Proximity‐Dependent Biotinylation for Identification of Interacting Proteins

Complex interaction networks orchestrate key cellular processes including but not limited to transcription, translation, metabolism, and cell signaling. Delineating these interactions will aid in deciphering the regulation and function of these pathways and potential for manipulation. Proximity-dependent biotin identification (BioID) is quickly gaining popularity as a powerful tool for identifying novel protein-protein and proximity-based interactions in live cells. This technique relies on a promiscuous biotin ligase, which is fused to a protein of interest and, upon expression in the desired cell, will biotinylate proximal endogenous proteins. In vivo protein-protein interactions can be very transient and occur momentarily to facilitate signaling or a metabolic function. BioID is useful in identifying these weak and/or transient interactions that are not detected by traditional methods such as yeast two-hybrid or affinity purification. Here, we outline a BioID protocol that can be used as a workflow to guide a new application. © 2016 by John Wiley & Sons, Inc.