kerstin.janisch at bbsrc.ac.uk schrieb:
> Hello,
>> i've got a problem with the fluorescence quenching of tryptophan in
> proteins due to binding other molecules. the assay is not
> reproducable.
What do you mean with that? What do you titrate with what, and what is
irreproducible?
> does anybody know something about this topic and can
> offer some help? the important bit is, is the binding and therefore
> the quenching temperature depending?
Binding of proteins to something - especially to macromolecules, is
usually rather temperature dependent in itself, regardless of the method
used. This is just because
RT ln (K_bind) = Delta G_bind = Delta H_bind - T * Delta S_bind,
and even if Delta S is small, H_bind is usually temperature dependent.
On the other hand, fluorescence in aqueous solution is highly
temperature dependent, since the solvent quenching increases with
temperature.
In a binding-quenching experiment you get both effects when you vary
temperature. I would extent Emir's point about controlling temperature
in that, to my opinion, _every_ experiment using fluorescence needs
thorough temperature control if you want to do quantitative analysis.
If you put an microtiter plate into an ELISA reader that can do
flourescence, it might be sufficient to make sure that room temperature
is constant during on set of experiments - but beware that experiments
done at 18°C in winter and at 27°C on a summer day may not be comparable
quantitatively.
If you use a real fluorescence spectrometer, it should always be
equipped with a thermostatted cell holder.
Bye, Frank
--
> But I don't really see running SETI at Home as practical as Folding at Home. What,
> exactly, would be the benefit of finding intelligent aliens on the other side
> of the galaxy?
Maybe they're broadcasting the principles of protein folding... [from bionet.*]
