Detection of thermal shift in cellular Keap1 by protein-protein interaction inhibitors using immunoblot- and fluorescence microplate-based assays
Sharadha Dayalan Naidu, Dina Dikovskaya, Terry W. Moore and 1 more
STAR Protocols · 2022
doi:10.1016/j.xpro.2022.101265 (opens in a new tab)
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Abstract
Summary: Pharmacologic inhibition of the protein-protein interaction (PPI) interface of the Keap1:Nrf2 complex, which leads to Nrf2 activation and cytoprotective gene expression, offers a promising strategy for disease prevention and treatment. To facilitate identification and validation of small-molecule Keap1:Nrf2 PPI inhibitors in the cellular environment in a low- and medium-throughput manner, we detail two adapted cellular thermal shift assay (CETSA) protocols, Keap1-CETSA, an immunoblotting-based methodology for detecting endogenous Keap1, and Keap1-Glow CETSA, a microtiter plate assay of overexpressed fluorescently-tagged Keap1.For an example of the use and execution of this protocol, please refer to Dayalan Naidu et al. (2021).
Abstract licence: CC BY-NC
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