>
Lab at Sutton Bonington<BR>
tel: +44 115 951 6156<BR>
fax: +44 115 951 6157<BR>
*************************<BR>
</FONT><BR>
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<BR>
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<BLOCKQUOTE><TT>John, <BR>
<BR>
--- John Philo <jphilo@mailway.com> wrote:<BR>
<BR>
> Ewa,<BR>
> <BR>
> Yes I think you have missed a key point. The point<BR>
> was NOT to estimate<BR>
> stoichiometry based on the actual Rh values and some<BR>
> calibration curve of Rh<BR>
> versus M. Rather, Peter's proposal was to get the<BR>
> molar mass by taking the<BR>
> ratio of the sedimentation coefficient (from SV of<BR>
> course) to the diffusion<BR>
> coefficient (measured directly by DLS), using the<BR>
> Svedberg equation. <BR>
> <BR>
thank you for the clarification. <BR>
<BR>
> For that approach in order to distinguish 21-mer<BR>
> from 24-mer you need only<BR>
> measure the diffusion coefficient to ~8% accuracy.<BR>
> (The sedimentation<BR>
> coefficient will easily be accurate to 0.5% or<BR>
> better.) The drawback to this<BR>
> approach is that it still requires an accurate vbar<BR>
> value to get an accurate<BR>
> true molar mass.<BR>
> <BR>
<BR>
wouldn't this be the starting point of the difficulty<BR>
that Chad described? <BR>
<BR>
> But actually yes I do find that the precision of the<BR>
> diffusion coefficients<BR>
> from DLS (and therefore Rh values too) can be about<BR>
> 2%. And as Hans<BR>
> Schoenfeld said, for homogeneous samples the<BR>
> diffusion coefficients from DLS<BR>
> do generally agree quite well with those from<BR>
> sedimentation velocity.<BR>
> <BR>
> But overall I would say static light scattering<BR>
> (after SEC or using Allen<BR>
> Minton's approach) is probably the easiest and most<BR>
> reliable approach to<BR>
> resolving this discrepancy.<BR>
> <BR>
<BR>
thank you, Ewa<BR>
<BR>
> John<BR>
> <BR>
> -----Original Message-----<BR>
> From: rasmb-bounces@rasmb.bbri.org<BR>
> [mailto:rasmb-bounces@rasmb.bbri.org] On<BR>
> Behalf Of Ewa Folta-Stogniew<BR>
> Sent: Wednesday, June 07, 2006 9:07 AM<BR>
> To: Schoenfeld, Hans-J.; Allen Minton;<BR>
> rasmb@server1.bbri.org<BR>
> Subject: RE: [RASMB] vbar<BR>
> <BR>
> <BR>
> Hi all,<BR>
> <BR>
> my point was that for such big oligomers (i.e.<BR>
> composed of many units) one should go after molar<BR>
> mass<BR>
> rather than size to distinguish 21-mer vs. 24-mer. <BR>
> The 24-mer differs by ~14.3% from 21-mer in mass<BR>
> while<BR>
> it differs only by ~5.8% in size. Thus, unless one<BR>
> can measure Rh with accuracy better than 2% (it this<BR>
> doable?), it seems virtually impossible to assign<BR>
> the stoichiometry with<BR>
> confidence.... <BR>
> <BR>
> Am I missing something?<BR>
> <BR>
> Ewa <BR>
> <BR>
> <BR>
> <BR>
> <BR>
> --- "Schoenfeld, Hans-J."<BR>
> <hans-j.schoenfeld@roche.com> wrote:<BR>
> <BR>
> > Static light scattering may be simpler as it saves<BR>
> > the sedimentation experiment.<BR>
> > However, to feel confident I would do both. Both<BR>
> > methods depend exclusively on absolute<BR>
> measurements<BR>
> > of parameters (M vers. D and s), should therefore<BR>
> > result in similar numbers for M and would in the<BR>
> > best case confirm each other...<BR>
> > <BR>
> > Hans-Joachim Schönfeld.<BR>
> > <BR>
> > -----Original Message-----<BR>
> > From: rasmb-bounces@rasmb.bbri.org <BR>
> > [mailto:rasmb-bounces@rasmb.bbri.org] On Behalf Of<BR>
> Allen Minton<BR>
> > Sent: Wednesday, June 07, 2006 3:21 PM<BR>
> > To: rasmb@server1.bbri.org<BR>
> > Subject: RE: [RASMB] vbar<BR>
> > <BR>
> > <BR>
> > I second Ewa's suggestion that static, not<BR>
> > dynamic, light scattering seems to be the method <BR>
> > of choice here, provided that you have an <BR>
> > accurate determination of concentration and <BR>
> > refractive increment. The ratio of scattering <BR>
