{"doi":"10.1002/cpz1.117","title":"O‐GlcNAc Engineering on a Target Protein in Cells with Nanobody‐OGT and Nanobody‐splitOGA","abstract":"The monosaccharide O-linked N-acetyl glucosamine (O-GlcNAc) is an essential and dynamic post-translational modification (PTM) that decorates thousands of nucleocytoplasmic proteins. Interrogating the role of O-GlcNAc on a target protein is crucial yet challenging to perform in cells. We recently reported a pair of methods to selectively install or remove O-GlcNAc on a target protein in cells using an engineered O-GlcNAc transferase (OGT) or split O-GlcNAcase (OGA) fused to a nanobody. Target protein O-GlcNAcylation and de-O-GlcNAcylation complements methods to interrogate the role of O-GlcNAc on a global scale or at individual glycosites. Herein, we describe a protocol for utilizing the nanobody-OGT and nanobody-splitOGA systems to screen for O-GlcNAc functionality on a target protein. We additionally include associated protocols for the detection of O-GlcNAc and cloning procedures to adapt the method for the user's target protein of interest. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Target protein O-GlcNAcylation of JunB using nanobody-OGT Basic Protocol 2: Target protein deglycosylation of Nup62 using nanobody-splitOGA Alternate Protocol: Verification of the O-GlcNAc state of a tagged target protein through chemoenzymatic labeling Support Protocol: Cloning of new nanobody-OGT/nanobody-splitOGA and target protein pairs.","journal":"Current Protocols","year":2021,"id":187319,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":18,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9561,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":635955,"name":"Yun Ge","orcid":"0000-0001-6769-5458","position":1,"is_corresponding":false},{"id":496356,"name":"Christina M. Woo","orcid":"0000-0001-8687-9105","position":2,"is_corresponding":false},{"id":635956,"name":"Daniel H. Ramirez","orcid":"0000-0003-3288-9951","position":0,"is_corresponding":true}],"reference_count":30,"raw_metadata":null,"created_at":"2026-07-18T23:48:51.315888Z","pmid":"33950562","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}