[FDS] A couple of observations

[Tom Laue]

Hi all-
I've had a chance to work with the FDS system a bit this fall, and have 
a couple of observations.

   1. Free fluorescein-type dyes seems to sediment at ~0.1 to 0.2. We
      see this in nearly every sample we analyze.
         1. To determine the amount of free dye using c(s) analysis, set
            the F-ratio to 0 (see attachment). Otherwise, the dye peak
            will be smeared into the 0 s hook.
         2. I've attached a snapshot from a c(s) analysis of a
            fluorescein-labeled protein. The sample had been passed over
            a desalting column prior to analysis. I'm not sure whether
            the dye has hydrolyzed from the protein, or if it is carried
            over the desalting column by non-covalent interactions.
            Anyone have an idea about this? There are a couple of
            comments on the web (e.g.
            http://www.microbiology.emory.edu/altman/jdaWebSite_v3/p_commOnRoederer.shtml#bufferX)
            indicating there may be incomplete free dye removal on
            desalting columns. I do not know if this is a problem generally.
   2. The object lens focus must be set near the right level before
      aligning the dichroic mirror for optimal signal.
         1. A symptom that the dichroic mirror is out of alignment is
            that a Focus scan (reached from Fluorescence/Fluorescence
            focusing...) produces a "double humped" intensity profile.
         2. The correct focus position is usually at the minimum between
            the two humps
         3. If you choose a lens position corresponding to one of the
            two "humps," alignment of the mirror for optimal intensity
            will not get rid of the double humps
         4. Instead, choose a lens position between the two humps, then
            align the dichroic mirror to optimize for intensity. You
            will wind up with a stronger signal and good radial resolution

Best wishes,
Tom

-- 
Department of Biochemistry and Molecular Biology
University of New Hampshire
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