> > intensity to concentration is proportional to <BR>
> > molar mass and can easily be determined to within <BR>
> > a few percent in less than 15 min. We do this <BR>
> > routinely. The only caveat is that special <BR>
> > attention must be paid to sample preparation: for <BR>
> > best results the sample must be filtered through <BR>
> > a 0.02 micron filter and degassed by<BR>
> centrifugation<BR>
> > just prior to measurement.<BR>
> > <BR>
> > Allen Minton<BR>
> > <BR>
> > At 03:32 AM 6/7/2006, you wrote:<BR>
> > >Ewa and Peter,<BR>
> > >I agree with both of you, that DLS is not<BR>
> > >precise in the characterization of single<BR>
> > >components of polydisperse samples. However, I <BR>
> > >also agree with Peter that the method is far <BR>
> > >better doing in the analysis of monodisperse <BR>
> > >samples. To my experience, diffusion <BR>
> > >coefficients as obtained by DLS are precise and <BR>
> > >highly reproducible (determination is absolute <BR>
> > >and is mainly based on measurement of time which <BR>
> > >can be done with very high precision...).<BR>
> > >Our experience was that the strategy to combine <BR>
> > >s from AUC with D from DLS resulted in molecular <BR>
> > >masses that were very close to values as <BR>
> > >obtained from sedimentation equilibrium runs and <BR>
> > >I therefore recommend to give it a trial (see <BR>
> > >table in: Schoenfeld, Poeschl, Mueller: <BR>
> > >"Quasi-elastic light scattering and analytical <BR>
> > >ultracentrifugation are indispensable tools...", <BR>
> > >Biochemical Society Transactions, 26, pp.753-758<BR>
> > (1998)).<BR>
> > >Best regards,<BR>
> > >Hans-Joachim.<BR>
> > > >============================================<BR>
> > > >Dr. Hans-Joachim Schönfeld<BR>
> > > >F. Hoffmann-La Roche Inc.<BR>
> > > >PRBD-E, B93/5.44<BR>
> > > >CH-4070 Basel<BR>
> > > >Switzerland<BR>
> > > ><BR>
> > > >Tel. (+41) 61 688 28 95<BR>
> > > >Fax. (+41) 61 688 90 60<BR>
> > >mailto:hans-j.schoenfeld@roche.com<BR>
> > ><BR>
> > ><BR>
> > >-----Original Message-----<BR>
> > >From: rasmb-bounces@rasmb.bbri.org <BR>
> > >[mailto:rasmb-bounces@rasmb.bbri.org] On Behalf<BR>
> Of<BR>
> > Peter Schuck<BR>
> > >Sent: Tuesday, June 06, 2006 11:08 PM<BR>
> > >To: Ewa Folta-Stogniew; Chad Brautigam<BR>
> > >Cc: rasmb@server1.bbri.org<BR>
> > >Subject: Re: [RASMB] vbar<BR>
> > ><BR>
> > ><BR>
> > >Ewa,<BR>
> > ><BR>
> > >I agree with you that one maybe should not use a<BR>
> > size-distribution<BR>
> > >model in that case. However, this limitation is<BR>
> > not one of DLS, but<BR>
> > >the analysis method used.<BR>
> > ><BR>
> > > From the sedimentation velocity, it should be<BR>
> > possible to assess very<BR>
> > >well how monodisperse the peak is. If it appears<BR>
> > to be pretty much a<BR>
> > >single species (without very large aggregates, or<BR>
> > those could perhaps<BR>
> > >be filtered out), then one can do the DLS<BR>
> analysis<BR>
> > nicely with a single<BR>
> > >species model. For example, both SEDFIT and<BR>
> > SEDPHAT allow you to use<BR>
> > >those discrete models and fit DLS autocorrelation<BR>
> > functions, and I<BR>
> > >believe some instrument makers also supply<BR>
> software<BR>
> > that can give a<BR>
> > >single diffusion coefficient, rather than a<BR>
> > distribution. That's what<BR>
> > >I would try to combine with s.<BR>
> > ><BR>
> > >Peter<BR>
> > ><BR>
> > ><BR>
> > >At 04:32 PM 6/6/2006, Ewa Folta-Stogniew wrote:<BR>
> > > >Peter and Chad,<BR>
> > > ><BR>
> > > >I maybe too pessimistic, but I do not believe<BR>
> > that DLS<BR>
> > > >has enough precision to distinguish 21 vs. 24<BR>
> configuration. The <BR>
> > > >standard fitting protocol for<BR>
> > Rh determination is<BR>
> > > >for Gaussian distribution of sizes with 15% SD,<BR>
> > which would encompass<BR>
