In our experimental set-up, upon addition of fluoropore the fluorescence intensity of our protein increases. Although with increase in temperature, the fluorescence intensities decrease for the same concentration of fluoropore. Can anybody explain the anamoly or suggest equations or formulas to reveal the nature of binding and complexation similar to Stern-Volmer that is used in fluorescence quenching.
Thanks.
it is absolutely normal to see decreasing fluroescence with rising temperature. this is due to preferral of non-radiative deactivation pathsways fore xcited states.
Generally, it is also normal behaviour to see a positive correlation of fluorophore amount and fluorescence intensity. So what is the unexpected aspect in your observations?
I agree with Peter, without the additional information it is impossible to say.
I can only suggest that if "your protein" has absorption/emission to the red of "your fluorophore" you could be seeing FRET from the fluorophore to the protein due to the fluorophore binding the protein. This would also be consistent with the decreased fluorescence from your protein upon heating, i.e. the fluorophore does not bind at higher temperatures and FRET is no longer possible.
This is a long shot, with more info we can provide a better answer.
To add to what has been said. Here is one possibility
Is there a possibility of aggregate formation? Aggregate formation can cause shifts in the wavelength and you might be exciting it giving you higher fluorescence. At higher temperatures, the aggregates might be breaking giving you less fluorescence.
it is absolutely normal to see decreasing fluroescence with rising temperature. this is due to preferral of non-radiative deactivation pathsways fore xcited states.
Generally, it is also normal behaviour to see a positive correlation of fluorophore amount and fluorescence intensity. So what is the unexpected aspect in your observations?
I am also facing the same problem .my ligand is polyphenolic compound.
and in some cases firstly fluorescence intensity decreases and then increases.
so how would I calculate the binding constant and explain this irregular pattern ?
Please anyone who can can help me out in doing my experiments further.
Thanks
Hi,In my experiment i have measured the emission intensity of the fluorophore which was decreasing with the aging time of the oil and viscosity was increasing.But in most of the papers I see a linearly increasing function of emission intensity and viscosity whereas I observe a linearly decreasing function between these two parameters.What could be the reason
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