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Plate-Based Assay for Measuring Direct Semaphorin–Neuropilin Interactions

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Semaphorin Signaling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1493))

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.

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Acknowledgements

This work was supported by National Institutes of Health grants R01GM094155 (C.W.V.K.) and T32HL072743 (M.W.P.).

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Authors and Affiliations

  1. Department of Molecular and Cellular Biochemistry, Center for Structural Biology, University of Kentucky, 741 South Limestone Avenue, BBSRB B263, Lexington, KY, 40536, USA

    Matthew W. Parker & Craig W. Vander Kooi

Authors
  1. Matthew W. Parker
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  2. Craig W. Vander Kooi
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Corresponding author

Correspondence to Craig W. Vander Kooi .

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Editors and Affiliations

  1. Departments of Neuroscience and Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Jonathan R. Terman

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Parker, M.W., Vander Kooi, C.W. (2017). Plate-Based Assay for Measuring Direct Semaphorin–Neuropilin Interactions. In: Terman, J. (eds) Semaphorin Signaling. Methods in Molecular Biology, vol 1493. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6448-2_5

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  • DOI: https://doi.org/10.1007/978-1-4939-6448-2_5

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6446-8

  • Online ISBN: 978-1-4939-6448-2

  • eBook Packages: Springer Protocols

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