BioID: Identifying Protein-Protein Interactions in Living Cells

The BioID (proximity-dependent biotin identification) method represents a powerful approach for identifying protein-protein interactions within living cells. This technique utilizes a promiscuous biotin ligase, BirA*, which is fused to a protein of interest (bait). Upon expression in cells, BirA* biotinylates proximate proteins (prey) in the vicinity of the bait, which can then be isolated using streptavidin affinity purification. Here, we describe in detail how to process stably expressing HEK cells containing BirA*-fusion protein and how to capture the biotinylated proteins which are to be subsequently identified through mass spectrometry (DIA).