Figure 1. A schematic overview of the steps involved in measuring peptide binding to MHCII molecules by fluorescence polarization.

(A) Set up the binding competition assay as shown in Table I and incubate at 37 °C for 72 hours. (B) Read FP on a 96-well microplate reader, which has been calibrated, and convert the FP values into Relative binding using the equation indicated. (C) Fit the competition curve to obtain IC₅₀. Peptide 1 represents a high affinity peptide, as IC₅₀ is 139nM; peptide 2 has no binding affinity as no competition is observed; peptide 3 has an intermediate affinity with an IC₅₀ of 1709nM.