Chapter title |
Measuring Sphingosine-1-Phosphate/Protein Interactions with the Kinetic Exclusion Assay
|
---|---|
Chapter number | 5 |
Book title |
Sphingosine-1-Phosphate
|
Published in |
Methods in molecular biology, March 2017
|
DOI | 10.1007/7651_2017_5 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7412-2, 978-1-4939-7413-9
|
Authors |
Jonathan K. Fleming, Jonathan M. Wojciak |
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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