> > > >sizes for both configurations making them<BR>
> > virtually identical.<BR>
> > > ><BR>
> > > >Static LS for MW determination maybe an option<BR>
> > given<BR>
> > > >the experiment is done with accuracy at least<BR>
> +/-<BR>
> > 3%.<BR>
> > > ><BR>
> > > ><BR>
> > > >Ewa<BR>
> > > ><BR>
> > > >--- Peter Schuck <pschuck@helix.nih.gov> wrote:<BR>
> > > ><BR>
> > > > > Hi Chad,<BR>
> > > > > if the oligomer is relatively pure, would it<BR>
> > be<BR>
> > > > > possible to do<BR>
> > > > > DLS? Perhaps taken together with s, that<BR>
> would<BR>
> > give another quick<BR>
> > > > > way of confirming M, instead of equilibrium<BR>
> > sedimentation.<BR>
> > > > > I don't think enclosed<BR>
> > > > > solvent adds to the buoyant molar mass,<BR>
> > therefore it<BR>
> > > > > would not contribute<BR>
> > > > > an error to the vbar. However, due to error<BR>
> > > > > propagation from vbar to the<BR>
> > > > > (1-vbar*rho) term, small errors in vbar are<BR>
> > > > > amplified; my feeling is that<BR>
> > > > > this usually could account for a few percent<BR>
> uncertainty (i.e. <BR>
> > > > > in the ballpark of 1/24).<BR>
> > > > > Peter<BR>
> > > > ><BR>
> > > > > At 02:45 PM 6/6/2006, you wrote:<BR>
> > > > > >Hello, All,<BR>
> > > > > ><BR>
> > > > > >Sorry if this is a rudimentary question. I<BR>
> > have<BR>
> > > > > been using velocity<BR>
> > > > > >sedimentation to examine the oligomeric<BR>
> > states of a<BR>
> > > > > protein and<BR>
> > > > > >mutants thereof. Some mutants are trimers,<BR>
> > and the<BR>
> > > > > molecular weight<BR>
> > > > > >estimates given by sedfit (either a c(M)<BR>
> > > > > distribution or a discrete<BR>
> > > > > >species model) are very reasonable.<BR>
> However,<BR>
> > based<BR>
> > > > > on a crystal<BR>
> > > > > >structure, we expect the wild-type to be a<BR>
> > 24-mer.<BR>
> > > > > Sedfit<BR>
> > > > > >consistently underestimates the molecular<BR>
> > weight (<BR>
> > > > > I get something<BR>
> > > > > >more akin to 21-mer).<BR>
> > > > > ><BR>
> > > > > >I assume that there are at least to<BR>
> > possibilites<BR>
> > > > > here:<BR>
> > > > > ><BR>
> > > > > >1. The crystal structure is wrong, and the<BR>
> > thing<BR>
> > > > > really is a 21-mer<BR>
> > > > > >in solution.<BR>
> > > > > ><BR>
> > > > > >2. The vbar calculated by Sednterp is<BR>
> > inaccurate-<BR>
> > > > > it is not<BR>
> > > > > >accounting for the fact that some of the<BR>
> > volume of<BR>
> > > > > the 24-mer is not<BR>
> > > > > >taken up by protein, but by solvent. The<BR>
> vbar<BR>
> > is<BR>
> > > > > therefore<BR>
> > > > > >significantly too low, with adverse effects<BR>
> > on the<BR>
> > > > > MW calculation.<BR>
> > > > > ><BR>
> > > > > >Does anyone know if there is a more<BR>
> accurate<BR>
> > way to<BR>
> > > > > estimate vbar in<BR>
> > > > > >cases of large macromolecular assemblies?<BR>
> Can<BR>
> > our<BR>
> > > > > crystal structure<BR>
> > > > > >help us out in any way?<BR>
> > > > > ><BR>
> > > > > >BTW, yes, I know that sed. equilibrium<BR>
> might<BR>
> > be the<BR>
> > > > > preferred<BR>
> > > > > >approach in this case, but instrument time<BR>
> is<BR>
> > > > > limited at the moment.<BR>
> > > > > ><BR>
> > > > > >Thanks,<BR>
> > > > > >Chad<BR>
> > > > > ><BR>
> > > > > ><BR>
> > > > > >==================================<BR>
> > > > > >Chad A. Brautigam, Ph.D.<BR>
> > > > > >Research Scientist<BR>
> > > > > >The University of Texas<BR>
> > > > > >Southwestern Medical Center at Dallas<BR>
> > > > > >5323 Harry Hines Blvd.<BR>
> > > > > >Dallas, TX 75390<BR>
> > > > > >Office: (214) 645-6384<BR>
> > > > > >Fax: (214) 645-5383<BR>
> > > > > ><BR>
> > > > > ><BR>
> > > > > ><BR>
> > > > > ><BR>
> > > > ><BR>
> > >_______________________________________________<BR>
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