[RASMB] vbar

Wed Jun 7 06:44:01 PDT 2006

Static light scattering may be simpler as it saves the sedimentation experiment.
However, to feel confident I would do both. Both methods depend exclusively on absolute measurements of parameters (M vers. D and s), should therefore result in similar numbers for M and would in the best case confirm each other...

Hans-Joachim Schönfeld.

-----Original Message-----
From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On Behalf Of Allen Minton
Sent: Wednesday, June 07, 2006 3:21 PM
To: rasmb at server1.bbri.org
Subject: RE: [RASMB] vbar

I second Ewa's suggestion that static, not 
dynamic, light scattering seems to be the method 
of choice here, provided that you have an 
accurate determination of concentration and 
refractive increment.  The ratio of scattering 
intensity to concentration is proportional to 
molar mass and can easily be determined to within 
a few percent in less than 15 min.  We do this 
routinely.  The only caveat is that special 
attention must be paid to sample preparation: for 
best results the sample must be filtered through 
a 0.02 micron filter and degassed by centrifugation just prior to measurement.

Allen Minton

At 03:32 AM 6/7/2006, you wrote:
>Ewa and Peter,
>I agree with both of you, that DLS is not
>precise in the characterization of single 
>components of polydisperse samples. However, I 
>also agree with Peter that the method is far 
>better doing in the analysis of monodisperse 
>samples. To my experience, diffusion 
>coefficients as obtained by DLS are precise and 
>highly reproducible (determination is absolute 
>and is mainly based on measurement of time which 
>can be done with very high precision...).
>Our experience was that the strategy to combine 
>s from AUC with D from DLS resulted in molecular 
>masses that were very close to values as 
>obtained from sedimentation equilibrium runs and 
>I therefore recommend to give it a trial (see 
>table in: Schoenfeld, Poeschl, Mueller: 
>"Quasi-elastic light scattering and analytical 
>ultracentrifugation are indispensable tools...", 
>Biochemical Society Transactions, 26, pp.753-758 (1998)).
>Best regards,
>Hans-Joachim.
> >============================================
> >Dr. Hans-Joachim Schönfeld
> >F. Hoffmann-La Roche Inc.
> >PRBD-E, B93/5.44
> >CH-4070 Basel
> >Switzerland
> >
> >Tel. (+41) 61 688 28 95
> >Fax. (+41) 61 688 90 60
>mailto:hans-j.schoenfeld at roche.com
>
>
>-----Original Message-----
>From: rasmb-bounces at rasmb.bbri.org
>[mailto:rasmb-bounces at rasmb.bbri.org] On Behalf Of Peter Schuck
>Sent: Tuesday, June 06, 2006 11:08 PM
>To: Ewa Folta-Stogniew; Chad Brautigam
>Cc: rasmb at server1.bbri.org
>Subject: Re: [RASMB] vbar
>
>
>Ewa,
>
>I agree with you that one maybe should not use a size-distribution 
>model in that case.  However, this limitation is not one of DLS, but 
>the analysis method used.
>
>  From the sedimentation velocity, it should be possible to assess very 
>well how monodisperse the peak is.  If it appears to be pretty much a 
>single species (without very large aggregates, or those could perhaps 
>be filtered out), then one can do the DLS analysis nicely with a single 
>species model.  For example, both SEDFIT and SEDPHAT allow you to use 
>those discrete models and fit DLS autocorrelation functions, and I 
>believe some instrument makers also supply software that can give a 
>single diffusion coefficient, rather than a distribution.  That's what 
>I would try to combine with s.
>
>Peter
>
>
>At 04:32 PM 6/6/2006, Ewa Folta-Stogniew wrote:
> >Peter and Chad,
> >
> >I maybe too pessimistic, but I do not believe that DLS
> >has enough precision to distinguish 21 vs. 24
> >configuration. The standard fitting protocol for Rh determination is 
