Dear Megha, I am puzzled by the results you presented. If you only see the effect in the presence of your protein, the protein must have something to do with it. The steep decline points to some highly cooperative effect, which might be aggregation/precipitation. Did you check that your protein is still in solution beyond the point of steep decline? Precipitation of your protein may explain what you are seeing.
My 2 cts, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von megha abbey Gesendet: Mittwoch, 21. Juni 2017 21:21 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] off-topic:fluorescence polarization displacement assay 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
