I'm studying the binding dynamics of an enzyme to its substrate by measuring the fluorescence anisotropy of a fluorophore attached to the protein. Since this substrate is a polymer (and/or maybe because of the presence of contaminating protein in the substrate), it exhibits strong light scattering and possibly the inner filter effect at high concentrations.
I was able to correct the light scattering effect by subtracting the emission spectrum of a sample in absence of the fluorophore (for both normal emission and anisotropy emission measurements). However, I don't really know if this is the most appropriate manner to correct this artifact.
I also don't know what is the most suitable way to correct the inner filter effect. So, I would like some help to correct these unwanted effects.
To correct for the inner filter effect experimentally, you can replace the fluorophore in your experiment with some other fluorophore with a similar spectrum that does not interact with the substrate and measure the effect of the substrate concentration on the fluorescence, using the same wavelengths as in your binding experiment. The inner filter effect will cause the fluorescence to decrease with substrate concentration. Calculate a correction factor multiplier for each substrate concentration as the ratio of the expected fluorescence to the measured fluorescence.
My go-to reference for details of fluorescence analysis, including inner filter effects and various ways to correct it:
Principles of Fluorescence Spectroscopy
By Joseph R. Lakowicz
All of the basic equations are in there, or will give you the right references.
Perhaps the definitive treatment of this effect of turbidity on polarization is the 1985 Biophysical Journal article by Eisinger and Flores. In this splendid paper they describe the depolarization caused by both scattering of the excitation and scattering of the emission. They also outline a procedure, based on varying the turbidity level, to correct for these effects.
Article Fluorometry of turbid and absorbant samples and the membrane...
The answer provided by Professor Jameson is gladly endorsed by me. It is lovely to hear from him after all these years of separation and I send him my fond regards!
Josef Eisinger ([email protected])
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