Detection of thermal shift in cellular Keap1 by protein-protein interaction inhibitors using immunoblot- and fluorescence microplate-based assays

TY - JOUR

T1 - Detection of thermal shift in cellular Keap1 by protein-protein interaction inhibitors using immunoblot- and fluorescence microplate-based assays

AU - Dayalan Naidu, Sharadha

AU - Dikovskaya, Dina

AU - Moore, Terry W.

AU - Dinkova-Kostova, Albena

N1 - We thank the Biotechnology and Biological Sciences Research Council (BB/L01923X/1), Tenovus Scotland (T17/14), and Cancer Research UK (C20953/A18644) for funding. SDN is a Reata Scholar.

PY - 2022/6/17

Y1 - 2022/6/17

N2 - 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).

AB - 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).

KW - Cell Biology

KW - Cell culture

KW - Cell-based Assays

KW - Molecular Biology

UR - https://www.scopus.com/pages/publications/85127517789

U2 - 10.1016/j.xpro.2022.101265

DO - 10.1016/j.xpro.2022.101265

M3 - Article

C2 - 35391936

SN - 2666-1667

VL - 3

JO - STAR Protocols

JF - STAR Protocols

IS - 2

M1 - 101265

ER -