I am trying to develop a fluorescence polarization assay. I have optimized the assay buffer conditions. The Kd for my protein-peptide interaction is around 5uM. I tried displacing FITC-peptide with unlabelled form for assay verification. There is no displacement at all. Why is there no displacement?
Hi Peter,
If the unlabelled peptide is displacing its labelled protein-bound counterpart, the FP signal should decrease. But I do not see any decrease with the signals over a wide range of unlabelled peptide conc. I am using Synergy4 instrument for my FP measurements. I have also attached my data sheet for the displacement assay.
It looks to me like you were getting competition by the unlabeled peptide in the range 0-30 uM, as judged by the concentration-dependent decrease in polarization over that range. At 60-120 uM peptide, however, the polarization increased again. This was possibly due to insufficient peptide solubility. The turbidity of the insoluble peptide would cause light scattering. Scattered light is highly polarized, causing the polarization to increase.
You may need to use a peptide with greater solubility. Perhaps you can add a few charged residues to the end.
But the Kd is 5uM and till 30uM of unlabelled peptide, the decrease in FP signal is not sufficient. Is this okay?
I have attached another file for displacement assay. There the peptide is diff but the protein is the same. I had optimized the assay conditions for that pair of interaction as well, with Kd=38nM. There, at higher unlabelled peptide conc. (above 3uM), the peptide tends to aggregate (checked with DLS) and so FP signal increases. But, given the Kd of 38nM, till 3uM of unlabelled peptide there is only 20% displacement observed, with the signal getting stabilized between 0.4-3uM. Is this normal?
Is this and the previous peptide displacement data a result of non-specific binding of FITC-peptide to my protein? or its just due to the unlabelled peptide solubility?
Thank you.
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