Articles
Identification Of False Positives In An FP Screen
October 5, 2006
•Application Note: Identification Of False Positives In An FP Screen
Fluorescence polarization (FP) provides a useful tool with which to screen for inhibitors of the interaction of proteins with defined peptides, and many examples have been cited in the literature.1-3 Fluorescence polarization gives a measure of the proportion of peptide found in the bound state in a homogeneous format. However compound interference and non-specific gross structural changes to the protein can give rise to a large number of false positives, which are only identified in later stage biochemical assays. Strategies to eliminate these at an early stage of screening will accelerate the hit to lead process.
Here we present data from an FP screen configured on the POLARstar Galaxy for the interaction of the retinoblastoma tumor suppressor protein (pRB) with E2F peptide. Fluorescein-tagged E2F peptide was used to screen 10,000 small drug like molecules. Hit confirmation strategies based on fluorescence interference and specificity were developed.
Based on the crystal structure, an FP screen was configured for the interaction of recombinant pRb A/B domains with E2F peptide (depicted in yellow in figure 1).4 In addition, a second peptide binding site (E7, depicted in green), distant from the E2F binding pocket, was utilized as an internal control for non-specific inhibitors. Fluorescein-E7 and rhodamine-E2F labelled peptides were synthesized and were used for hit confirmation.
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•Application Note: Identification Of False Positives In An FP Screen
SOURCE: BMG LABTECH, GmbH