> >for Gaussian distribution of sizes with 15% SD, which would encompass 
> >sizes for both configurations making them virtually identical.
> >
> >Static LS for MW determination maybe an option given
> >the experiment is done with accuracy at least +/- 3%.
> >
> >
> >Ewa
> >
> >--- Peter Schuck <pschuck at helix.nih.gov> wrote:
> >
> > > Hi Chad,
> > > if the oligomer is relatively pure, would it be
> > > possible to do
> > > DLS? Perhaps taken together with s, that would give another quick 
> > > way of confirming M, instead of equilibrium sedimentation.
> > > I don't think enclosed
> > > solvent adds to the buoyant molar mass, therefore it
> > > would not contribute
> > > an error to the vbar. However, due to error
> > > propagation from vbar to the
> > > (1-vbar*rho) term, small errors in vbar are
> > > amplified; my feeling is that
> > > this usually could account for a few percent
> > > uncertainty (i.e. in the
> > > ballpark of 1/24).
> > > Peter
> > >
> > > At 02:45 PM 6/6/2006, you wrote:
> > > >Hello, All,
> > > >
> > > >Sorry if this is a rudimentary question. I have
> > > been using velocity
> > > >sedimentation to examine the oligomeric states of a
> > > protein and
> > > >mutants thereof. Some mutants are trimers, and the
> > > molecular weight
> > > >estimates given by sedfit (either a c(M)
> > > distribution or a discrete
> > > >species model) are very reasonable. However, based
> > > on a crystal
> > > >structure, we expect the wild-type to be a 24-mer.
> > > Sedfit
> > > >consistently underestimates the molecular weight (
> > > I get something
> > > >more akin to 21-mer).
> > > >
> > > >I assume that there are at least to possibilites
> > > here:
> > > >
> > > >1. The crystal structure is wrong, and the thing
> > > really is a 21-mer
> > > >in solution.
> > > >
> > > >2. The vbar calculated by Sednterp is inaccurate-
> > > it is not
> > > >accounting for the fact that some of the volume of
> > > the 24-mer is not
> > > >taken up by protein, but by solvent. The vbar is
> > > therefore
> > > >significantly too low, with adverse effects on the
> > > MW calculation.
> > > >
> > > >Does anyone know if there is a more accurate way to
> > > estimate vbar in
> > > >cases of large macromolecular assemblies? Can our
> > > crystal structure
> > > >help us out in any way?
> > > >
> > > >BTW, yes, I know that sed. equilibrium might be the
> > > preferred
> > > >approach in this case, but instrument time is
> > > limited at the moment.
> > > >
> > > >Thanks,
> > > >Chad
> > > >
> > > >
> > > >==================================
> > > >Chad A. Brautigam, Ph.D.
> > > >Research Scientist
> > > >The University of Texas
> > > >Southwestern Medical Center at Dallas
> > > >5323 Harry Hines Blvd.
> > > >Dallas, TX 75390
> > > >Office:  (214) 645-6384
> > > >Fax:      (214) 645-5383
> > > >
> > > >
> > > >
> > > >
> > > >_______________________________________________
> > > >RASMB mailing list
> > > >RASMB at rasmb.bbri.org http://rasmb.bbri.org/mailman/listinfo/rasmb
> > > _______________________________________________
> > > RASMB mailing list
> > > RASMB at rasmb.bbri.org http://rasmb.bbri.org/mailman/listinfo/rasmb
> > >
> >
> >
> >__________________________________________________
> >Do You Yahoo!?
> >Tired of spam? Yahoo! Mail has the best spam protection around 
> >http://mail.yahoo.com
>_______________________________________________
>RASMB mailing list
>RASMB at rasmb.bbri.org http://rasmb.bbri.org/mailman/listinfo/rasmb
>_______________________________________________
>RASMB mailing list
>RASMB at rasmb.bbri.org http://rasmb.bbri.org/mailman/listinfo/rasmb
_______________________________________________
RASMB mailing list
RASMB at rasmb.bbri.org http://rasmb.bbri.org/mailman/listinfo/rasmb