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

ABSTRACT: 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).

SUBMITTER: Ahmed O

PROVIDER: S-EPMC8784402 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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

Ahmed Oumaima O Affar Malik M Masclef Louis L Echbicheb Mohamed M Gushul-Leclaire Mila M Estavoyer Benjamin B Vocadlo David J DJ Affar El Bachir EB

STAR protocols 20220119 1

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). ...[more]

PMID: 35106498

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