This application is to request fund for purchasing an Octet R2 Biolayer Interferometry (BLI) system. This BLI system is capable of quantitative measurements of binding kinetics and affinity with very small amounts of proteins. The project supported by the parent R35 grant is focused on study the structures and regulatory mechanisms of transmembrane receptors critical for the development and functions of the nervous system, cardiovascular system and immune system, including but not limited to plexin and VEGF receptor. The activation of these receptors by their ligands and co-receptors as well as heparin sulfate proteoglycans are very complex, involving multiple binding interfaces mediated by two or more proteins as well as glycans. The binding affinities span a very broad range, from nanomolar to high micromolar. Quantitative measurements of these binding events are critical for validating our cryo-electron microscopic (cryo-EM) structures and gaining deeper understanding of the underlying mechanisms. However, Such measurements are difficult to do with techniques that are currently available in the lab or accessible from the core facilities at UT Southwestern medical center, such as isothermal titration calorimetry, which requires large amounts of proteins that are not practical for us to produce in human cell lines. We therefore request to purchase an Octet R2 BLI system, which uses surface- based binding measurements based on light interference that requires very small amounts of proteins and no labelling. In addition, the system can be expanded in future to allow simultaneous measurements of 8 samples in the 96-well format, with the capacity of high- throughput screen of many mutants and libraries of antibodies or small molecules. This system will greatly accelerate the progress of the parent project and may even make it possible for us to pursue exciting new directions.