I am using a chemical tagged with fluorophore for FP assay. The chemical is a known binder for the protein. After optimizing G-factor to 0.91 for improving the FP values, I observed in subsequent experiments that the intensity values for both parallel and vertical are becoming half in presence of the protein and the trend continues with concentration.
Also, when titrated against a inhibitor for interaction, the intensities increase with increase in the inhibitors concentration.
Although my dose-response curve is looking good, I doubt it is not correct in principle because of variation in intensities. Please suggest troubleshooting ideas for it.
Thanks
One cause for a fluorescence decrease with increasing concentration of titrant is the inner filter effect, which is a decrease in fluorescence due to absorbance of light by the the titrant.
Another cause is dynamic quenching, whereby the titrant deactivates the excited state by a transient molecular collision.
A third possibility is that the quantum yield of the fluorophore in the bound state is lower than in the free state (this could be viewed as saturable static quenching). This effect could be used to measure the affinity of the interaction, once the other 2 effects are ruled out, instead of the polarization or anisotropy change.
It is also possible to correct the anisotropy change for the difference in fluorescence intensities between bound and free fluorophores, as long as you know the ratio between them.
Thank you Adam B Shapiro
I was wondering if I can titrate a non-specific protein to check the binding-induced effects. In that case it should give me opportunity to measure the affinity in the third approach mentioned by you.
That might be helpful, if the nonspecific protein has similar characteristics as the protein of interest. However, an even better method would be to include a highly saturating concentration of a high-affinity, non-fluorescent ligand to block the active site. Then, any effect you observe on the fluorescence would be attributable to effects other than binding.
In general, because fluorescence-based binding assays can be affected by so many confounding effects, you should always check that the signal is due to specific binding by competing it with a ligand known to bind to the site of interest on the protein.
So, when I titrated a non-fluorescent competing ligand with nM affinity, the loss of intensity was restored at highest concentration! Is this effect then attributable to binding-independent sources or events.
If I understand correctly, the fluorescent ligand had lower fluorescence intensity in the presence of the receptor than in its absence, and when the high-affinity, non-fluorescent competing ligand was added, the fluorescence intensity of the fluorescent ligand returned to the level it had in the absence of the receptor. This result is consistent with the fluorescence intensity being lower in the bound state than in the free state. This phenomenon allows you to measure the affinity of binding of the fluorescent ligand by means of its fluorescence intensity change. You can also use the effect to measure the affinity of competing, non-fluorescent ligands.
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