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 T. Dinkova-Kostova

doi:10.1016/j.xpro.2022.101265

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 T. Dinkova-Kostova^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

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 language	English
Article number	101265
Journal	STAR Protocols
Volume	3
Issue number	2
DOIs	https://doi.org/10.1016/j.xpro.2022.101265
Publication status	Published - 17 Jun 2022
Externally published	Yes

ASJC Scopus subject areas

General Neuroscience
General Biochemistry,Genetics and Molecular Biology
General Immunology and Microbiology

Keywords

Cell Biology
Cell culture
Cell-based Assays
Molecular Biology

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10.1016/j.xpro.2022.101265

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

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).",

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author = "\{Dayalan Naidu\}, Sharadha and Dina Dikovskaya and Moore, \{Terry W.\} and Dinkova-Kostova, \{Albena T.\}",

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year = "2022",

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doi = "10.1016/j.xpro.2022.101265",

language = "English",

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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 T.

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

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ER -

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

Abstract

ASJC Scopus subject areas

Keywords

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