Fluorescence polarization assays are useful tools to screen inhibitors of protein-peptide interactions. However, false positives that arise due to compound interference or non-specific structural changes necessitate counter-screens. Here, we present a method to identify false positives of a primary screen for pRb-E2F interaction. It uses a rhodamine-coupled E2F peptide and a second Rb-binding peptide, E7, linked to fluorescein as internal control for non-specific inhibitors.
Screening of the hits against the second peptide site, E7, identified non-specific inhibitors, which caused gross structural changes to the protein. The combination of rhodamine-E2F and fluorescein-E7 with pRb in the same well allowed rapid selection of specific inhibitors, and minimized reagent usage. Identification of these non-specific inhibitors dramatically reduced the downstream work load.