Abstract
Membrane proteins contain hydrophobic sequences that must be chaperoned during protein folding and transport to the lipid bilayer. Failures in this essential process result in the formation of potentially toxic aggregates. Our mechanistic understanding of how chaperones interact with hydrophobic sequences is limited due to the technical challenges of measuring interactions between aggregation prone proteins in a detergent-free environment. As hydrophobic substrates are prone to non-specific interactions, traditional binding assays that utilize one substrate and one chaperone per assay struggle to demonstrate that an interaction is specific and physiological. To address these technical challenges, we developed the barcoded binding assay, which allows for the measurement of chaperone binding selectivity to a complex pool of substrates in a detergent-free environment. In this assay, a pool of 35S-labeled barcoded substrates is produced by in vitro translation in the presence of the chaperone of interest and chaperone selectivity is quantified by immunoprecipitation, SDS PAGE, and autoradiography. We previously used this assay to identify novel substrate binding preferences for the Ubiquilin family of chaperones. Here, we provide an overview of the assay, demonstrate the benefits of the multiplexed barcoded binding assay over a single substrate approach, and provide a detailed protocol for how this assay can be adapted to work with a broad array of chaperones.