I have encountered consistent issues with fluorescence anisotropy measurement. I attached Alexa 488 to my protein and want to study the binding of my protein to lipid vesicles. I did titration which means adding lipids into protein solution and measure the anisotropy value change. The question is I can see anisotropy value changes when high concentration of lipid vesicles were added. The anisotropy value never reaches plateau and the shape of the curve is like a line. Not like a Hill1 equation curve. Anyone knows the reason?
Consider the possibility that the increase in anisotropy is caused by light scattering off the lipid vesicles. Scattered light is highly polarized, so the more vesicles you add, the more highly polarized the light that you detect.
You can check for this by repeating the titration of the lipid vesicles without adding the labeled protein. You can also use that data to correct the anisotropy measurements made with protein + lipids. To do this, you should subtract each of the two fluorescence intensity measurements (parallel and perpendicular) made with lipids alone from those made with lipids + protein at each lipid concentration. Then use those subtracted intensity measurements to calculate the anisotropies.
Could see anything without fluorescence? If I do not add labeled proteins, I do not think anything will be detected.
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