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
Description: MS-Excel 2007 spreadsheet
