Hi, I want to determine the binding affinity between a shot peptide (10 residues) labelled with fluorescein and a protein (dimeric alpha-beta tubulin). I am doing this by MicroScale Thermophoresis. In practice I am titrating a fixed concentration of the fluorescent peptide (about 350 nM) with increasing concentration of tubulin (from 3.5 nM to 115 microM). 350 nM of peptide alone gives 200 fluorescence unit emission in blu light, however in presence of tubulin the peptide emission decreases with the DECREASING of tubule's concentration. Does anyone have physicochemical explanations for this behaviour? Thank you in advance.
The quantum yield of a fluorophore can be affected by its environment. The environment of the fluorescein dye when the peptide binds tubulin is one in which the quantum yield of the dye is higher than when the peptide is not bound to tubulin. Since the quantum yield of fluorescein is already very high in water at neutral pH or above, I would guess that the fluorescein fluorescence is quenched due to an interaction with the peptide to which it is attached, and that this quenching interaction is eliminated when the peptide binds to tubulin.
Another possibility has to do with pH. Fluorescein loses fluorescence at acidic pH. If the free peptide solution is at an acidic pH, the fluorescein quantum yield will be low. If when tubulin is added the pH rises, the fluorescein quantum yield will increase.
Assuming the observed effect is not an artifact of a pH shift, you can use it measure the Kd of the peptide-tubulin interaction directly by a fluorescence intensity measurement, without microscale thermophoresis.
The quantum yield of a fluorophore can be affected by its environment. The environment of the fluorescein dye when the peptide binds tubulin is one in which the quantum yield of the dye is higher than when the peptide is not bound to tubulin. Since the quantum yield of fluorescein is already very high in water at neutral pH or above, I would guess that the fluorescein fluorescence is quenched due to an interaction with the peptide to which it is attached, and that this quenching interaction is eliminated when the peptide binds to tubulin.
Another possibility has to do with pH. Fluorescein loses fluorescence at acidic pH. If the free peptide solution is at an acidic pH, the fluorescein quantum yield will be low. If when tubulin is added the pH rises, the fluorescein quantum yield will increase.
Assuming the observed effect is not an artifact of a pH shift, you can use it measure the Kd of the peptide-tubulin interaction directly by a fluorescence intensity measurement, without microscale thermophoresis.
Hello Adam, I found your comment very helpful, thank you very much.
Dear sir .. you must attention on condition like pH , environment ...
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