[RASMB] High sucrose concentrations in sed velocity experimen ts

Stoner, Michael mstoner at amgen.com
Mon Nov 14 09:39:02 PST 2005


Matthew-

I agree with Drs. Laue and Philo that you have significant density gradients
and that you need to gather sedimentation, viscosity, and density data for
your sucrose buffer.

Assuming you have those data, I would suggest changing SEDFIT to the
inhomogeneous solvent mode and then generating simulated SV scans that are
representative of your system.  You can then fit these scans both with and
without invoking the inhomo. solvent options in SEDFIT.  By comparing those
results you will gain some insight as to how dramatically the gradients are
affecting the accuracy of your response(s).  (Be prepared to spend
approximately 10x more computation time when fitting with inhomo. solvent
options turned on.)  

If you do not have sedimentation/viscosity/density data for your buffer, it
may still be worthwhile to go through the simulation exercise using
estimates.

I would also like to echo Dr. Laue's question about ionic strength; the
primary charge effect may also be contributing to the lower-than-expected
sedimentation coefficients you observed.

Regards,

Michael Stoner


-----Original Message-----
From: rasmb-bounces at server1.bbri.org
[mailto:rasmb-bounces at server1.bbri.org]On Behalf Of Tom Laue
Sent: Monday, November 14, 2005 9:22 AM
To: Parker, Matthew
Cc: rasmb at server1.bbri.org
Subject: Re: [RASMB] High sucrose concentrations in sed velocity
experiments


Hi Matt-
At 10% sucrose, significant density and viscosity gradients will 
develop. I believe that Sedfit allows for this possibility. On the other 
hand, it is less likely that the areas under the peaks will be shifted 
significantly (unless the peaks overlap, and you are forced to estimate 
the curve shapes) by the density/viscosity gradients. It is possible 
that the relative abundance of the different species/components are 
affected by sucrose. What are the other solvent components? In 
particular, what is the ionic strength of your solution?
I would recommend that you run a series of experiments with increasing 
sucrose concentrations to check if the effects are merely due to bulk 
hydrodynamics, or if there are other interactions occurring. Finally, if 
there continues to be disagreement between your sedimentation and GPC 
results, you should become suspicious of the GPC results.
Best wishes,
Tom Laue

Parker, Matthew wrote:

> Hi. We recently ran sedimentation velocity on some samples that 
> contained 10% sucrose in the buffer. We obtained results for the c(s) 
> distribution that didn’t agree well with what we saw by SEC, and the 
> s-values were also shifted to significantly lower values than what we 
> would expect (and that we have seen in other formulations) for an IgG. 
> It was suggested to us that our sucrose concentration was high enough 
> to create a sucrose gradient in the cell during the course of the run, 
> and that this could cause the relative peak areas in the c(s) plot to 
> be inaccurate.
>
> Does anybody have any comments about this, or suggestions about how 
> high in sucrose content you can go before this starts to become a problem?
>
> Thanks,
>
> Matt
>
> Matthew Parker, Ph.D.
>
> Analytical and Pharmaceutical Sciences
>
> Immunogen, Inc.
>
> Cambridge, MA
>
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-- 
Department of Biochemistry and Molecular Biology
University of New Hampshire
Durham, NH 03824-3544
Phone: 603-862-2459
FAX:   603-862-0031
E-mail: Tom.Laue at unh.edu
www.bitc.unh.edu
www.camis.unh.edu

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