title: Plate-Based Assay for Measuring Direct Semaphorin-Neuropilin Interactions.
abstract: 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.
authors: Department of Molecular and Cellular Biochemistry, Center for Structural Biology, University of Kentucky, 741 South Limestone Avenue, BBSRB B263, Lexington, KY, 40536, USA.
authors: Matthew W
authors: MW
authors: Parker
authors: Department of Molecular and Cellular Biochemistry, Center for Structural Biology, University of Kentucky, 741 South Limestone Avenue, BBSRB B263, Lexington, KY, 40536, USA. craig.vanderkooi@uky.edu.
authors: Craig W
authors: CW
authors: Vander Kooi
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D003412:Cricetulus; D006801:Humans; D008024:Ligands; D039921:Neuropilins / Q000378:metabolism*; D039961:Semaphorins / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D008024:Ligands; D039921:Neuropilins; D039961:Semaphorins; D000469:Alkaline Phosphatase
keywords: Assay; Binding; Competition; Interaction; Ligand; Neuropilin; Plate-based; Receptor; Semaphorin