To validate the ability of SH2 to induce ternary complex formation, we turned to size exclusion chromatography (SEC) as orthogonal method to the previously performed native gel electrophoresis assay. Using CM11 as benchmark, we confirmed the formation of a VCB2:SH2 ternary complex, which migrated quicker than the VCB vehicle control (Figure ). Furthermore, we synthesized a negative control compound (SH2ct) featuring the VH032 binder as one ligand and its non-binding derivative cis-VH032, which abrogates binding through inverted stereochemistry at the hydroxyl group of hydroxyproline, as second ligand (see synthesis overview in Scheme S1). As anticipated, SH2ct did not affect the retention volume of VCB during SEC, confirming the requirement of two binding ligands in SH2 for ternary complex formation.