High-Throughput Screening for LC3/GABARAP Binders Utilizing the Fluorescence Polarization Assay

The characterization of interactions between autophagy modifiers (Atg8-family proteins) and their natural ligands (peptides and proteins) or small molecules is important for a detailed understanding of selective autophagy mechanisms and for the design of potential Atg8 inhibitors that affect the autophagy processes in cells. The fluorescence polarization (FP) assay is a rapid, cost-effective, and robust method that provides affinity and selectivity information for small molecules and peptide ligands targeting human Atg8 proteins. This chapter introduces the basic principles of FPFluorescence polarization (FP) assays. In addition, a case study onFluorescence polarization (FP) peptide interaction with human Atg8 proteins (LC3LC3/GABARAPsGABARAPs) is described. Finally, data analysis and quality control of FPFluorescence polarization (FP) assays are discussed for the proper calculation of Ki values for the measured compounds.