Abstract
Proximity-dependent labeling methods for detecting candidate protein-protein interactions (PPIs) or mapping the protein constituency of subcellular domains have become increasingly utilized by the scientific community. One such method, BioID, allows for the identification of not only strong interactions but also weak and transient associations between a protein of interest (POI) or targeting motif and adjacent proteins. A promiscuous biotin ligase is fused to a POI or targeting motif, expressed in living cells, and induced to biotinylate proximal proteins during a defined labeling period by biotin supplementation. This generates a history of protein-protein associations that occurred with the POI or the protein constituency within a discrete subcellular domain during the labeling period. Biotinylated proteins are subsequently isolated, identified via mass spectrometry, and investigated as candidate interactors with the POI or as constituents within a subcellular domain. The BioID method has been utilized by numerous research groups and is continually being optimized, applied to new models, and modified for use in novel applications. Here we describe a protocol by which a BioID fusion protein can be validated and utilized for BioID pull-downs.