Technical Considerations for Detecting Protein–Protein Interactions Using Proximity Ligation Assay

Proximity ligation assay has been widely used to detect protein-protein interaction in cells and tissues. While with great sensitivity, its specificity was often neglected. Here, we report the existence of varying levels of false positives observed with this assay, most likely due to its high sensitivity. We also provide suggestions to minimize false positives for more accurate detection of protein-protein interactions, especially for membrane proteins. These suggestions include co-staining target proteins, using various negative controls and suitable antibodies, avoiding detergents if possible, and validating interactions with complementary methods.