Hi! I am doing FP/FA to study competitive binding interactions. I am currently doing experiments to determine the optimal buffer for my protein. From the literature, I am trying HEPES and tris buffers (I'm trying both with and without BSA). When I plot the mP values based on protein concentration, even at my lowest protein concentration, I am not getting an mP value of zero; they mostly start at 200mP and go higher.
Is there a reason why my mP value will not reach zero? Do I need to just continue to lower my protein concentration, or is there an issue with my buffer?
Thank you in advance!
Hi Chinyere,
For determining if the fluorescence polarization is working, you have to do saturation binding assay i.e., titrating fixed concentration of fluorescence probe with increasing concentration of protein. I am not sure if you are currently measuring the polarization in presence of probe. If there is no probe, the fluorescence signal will be too low and the calculated polarization will be always around 200-300 mP (regardless of buffer used). If you are using probe, then you should try at lower protein concentration. At this point, I don't think there is any issue with the buffer. You can also compare the fluorescence signal of buffer alone, buffer+probe and protein+probe mixture. If the system worked well, you can see a nice looking saturation curve.
Good luck with your experiments.
There are two ways to measure fluorescence polarization (FP) or anisotropy (FA): absolute and relative.
To measure absolute FP or FA, you must apply the G-factor correction, which takes care of the differential sensitivity of detecting horizontal and vertical intensities by the instrument. Measurements of absolute FP can range from -0.33 to +0.5. Measurements of absolute anisotropy can range from -0.25 to +0.4. There is no particular reason why the FP or FA measurement of the probe should be zero in the absence of protein. For an organic fluorophore, it is likely to be greater than zero.
To use FP or FA for a binding measurement, it is sufficient to measure relative FP or FA, since you only need to know the change in the value. It is therefore not necessary to apply a G-factor correction. Some plate readers do not even have the capability to make this correction, since it is unnecessary for the purpose for which the instrument is normally used, namely ligand competition assays.
So don't worry that your measurement is not zero when there is no protein there to bind the fluorescent ligand.
However, you should worry about whether the protein itself contributes to the FP or FA because of light scattering. I suggest that when you do your protein titration, you do it in the presence and in the absence of the fluorescent ligand to see whether there is any signal coming from the protein alone. Look at the individual fluorescence measurements (horizontal and perpendicular), not just the FP or FA.
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