Plate-Based Assay for Measuring Direct Semaphorin-Neuropilin Interactions.

The semaphorins are an essential family of axon guidance molecules that can be either secreted or are transmembrane proteins. Class 3 semaphorin (Sema3) family members are secreted and provide long-range guidance cues through two receptor families: neuropilins (Nrp) and plexins. Nrp is uniquely required for high-affinity Sema3 binding and signaling. Therefore, characterizing the molecular details of the Sema3/Nrp interaction is important for understanding the broader physiological and pathological role of the Sema3 family of proteins. Here we describe an in vitro plate-based binding assay for characterization of the Sema3/Nrp interaction. This assay utilizes Nrp-affinity plates and an alkaline phosphatase (AP)-Sema3 fusion to rapidly measure direct Sema3/Nrp binding. This assay can be used to measure receptor-ligand binding, the contribution of different domains, and exogenous factors, and to characterize competitive ligand binding.