Author:

Keywords:

Biochemical Research Methods, Biochemistry & Molecular Biology, CATALYTIC SUBUNIT, Life Sciences & Biomedicine, Science & Technology, SPECIFICITY, STRUCTURAL BASIS, Cell Biology, Molecular/Chemical Probes, Protein Biochemistry, Humans, HEK293 Cells, Luciferases, Protein Interaction Mapping, Protein Phosphatase 1, High-Throughput Screening Assays, Protein Binding

Abstract:

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.