A set of 203 extracellular and transmembrane proteins containing IgSF, LRR or FnIII domains was chosen for analysis. Predicted extracellular domains (between the signal peptide and any predicted transmembrane domains or GPI-linkage sites) were successfully cloned for 202 of these genes. Each of these regions was cloned into a bait vector (pECIA2, Fc tag fusion) and prey vector (pECIA14, alkaline phosphatase fusion) to provide copper-inducible expression and protein secretion. Notably, bait and prey proteins were designed to oligomerize via their Fc-IgG or COMP motifs, respectively, to increase avidity and thus facilitate the detection of low-affinity interactions that are known to dominate the extracellular interactome. Plasmids were introduced into S2 cells by transient transfection and copper induced at 18hr post-transfection for 3 days, at which point conditioned medium was collected.

All bait and prey constructs were collected from conditioned media without further purification.

Fc-tagged bait proteins were captured on Protein-A-coated plates, incubated with prey proteins from conditioned media, and binding was detected by alkaline phosphatase activity using a colorigenic substrate and microplate reader.