Interactions between extracellular domains (ECDs) are crucial for many physiological processes in the cell, most importantly perception of its environment. However, studying these often-transient interactions can be challenging. Here we describe a method that allows for in vitro detection of extracellular domain interactions through an oligomerization-based cell surface interaction (CSI) assay. In a CSI, bait- and prey-tagged proteins are produced and secreted by Drosophila S2 cells to ensure proper folding and post-translational modifications. Subsequently, Bait (FC fragment) and Prey (pentamer domain and alkaline phosphatase) tags allow the detection of interactions in protein A-coated 96 wells plates through a colorimetric readout. Due to the easy detection of interactions this approach can be used for high-throughput screening and mapping of extracellular interaction networks.
Keywords: ECDs; High-throughput screen; Interactome assay; Protein–protein interactions; Receptor kinases; S2 cell protein expression; Signaling.
© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.