biostudies-literatureKesari ASNIAID NIH HHSNIH HHS37https://www.ebi.ac.uk/biostudies/studies/S-EPMC7709110biostudies-literatureUnknown8(4)Proximity biotinylation was developed to detect physiologically relevant protein-protein interactions in living cells. In this method, the protein of interest is tagged with a promiscuous biotin ligase, such as BioID or BioID2, which produces activated biotin that reacts with nearby proteins; these proteins can subsequently be purified and identified by mass spectrometry. Here we report a novel modification of this technique by combining it with a self-associating split-GFP system in which we exploit the high-affinity interaction between GFP1-10 and GFP11 to recruit BioID2 to the protein of interest. As a test case, we fused GFP11 to clathrin light chain (CLTB) and BioID2 to GFP1-10. Co-expression of GFP11-CLTB and BioID2-GFP1-10 yielded a green fluorescent complex that co-localized with cProteomesA Novel Proximity Biotinylation Assay Based on the Self-Associating Split GFP1-10/11.PMC7709110AI114814P01 AI120943AI120943R01 AI114814LaCount DJAryal UKKesari ASfalseA Novel Proximity Biotinylation Assay Based on the Self-Associating Split GFP1-10/11.Proximity biotinylation was developed to detect physiologically relevant protein-protein interactions in living cells. In this method, the protein of interest is tagged with a promiscuous biotin ligase, such as BioID or BioID2, which produces activated biotin that reacts with nearby proteins; these proteins can subsequently be purified and identified by mass spectrometry. Here we report a novel modification of this technique by combining it with a self-associating split-GFP system in which we exploit the high-affinity interaction between GFP1-10 and GFP11 to recruit BioID2 to the protein of interest. As a test case, we fused GFP11 to clathrin light chain (CLTB) and BioID2 to GFP1-10. Co-expression of GFP11-CLTB and BioID2-GFP1-10 yielded a green fluorescent complex that co-localized with c2020-01-01T00:00:00Z2020 Dec2025-04-04T19:21:23.027Z2025-04-04T19:21:23.027ZS-EPMC77091103327649410.3390/proteomes8040037