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

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 Dinkova-Kostova (Lead / Corresponding author)

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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
Number of pages	17
Journal	STAR Protocols
Volume	3
Issue number	2
Early online date	1 Apr 2022
DOIs	https://doi.org/10.1016/j.xpro.2022.101265
Publication status	Published - 17 Jun 2022

Keywords

Cell Biology
Cell culture
Cell-based Assays
Molecular Biology

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10.1016/j.xpro.2022.101265Licence: CC BY

Final Published VersionFinal published version, 2.52 MBLicence: CC BY

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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).",

keywords = "Cell Biology, Cell culture, Cell-based Assays, Molecular Biology",

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

note = "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.",

year = "2022",

month = jun,

day = "17",

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

language = "English",

volume = "3",

journal = "STAR Protocols",

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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

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

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DO - 10.1016/j.xpro.2022.101265

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JF - STAR Protocols

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

Abstract

Keywords

Access to Document

Other files and links

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Method Development for Quantitative Assessment of Protein Turnover in Live Cells at Subcellular Resolution

The Role of the Keap1/Nrf2 Pathway in Tumour Metabolic Adaptation (Joint with University of Cambridge and University College London)

The Spatiotemporal Regulation of the Keap1/Nrf2 Pathway (Joint with University College London)

Cite this

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

Abstract

Keywords

Access to Document

Other files and links

Fingerprint

Projects

Method Development for Quantitative Assessment of Protein Turnover in Live Cells at Subcellular Resolution

The Role of the Keap1/Nrf2 Pathway in Tumour Metabolic Adaptation (Joint with University of Cambridge and University College London)

The Spatiotemporal Regulation of the Keap1/Nrf2 Pathway (Joint with University College London)

Cite this