Probing Pairwise Protein-Protein Interactions with the Bacterial POLAR Assay

The PopZ-linked apical recruitment (POLAR) assay is an in vivo two-hybrid assay performed in Escherichia coli, using epifluorescence microscopy to visualize the recruitment of a prey protein fusion by a bait protein fusion tethered artificially at the cell pole. Here, we present a standardized protocol of the POLAR assay, specifically designed to investigate protein-protein interactions (PPIs) in the cytoplasm. Notably, the POLAR assay has been successfully used to validate potential PPIs identified through proximity labeling, highlighting its relevance as a complementary tool within the broader context of studying protein interaction networks.