High-Throughput Detection of Ligand-Protein Binding Using a SplitLuc Cellular Thermal Shift Assay

Abstract

The confirmation of a small molecule binding to a protein target can be challenging when switching from biochemical assays to physiologically relevant cellular models. The cellular thermal shift assay (CETSA) is an approach to validate ligand-protein binding in a cellular environment by examining a protein's melting profile which can shift to a higher or lower temperature when bound by a small molecule. Traditional CETSA uses SDS-PAGE and Western blotting to quantify protein levels, a process that is both time consuming and low-throughput when screening multiple compounds and concentrations. Herein, we outline the reagents and methods to implement split Nano Luciferase (SplitLuc) CETSA, which is a reporter-based target engagement assay designed for high-throughput screening in 384- or 1536-well plate formats.

Keywords: CETSA; Cellular Thermal Shift Assay; High-throughput screening; Ligand binding; Luciferase; SplitLuc; Target engagement; Thermal stability; qHTS CETSA.