[RASMB] one or two components

[Peter Schuck]

Judith,

I think this has to do with limited information in the sedimentation data 
from not enough separation.

Assuming that there's really most of the material sedimenting at 4.2 S, 
after the first 4.5 hours a lot of this will be gone, but there is still 
significant sedimentation of the slower sedimenting tail from the 3 S 
component.  Also, the later data have a lot of information on the trailing 
part of the diffusion boundary, which can help to determine f/f0 and to 
discriminate diffusion from sedimentation. Finally, including scans where 
everything is gone will remove the correlation with very low s-values (< 
1S) and the baseline offsets.

Therefore, including all the scans so that everything is depleted is 
certainly a good idea and will improve the information a lot, even though 
it seems to the eye that some or most of the material has depleted and the 
scans are 'only' baselines.

There's a fundamental limitation of looking at relatively small molecules 
at low rotor speed.  For the same experiment, simulating the profiles one 
would expect at 50,000 rpm instead of 42,000 rpm, there's significantly 
better separation for an equivalent time-point (i.e. major boundary 
starting to disappear).  This is even better at 60,000 rpm, where one would 
expect baseline-separated peaks for these conditions.   That means that one 
can get away with not including all the scans, if for the scans to be 
considered there's already sufficient separation.  In your case, 
fortunately the tail of the data set just seems to make the difference.

Despite Beckman's 42,000 rpm rating of the standard charcoal-filled Epon 
centerpieces, we regularly use them at 50 and 60,000 rpm for years now 
without any noticeable detrimental effect.  The resolution goes with the 
square of the rotor speed, and that helps a lot.  One just has to watch for 
leaks (which can be done by prior testing at lower rpm).

With the given experimental data, I would try to improve the analysis by 
including knowledge of the molar mass (it seems you know that there are 
45000 and 90000 Da species).  I would try that with the hybrid 
discrete/continuous model in SEDPHAT, using the continuous segments to 
describe trace components outside the species of interest.  That will 
pre-determine the diffusional spread, and might help unraveling the 
underlying s-values.

Regards,
Peter



At 09:01 AM 8/1/2005, you wrote:
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>I've been analyzing some velocity runs that were done at 42 000 rpm, 15 C,
>for about 14 hours. When Iuse Sedfit and analyze the first 49 scans only
>(the first 4.5 hours of the run), I find one, slightly asymmetric peak,
>with S = 3.98 and a lot of material at less that 1.0 S. If, however, I
>use the first 100 scans (9 hours of centrifuging) or all 150 scans, I have
>two peaks, not totally separated, with S values of 2.93 (about 20% of the
>total) and 4.19. In addition, the material at less than 1.0 S has almost
>disappeared. Is this the way you expect species of about 45000 and 90000
>to behave during a velocity run? Does this mean that one should never try
>to use Sedfit unless the run has gone long enough that everything is at
>the botton of the cell? Judith
>
>Judith Kornblatt
>Dept of Chemistry and Biochemistry
>Concordia University
>7141 Sherbrooke Ouest
>Montreal, Qc H4B 1R6
>Tel: 1 514 848 2424, ext 3384  FAX: 1 514 848 2868
>
>
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***********************************************************
Peter Schuck, PhD
Division of Bioengineering & Physical Science
National Institutes of Health
Bldg. 13 Rm. 3N17
13 South Drive
Bethesda, MD 20892 - 5766
Tel: (301) 435-1950
Fax: (301) 480-1242
email: Peter_Schuck at nih.gov
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