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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.

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Probing Pairwise Protein–Protein Interactions with the Bacterial POLAR Assay

  • 实验方案

  • 发表日期：

    2025年

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  • Coralie Tesseur,
  • Géraldine Laloux

摘要

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.