From: John Philo <jphilo@mailway.com>
  To  : Rasmb <rasmb@alpha.bbri.org>
  Date: Sun, 21 Nov 1999 14:44:26 -0800

RE: Help... what's going on?

Art,

What you are seeing is a consequence of the fact that the monochromator is
not coming back to the same wavelength (there is an uncertainty of +/- 1 nm
or so every time it moves). The different extinction coefficients cause the
amplitudes of the scans to jump up and down, particularly when you are on a
steep part of the absorbance spectrum. It only *looks* like the scans are
being taken at the wrong times.

Basically, for this reason you should never do velocity runs at multiple
wavelengths. If you need multiple wavelengths you simply have to do separate
runs. In principle, if all scans for any run are at the same wavelength then
the monochromator doesn't move and this problem goes away (but see my recent
message to RASMB and the responses to that).

Because of this problem you really won't be able to analyze your data by any
method that uses global analysis of multiple scans, since really no two
scans are at the same wavelength. You can, however, analyze a *single* scan
using the second moment or transport methods, SVEDBERG, etc. and at least
get an approximate value of sedimentation coefficient.

John Philo, Alliance Protein Laboratories
www.ap-lab.com


-----Original Message-----
From: Arturo Morales [art@scripps.edu]">mailto:art@scripps.edu]
Sent: Saturday, November 20, 1999 1:55 PM
To: rasmb@alpha.bbri.org
Subject: Help... what's going on?



Dear RASMBers...

I just did a velocity run and something annoying happened...

I scanned 2 wavelengths in three cells, with 2 minute separations between
scans (at 50K) If I plot every tenth scan (even numbers for wavelenght #2)
I get the following: (at 235 nm)

http://schimmel.scripps.edu/art/velocity.jpg

those scans are not evenly spaced as I would expect...

at 280, it is way too noisy, but that's expected since there's no Trp's in
the sample, and the protein is at 30 uM concentration only...I got similar
behaviour from the other 2 cells... (even though they are independently
purified mutants)

We just cleaned the lamp and it showed intensities of about 8-12 thousand
in the scan...

Any ideas? can I trust a fit from this data? is there something wrong with
the machine? can my sample have this effect?  (I did runs about a year ago
on the same protein (different sample, of course) and it did not show this
behaviour...

Thanks!

Art


--
Arturo J. Morales    (RPI '94)       --       art@scripps.edu
Department of Biology - Massachusetts Institute of Technology &
Skaggs Institute for Chemical Biology - Scripps Research Institute
                    http://schimmel.scripps.edu/~art