in the well known formula of inner filter effect the Aexe and Aem are the absorbance value of only drug or both protein and drug solution ?
You already mentioned the required input values to calculated the inner-filter compensated fluorescence intensity. To repeat, the corrected intensity is
Icorr=Iobs*10((Aex+Aem)/2)
Please go through Lakowicz's book. Chapter 2.
This equation is strongly dependent on the instrument. As a result a calibration curve should be used. Please go through this paper http://pubs.rsc.org/en/content/articlehtml/1994/an/an9941900417
When both your dye and quencher absorbs at the same excitation wavelength a quantitative calculation is problematic. Ideally the quencher should not absorb at the excitation wavelength. If it is collisional quenching then lifetime should be measured.
In any titration, the concentration of only one of the two substances is varied. For example, if you are measuring the effect of a drug on the intrinsic Trp fluorescence of a protein, you keep the protein concentration constant and titrate the drug concentration. It is only the increasing absorbance of the drug as its concentration increases that is the cause for concern about the inner filter effect in that experiment.
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