Immunoprecipitation and Western blot-based detection of protein O-GlcNAcylation in cells

Detection of protein O-GlcNAcylation could be challenging. By using the host-cell factor 1 (HCF-1), a known O-GlcNAcylated protein, we immunoprecipitated HCF-1 from transfected HEK293T cells or endogenous HCF-1 from HeLa cells to detect its O-GlcNAc levels by Western blotting. We also take advantage...

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Autores principales: Ahmed, Oumaima, Affar, Malik, Masclef, Louis, Echbicheb, Mohamed, Gushul-Leclaire, Mila, Estavoyer, Benjamin, Vocadlo, David J., Affar, El Bachir
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8784402/
https://www.ncbi.nlm.nih.gov/pubmed/35106498
http://dx.doi.org/10.1016/j.xpro.2021.101108
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author Ahmed, Oumaima
Affar, Malik
Masclef, Louis
Echbicheb, Mohamed
Gushul-Leclaire, Mila
Estavoyer, Benjamin
Vocadlo, David J.
Affar, El Bachir
author_facet Ahmed, Oumaima
Affar, Malik
Masclef, Louis
Echbicheb, Mohamed
Gushul-Leclaire, Mila
Estavoyer, Benjamin
Vocadlo, David J.
Affar, El Bachir
author_sort Ahmed, Oumaima
collection PubMed
description Detection of protein O-GlcNAcylation could be challenging. By using the host-cell factor 1 (HCF-1), a known O-GlcNAcylated protein, we immunoprecipitated HCF-1 from transfected HEK293T cells or endogenous HCF-1 from HeLa cells to detect its O-GlcNAc levels by Western blotting. We also take advantage of RNAi or chemical inhibitors to modulate OGT and OGA activities before HCF-1 immunoprecipitation. For complete details on the use and execution of this protocol, please refer to Daou et al. (2011).
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spelling pubmed-87844022022-01-31 Immunoprecipitation and Western blot-based detection of protein O-GlcNAcylation in cells Ahmed, Oumaima Affar, Malik Masclef, Louis Echbicheb, Mohamed Gushul-Leclaire, Mila Estavoyer, Benjamin Vocadlo, David J. Affar, El Bachir STAR Protoc Protocol Detection of protein O-GlcNAcylation could be challenging. By using the host-cell factor 1 (HCF-1), a known O-GlcNAcylated protein, we immunoprecipitated HCF-1 from transfected HEK293T cells or endogenous HCF-1 from HeLa cells to detect its O-GlcNAc levels by Western blotting. We also take advantage of RNAi or chemical inhibitors to modulate OGT and OGA activities before HCF-1 immunoprecipitation. For complete details on the use and execution of this protocol, please refer to Daou et al. (2011). Elsevier 2022-01-19 /pmc/articles/PMC8784402/ /pubmed/35106498 http://dx.doi.org/10.1016/j.xpro.2021.101108 Text en Crown Copyright © 2022. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Ahmed, Oumaima
Affar, Malik
Masclef, Louis
Echbicheb, Mohamed
Gushul-Leclaire, Mila
Estavoyer, Benjamin
Vocadlo, David J.
Affar, El Bachir
Immunoprecipitation and Western blot-based detection of protein O-GlcNAcylation in cells
title Immunoprecipitation and Western blot-based detection of protein O-GlcNAcylation in cells
title_full Immunoprecipitation and Western blot-based detection of protein O-GlcNAcylation in cells
title_fullStr Immunoprecipitation and Western blot-based detection of protein O-GlcNAcylation in cells
title_full_unstemmed Immunoprecipitation and Western blot-based detection of protein O-GlcNAcylation in cells
title_short Immunoprecipitation and Western blot-based detection of protein O-GlcNAcylation in cells
title_sort immunoprecipitation and western blot-based detection of protein o-glcnacylation in cells
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8784402/
https://www.ncbi.nlm.nih.gov/pubmed/35106498
http://dx.doi.org/10.1016/j.xpro.2021.101108
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