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

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 Ke...

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Autores principales: Dayalan Naidu, Sharadha, Dikovskaya, Dina, Moore, Terry W., Dinkova-Kostova, Albena T.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8980889/
https://www.ncbi.nlm.nih.gov/pubmed/35391936
http://dx.doi.org/10.1016/j.xpro.2022.101265
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author Dayalan Naidu, Sharadha
Dikovskaya, Dina
Moore, Terry W.
Dinkova-Kostova, Albena T.
author_facet Dayalan Naidu, Sharadha
Dikovskaya, Dina
Moore, Terry W.
Dinkova-Kostova, Albena T.
author_sort Dayalan Naidu, Sharadha
collection PubMed
description 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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spelling pubmed-89808892022-04-06 Detection of thermal shift in cellular Keap1 by protein-protein interaction inhibitors using immunoblot- and fluorescence microplate-based assays Dayalan Naidu, Sharadha Dikovskaya, Dina Moore, Terry W. Dinkova-Kostova, Albena T. STAR Protoc Protocol 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). Elsevier 2022-04-01 /pmc/articles/PMC8980889/ /pubmed/35391936 http://dx.doi.org/10.1016/j.xpro.2022.101265 Text en © 2022 The Author(s) https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Protocol
Dayalan Naidu, Sharadha
Dikovskaya, Dina
Moore, Terry W.
Dinkova-Kostova, Albena T.
Detection of thermal shift in cellular Keap1 by protein-protein interaction inhibitors using immunoblot- and fluorescence microplate-based assays
title Detection of thermal shift in cellular Keap1 by protein-protein interaction inhibitors using immunoblot- and fluorescence microplate-based assays
title_full Detection of thermal shift in cellular Keap1 by protein-protein interaction inhibitors using immunoblot- and fluorescence microplate-based assays
title_fullStr Detection of thermal shift in cellular Keap1 by protein-protein interaction inhibitors using immunoblot- and fluorescence microplate-based assays
title_full_unstemmed Detection of thermal shift in cellular Keap1 by protein-protein interaction inhibitors using immunoblot- and fluorescence microplate-based assays
title_short Detection of thermal shift in cellular Keap1 by protein-protein interaction inhibitors using immunoblot- and fluorescence microplate-based assays
title_sort detection of thermal shift in cellular keap1 by protein-protein interaction inhibitors using immunoblot- and fluorescence microplate-based assays
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8980889/
https://www.ncbi.nlm.nih.gov/pubmed/35391936
http://dx.doi.org/10.1016/j.xpro.2022.101265
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