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, Albena Dinkova-Kostova (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)
30 Downloads (Pure)

Abstract

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).

Original languageEnglish
Article number101265
Number of pages17
JournalSTAR Protocols
Volume3
Issue number2
Early online date1 Apr 2022
DOIs
Publication statusPublished - 17 Jun 2022

Keywords

  • Cell Biology
  • Cell culture
  • Cell-based Assays
  • Molecular Biology

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