Fluorescence polarization binding assays for the E3 ligase FEM1C

Feminization-1 homolog C (FEM1C) is a substrate-recognition component of the Cullin 2-RING E3 ubiquitin ligases (CRL2), selectively binding the C-degron containing arginine-terminated motifs to mediate proteasomal degradation of target proteins. Owing to its wide expression, FEM1C holds promise for targeted protein degradation. This chapter describes fluorescence polarization-based binding assays for FEM1C, as well as preparation of the required reagents including the recombinant protein and fluorescent probe. These methods can be applied to identify ligands for FEM1C or adapted for other E3 ligases, facilitating the development of new handles for targeted protein degradation.