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 K d for the lipid binding the protein and the antibody can be determined simultaneously. Herein, we describe this label-free approach for determining the K d for S1P-binding serum albumin, which chaperones ~30% of the S1P in human plasma.
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Acknowledgment
The authors thank Tom Glass and Steve Lackie for their extensive advice and technical support.
Conflicts of Interest
KinExA is a registered trademark of Sapidyne Instruments Inc.
LT1009 is a proprietary agent of Lpath Inc. but may be provided upon request.
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Fleming, J.K., Wojciak, J.M. (2017). Measuring Sphingosine-1-Phosphate/Protein Interactions with the Kinetic Exclusion Assay. In: Pébay, A., Turksen, K. (eds) Sphingosine-1-Phosphate. Methods in Molecular Biology, vol 1697. Humana Press, New York, NY. https://doi.org/10.1007/7651_2017_5
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DOI: https://doi.org/10.1007/7651_2017_5
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7412-2
Online ISBN: 978-1-4939-7413-9
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