Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Frankfurt, Germany.
Structural Genomics Consortium, BMLS, Goethe University Frankfurt, Frankfurt, Germany.
Methods Mol Biol. 2024;2845:203-218. doi: 10.1007/978-1-0716-4067-8_17.
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 FP assays. In addition, a case study on peptide interaction with human Atg8 proteins (LC3/GABARAPs) is described. Finally, data analysis and quality control of FP assays are discussed for the proper calculation of K values for the measured compounds.
自噬修饰物（Atg8 家族蛋白）与其天然配体（肽和蛋白质）或小分子之间相互作用的特性对于深入了解选择性自噬机制以及设计可能影响细胞自噬过程的 Atg8 抑制剂非常重要。荧光偏振（FP）测定法是一种快速、经济高效且强大的方法，可提供针对人类 Atg8 蛋白的小分子和肽配体的亲和力和选择性信息。本章介绍了 FP 测定法的基本原理。此外，还描述了一个关于肽与人类 Atg8 蛋白（LC3/GABARAPs）相互作用的案例研究。最后，讨论了 FP 测定法的数据分析和质量控制，以正确计算所测化合物的 K 值。
