Looks like positive cooperativity to me. If binding at one site increases affinity at the second site, then intermediate concentrations of your unlabelled ligand can (seemingly paradoxically) *increase* binding of your tracer. At higher levels still, the unlabelled ligand outcompetes the tracer as expected. See figure 1 in http://molpharm.aspetjournals.org/content/70/5/1783.
Hope this helps, Tristan Tristan Croll Research Fellow Cambridge Institute for Medical Research University of Cambridge CB2 0XY > On 21 Jun 2017, at 20:20, megha abbey <abbey...@gmail.com> wrote: > > Hello All, > > This is an off-topic question. I have some issues regarding Fluorescence > Polarization competitive displacement assay and would need some advice. > > I have developed an in vitro fluorescence polarization based assay using a > N-terminus labelled FITC peptide. The peptide is 21 amino acids long and the > binding protein is 50 Kda in size. The Kd for interaction is 1 uM. I am > further running a competitive displacement assay using the exactly same > unlabelled peptide. Here, with increasing concentration of the unlabeled > peptide, the polarization signal first increases and then shows a sharp > decline. Attached is the raw data file for the above. > > I believe that if the unlabelled peptide had been aggregating, the > polarization signal would increase but not drop. If the binding would have > been non-specific, then the unlabelled peptide should not displace at all, > but here I see an increase followed by a decrease in the signal. What does > this increase and sharp drop in polarization signify and how do I fix this? > Please help. > > I have checked the polarization for titration of the unlabelled peptide mixed > with fixed conc. of FITC-peptide (no protein added). Here, the polarization > signals are the same for the entire range of unlabeled peptide. I have also > tried incubating the unlabelled peptide with the protein (for ~15min) first > followed by addition of FITC-peptide, but the results are the same. > > Thank you, > Megha > <unlabelled displ..xlsx>
