[RASMB] Multi-wavelength velocity

[Peter Schuck]

Hi Andrew,

yes, that would be nice, in particular for sedimentation 
equilibrium.  However, it would not solve the problem completely because of 
the need to repeatedly scan each cell at each wavelength, either for 
multiple rotor speeds in sedimentation equilibrium, or to repeatedly 
observe the migration at each wavelength.  Essentially, in this case the 
shifts in wavelength for all cells would be in sync, but not necessarily 
absent.

Fortunately, at least with our centrifuges we have not seen that to be too 
much of a problem, as long as you can stick with wavelengths that are on a 
minimum or maximum of the extinction profile, since there a few nm movement 
doesn't matter too much.  Also, we've had good success using 230 nm, and I 
believe that this is due to the fact that we have a relatively sharp 
emission line in combination with a wide monochomator bandwidth, for which 
also the choice of particular nominal wavelength may not affect too much 
the amount of light going through, as long as the major line is still 
within the window.  [That means the nominal wavelength may not necessarily 
be the wavelength of most of the light... and consequently the wavelength 
of light that we're really observing the absorption of ... ]  We've 
certainly got bad results when using 235 nm in the context of a 
multi-wavelength experiment !  (But can be OK in combination of 235 nm 
(only wavelength) and interference signal).

In the paper
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15613487&query_hl=1
we've described that somewhat, but due to space restrictions not in too 
much detail.  There is one example of data collected at 280 nm, 250 nm, and 
interference, and the global analysis of it.  In some of the scans, the 
wavelength was changed by 1 nm.  What could not be shown are the residuals 
of the fit, which look great and give confidence that the scans are OK.

In any case, we've used that multi-wavelength approach on 4 of our 
centrifuges, with reasonably good result on all.  I should say that in most 
cases on our machines, I don't see the wavelength move by more than 1 
nm.  Also, there is another caution - when using FITC or rhodamine labeled 
proteins, I've observed that the apparent peak wavelength is different on 
different centrifuges, pointing to imperfections in the wavelength 
calibration.  Therefore, we usually take a wavelength scan of the material 
in the particular centrifuge that is being used later in sedimentation 
velocity, and figure out which wavelength setting will bring us to the 
middle of the peak.

Also, in sedimentation equilibrium analysis we don't see a sign that there 
is a problem with that, again, using the precautions for wavelength 
selection indicated above.

Again, I don't know if that's generally true or just for our machines, 
which apparently shift no more than 1 nm during a run.

Best,
Peter




At 06:12 AM 6/13/2005, Leech, AP wrote:
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>Peter Schuck wrote:
>
>>Hi Dror,
>...
>>We've been doing a lot of sedimentation velocity runs scanning 7 cells, 
>>even at 2 or 3 wavelengths simultaneously. We use a radial incrmement of 
>>0.003, and no averages, in the continuous mode. Yes, if you are running 
>>at 50,000 rpm, and have 7 cells and 2 absorbance wavelenghts (+ 1 set if 
>>IF scans in between), you get time intervals of scans of about 15 min.
>...
>>Regards,
>>Peter
>
>Hello Peter, RASMBers,
>
>Do you have trouble with the wavelength re-setting when you use
>multiple wavelengths in velocity experiments?
>
>The XL/A software for both equilibrium and velocity seems to
>collect data at all selected wavelengths for a given cell then
>pass to the next cell; I think it would be nice to have an
>option to collect data from all cells at one wavelength then
>move the monochromator, which would decrease the probability of
>wavelength errors. What do other people think?
>
>Best regards,
>
>Andrew
>
>--
>Dr Andrew Leech                  * Laboratory Manager
>Technology Facility              * Molecular Interactions Laboratory
>Department of Biology (Area 15)  * Tel  : +44 (0)1904 328723
>University of York               * Fax  : +44 (0)1904 328804
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