[Protein-analysis] Re: Drug binding: When fluorescence polarization fails

Marvin via proteins%40net.bio.net (by physchem from verizon.net)
Tue Oct 2 10:50:27 EST 2007


Dominic-Luc Webb wrote:
> Thanks Marvin for your response, and sorry about any confusion I
> might have created in the use of the word "strangely". The
> "strangeness" had only to do with the disjunct between the
> unexpected glycerol data and the expected protein binding data
> using the same labelled drug. Until now, I regarded both of
> these as positive control tests.
> 
> Regarding 50% glycerol, I could be convinced that the fluorescence
> lifetime was changed, but I would need to bring this in line with
> recent experiments. I went back and made some further measurements
> and quantifications together with data I collected earlier. I
> calculate a 1.4 fold increase in fluorescence intensity (FI) when
> measured with normal FI filter set. Using the FP filters to measure
> same samples, there was also an increase in fluorescence intensity in
> both of the two channels (1.35 in the static channel, 1.41 in the
> perpendicular channel). Fluorescence yield must have increased
> concommitantly with a differential shift towards the perpendicular
> channel.
> 
> Considering that last sentence, this assay has thus far not been
> very sensitive to different intensities (e.g., fluorescein over a
> few orders of magnitude). However, the differential shift towards
> the perpendicular channel (1.41/1.35), although small, approaches
> significance and seems in line with your suggestion that fluorescence
> lifetime was decreased. Does this make sense?
> 
> Dominic
When you change the environment around a molecule, energy 
levels change.  Try scanning the absorption and emission 
spectra, which should be different in 50 glycerol vs. water. 
  It isn't easy to generalize about specific changes in 
optical properties.


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