Protocol for analyzing protein-protein interactions by split-luciferase complementation assays in human cell lysates

Summary: Here, we present a lysate-based split-luciferase assay for examining protein-protein interactions (PPIs) in HEK293T cell lysates, exemplified by interactions between subunits of protein phosphatase PP1. We describe steps for storing and re-using lysates, sensor design, assay setup/optimization, and high-throughput screening of compound libraries. We then detail procedures for applying the assay as a research tool to characterize the dynamics of PPIs, which we illustrate with specific examples.For complete details on the use and execution of this protocol, please refer to Claes and Bollen.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.