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Measuring Sphingosine-1-Phosphate/Protein Interactions with the Kinetic Exclusion Assay
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.
KeywordsAnti-lipid antibody Bovine serum albumin Competitive affinity analysis Human serum albumin Kinetic exclusion assay Physical biochemistry Sphingosine-1-phosphate
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