[RASMB] High sucrose concentrations in sed velocity experiments

[Tom Laue]

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
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