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Measuring Sphingosine-1-Phosphate/Protein Interactions with the Kinetic Exclusion Assay

Protocol
Part of the Methods in Molecular Biology book series

Abstract

By directly detecting the ligand-free binding sites in a sample, the kinetic exclusion assay (KinExA®) provides a compelling alternative to SPR-based techniques for determining equilibrium dissociation constants of protein-ligand interactions. It is especially useful for observing protein-lipid interactions, as binding of native lipids occurs entirely in solution, and monoclonal antibodies can be used to directly compete with a protein of interest for lipid binding. By measuring the antigen-free binding sites on the antibody and using competition affinity analysis, the Kd for the lipid binding the protein and the antibody can be determined simultaneously. Herein, we describe this label-free approach for determining the Kd for S1P-binding serum albumin, which chaperones ~30% of the S1P in human plasma.

Keywords

Anti-lipid antibody Bovine serum albumin Competitive affinity analysis Human serum albumin Kinetic exclusion assay Physical biochemistry Sphingosine-1-phosphate 

References

  1. 1.
    Fleming JK, Glass TR, Lackie SJ, Wojciak JM (2016) A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the kinetic exclusion assay. J Lipid Res 57(9):1737–1747. doi:10.1194/jlr.D068866 CrossRefPubMedGoogle Scholar
  2. 2.
    Rosen H, Gonzalez-Cabrera PJ, Sanna MG, Brown S (2009) Sphingosine 1-phosphate receptor signaling. Annu Rev Biochem 78:743–768. doi:10.1146/annurev.biochem.78.072407.103733 CrossRefPubMedGoogle Scholar
  3. 3.
    O'Brien N, Jones ST, Williams DG, Cunningham HB, Moreno K, Visentin B, Gentile A, Vekich J, Shestowsky W, Hiraiwa M, Matteo R, Cavalli A, Grotjahn D, Grant M, Hansen G, Campbell MA, Sabbadini R (2009) Production and characterization of monoclonal anti-sphingosine-1-phosphate antibodies. J Lipid Res 50(11):2245–2257. doi:10.1194/jlr.M900048-JLR200 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Lpath Incorporated4025 Sorrento Valley BlvdSan DiegoUSA

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