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

Dominic-Luc Webb via proteins%40net.bio.net (by dlwebb from canit.se)
Tue Oct 2 04:11:06 EST 2007

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

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?


> > Strangely, when a protein that is known to be a drug target is
> > present, there is a strong increase in polarization (100-300 mP).
> That is what I'd expect.  Binding to a large molecule (i.e.,
> the protein) reduces the rotation rate of the labeled drug,
> thus increasing the polarization.
> > The glycerol data has created an uncertainty about negative
> > data for test proteins that I am screening for drug binding.
> >
> > Is there anyone in here that might have some hints how this
> > lack of change in polarization in glycerol might have come
> > about, maybe some suggestions how to troubleshoot this?
> It could be that the change of solvent to 50% glycerol
> reduces the fluorescence lifetime, thus increasing the
> measured polarization.
> >
> > Dominic
> >

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