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. I am 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.
I believe that if the unlabelled peptide had been aggregating, the polarization signal would increase but not drop. What does this increase and sharp drop in polarization signify and how do I fix this? Please help.
Attached is a raw data file for the above.
Thank you.
Hi Megha, that is indeed a sharp drop. If it were salt, solvent or quenchers, you would expect a gradual drop.
The only things I can think of is that you reached the pI of the protein or the fluorophore. If there is no apparent precipitation, I would look at the pH of the solutions.
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