[RASMB] high salt in sedimentation velocity

[Peter Schuck]

Hi Chin,

I doubt that 0.5M NaCl would require it, and I dont' know about the 0.5M 
Na2SO4. Unless somebody else has more specific knowledge about this, to be 
sure I suggest the following experiment:

Fill a cell with your buffer in the sample side, and water in the reference 
side. Run it at the rotor speed of your later experiment with protein 
(assuming it is very high), use the interference optics to visualize and 
acquire data on the buffer redistribution. Use the single species model in 
sedfit to determine an average s and D value for the buffer component. (You 
might need that later for the density gradient model.) We're assuming all 
buffer components sediment similarly, which may not be exactly true, but 
that's the approximation we have to make. I think it's actually reasonably 
good for this purpose.

In any case, in sedfit, you'll get the estimated total signal of the 
sedimenting material (which will be the buffer component). After having 
subtracted all systematic noise components, I would compare that with the 
gradient you see (fringes at the bottom minus fringes at the meniscus). For 
0.15M NaCl I seem to remember something like a gradient of 10 fringes out 
of 100 or so. For example, with these values, it would mean that we'd have 
a 10% concentration difference, or less than from about 140 to 160 mM, 
across the cell. Now use sednterp and calculate what density difference 
that would correspond to, and what the maximal difference in s would be 
near to the meniscus and bottom.

If your concern is not the precision of s-values, but perhaps faithful 
recovery of trace components, I would do a simulation to mimic 
sedimentation of some macromolecular mixture using the experimentally 
measured (average) s and D values, along with the density and viscosity 
increments of your buffer (which one could take from sednterp, or perhaps 
determine experimentally by viscosimetry and densitometry of buffer at 
different dilutions).

Regards,
Peter



At 08:53 PM 12/6/2005, you wrote:
>Dear all,
>What salt concentrations are considered high enough that the density 
>gradient has to be used in sedfit (dynamic gradient)? For example, 0.4M 
>Na2SO4 or 0.5 NaCl? Thanks.
>
>Qin "Chin" Zou
>Eli Lilly and Co.
>_______________________________________________
>RASMB mailing list
>RASMB at rasmb.bbri.org
>http://rasmb.bbri.org/mailman/listinfo/rasmb
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