Co-immunoprecipitation assay using endogenous nuclear proteins from cells cultured under hypoxic conditions

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. HIF-1 consists of a heterodimeric oxygen-regulated α subunit (HIF-1α) and constitutively expressed β subunit (HIF-1β) also known as aryl hydrocarbon r...

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Main Authors: Zheng, Xiaofeng, Ho, Calvin Qing Wei, Zheng, Xiaowei, Lee, Kian Leong, Gradin, Katarina, Pereira, Teresa S., Berggren, Per-Olof, Yusuf Ali
Other Authors: Lee Kong Chian School of Medicine (LKCMedicine)
Format: Article
Language:English
Published: 2020
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Online Access:https://hdl.handle.net/10356/138172
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Institution: Nanyang Technological University
Language: English
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spelling sg-ntu-dr.10356-1381722020-11-01T05:21:13Z Co-immunoprecipitation assay using endogenous nuclear proteins from cells cultured under hypoxic conditions Zheng, Xiaofeng Ho, Calvin Qing Wei Zheng, Xiaowei Lee, Kian Leong Gradin, Katarina Pereira, Teresa S. Berggren, Per-Olof Yusuf Ali Lee Kong Chian School of Medicine (LKCMedicine) Science::Medicine Co-immunoprecipitation Molecular Biology Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. HIF-1 consists of a heterodimeric oxygen-regulated α subunit (HIF-1α) and constitutively expressed β subunit (HIF-1β) also known as aryl hydrocarbon receptor nuclear translocator (ARNT), regulating genes involved in diverse processes including angiogenesis, erythropoiesis and glycolysis. The identification of HIF-1 interacting proteins is key to the understanding of the hypoxia signaling pathway. Besides the regulation of HIF-1α stability, hypoxia also triggers the nuclear translocation of many transcription factors including HIF-1α and ARNT. Notably, most of the current methods used to study such protein-protein interactions (PPIs) are based on systems where protein levels are artificially increased through protein overexpression. Protein overexpression often leads to non-physiological results arising from temporal and spatial artifacts. Here we describe a modified co-immunoprecipitation protocol following hypoxia treatment using endogenous nuclear proteins, and as a proof of concept, to show the interaction between HIF-1α and ARNT. In this protocol, the hypoxic cells were harvested under hypoxic conditions and the Dulbecco's Phosphate-Buffered Saline (DPBS) wash buffer was also pre-equilibrated to hypoxic conditions before usage to mitigate protein degradation or protein complex dissociation during reoxygenation. In addition, the nuclear fractions were subsequently extracted to concentrate and stabilize endogenous nuclear proteins and avoid possible spurious results often seen during protein overexpression. This protocol can be used to demonstrate endogenous and native interactions between transcription factors and transcriptional co-regulators under hypoxic conditions. MOE (Min. of Education, S’pore) Published version 2020-04-28T01:11:35Z 2020-04-28T01:11:35Z 2018 Journal Article Zheng, X., Ho, C. Q. W., Zheng, X., Lee, K. L., Gradin, K., Pereira, T. S., . . . Yusuf Ali. (2018). Co-immunoprecipitation assay using endogenous nuclear proteins from cells cultured under hypoxic conditions. Journal of Visualized Experiments, 2018(138), e57836-. doi:10.3791/57836 1940-087X https://hdl.handle.net/10356/138172 10.3791/57836 30124647 2-s2.0-85054507119 138 2018 en Journal of Visualized Experiments © 2018 Journal of Visualized Experiments. All rights reserved. This paper was published in Journal of Visualized Experiments and is made available with permission of Journal of Visualized Experiments. application/pdf
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic Science::Medicine
Co-immunoprecipitation
Molecular Biology
spellingShingle Science::Medicine
Co-immunoprecipitation
Molecular Biology
Zheng, Xiaofeng
Ho, Calvin Qing Wei
Zheng, Xiaowei
Lee, Kian Leong
Gradin, Katarina
Pereira, Teresa S.
Berggren, Per-Olof
Yusuf Ali
Co-immunoprecipitation assay using endogenous nuclear proteins from cells cultured under hypoxic conditions
description Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. HIF-1 consists of a heterodimeric oxygen-regulated α subunit (HIF-1α) and constitutively expressed β subunit (HIF-1β) also known as aryl hydrocarbon receptor nuclear translocator (ARNT), regulating genes involved in diverse processes including angiogenesis, erythropoiesis and glycolysis. The identification of HIF-1 interacting proteins is key to the understanding of the hypoxia signaling pathway. Besides the regulation of HIF-1α stability, hypoxia also triggers the nuclear translocation of many transcription factors including HIF-1α and ARNT. Notably, most of the current methods used to study such protein-protein interactions (PPIs) are based on systems where protein levels are artificially increased through protein overexpression. Protein overexpression often leads to non-physiological results arising from temporal and spatial artifacts. Here we describe a modified co-immunoprecipitation protocol following hypoxia treatment using endogenous nuclear proteins, and as a proof of concept, to show the interaction between HIF-1α and ARNT. In this protocol, the hypoxic cells were harvested under hypoxic conditions and the Dulbecco's Phosphate-Buffered Saline (DPBS) wash buffer was also pre-equilibrated to hypoxic conditions before usage to mitigate protein degradation or protein complex dissociation during reoxygenation. In addition, the nuclear fractions were subsequently extracted to concentrate and stabilize endogenous nuclear proteins and avoid possible spurious results often seen during protein overexpression. This protocol can be used to demonstrate endogenous and native interactions between transcription factors and transcriptional co-regulators under hypoxic conditions.
author2 Lee Kong Chian School of Medicine (LKCMedicine)
author_facet Lee Kong Chian School of Medicine (LKCMedicine)
Zheng, Xiaofeng
Ho, Calvin Qing Wei
Zheng, Xiaowei
Lee, Kian Leong
Gradin, Katarina
Pereira, Teresa S.
Berggren, Per-Olof
Yusuf Ali
format Article
author Zheng, Xiaofeng
Ho, Calvin Qing Wei
Zheng, Xiaowei
Lee, Kian Leong
Gradin, Katarina
Pereira, Teresa S.
Berggren, Per-Olof
Yusuf Ali
author_sort Zheng, Xiaofeng
title Co-immunoprecipitation assay using endogenous nuclear proteins from cells cultured under hypoxic conditions
title_short Co-immunoprecipitation assay using endogenous nuclear proteins from cells cultured under hypoxic conditions
title_full Co-immunoprecipitation assay using endogenous nuclear proteins from cells cultured under hypoxic conditions
title_fullStr Co-immunoprecipitation assay using endogenous nuclear proteins from cells cultured under hypoxic conditions
title_full_unstemmed Co-immunoprecipitation assay using endogenous nuclear proteins from cells cultured under hypoxic conditions
title_sort co-immunoprecipitation assay using endogenous nuclear proteins from cells cultured under hypoxic conditions
publishDate 2020
url https://hdl.handle.net/10356/138172
_version_ 1683493786570719232