Protocol

Part of the series Methods in Molecular Biology pp 1-8

Date:

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

  • Jonathan K. FlemingAffiliated withLpath Incorporated
  • , Jonathan M. WojciakAffiliated withLpath Incorporated Email author 

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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.

Keywords

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