Case Study: Using Steady-State Flourescence Polarization

In our article on WD40 proteins (Moody, Wolfe, et al., 2021), Steady state flourescence polarization assays were conducted in triplicate 24-point serial dilutions for all six SET1Win peptides against WDR5. Measurements were taken using a SpectraMax i3x plate reader with target peptides tagged with N-terminal sulforhodamine B and C-terminal amides. Steady State FP anisotropy was measured on plates after one hour of incubation in a dark room. Dose response data was graphed and binding affinity for each interaction was determined. This data allowed us to determine the fractional occupancy of binding sites for each of the SET1Win peptides against concentration.