期刊
ANALYTICAL CHEMISTRY
卷 86, 期 6, 页码 3181-3187出版社
AMER CHEMICAL SOC
DOI: 10.1021/ac500093m
关键词
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资金
- National Institute of Biomedical Imaging and Bioengineering
- National Institute of Child Health and Human Development, National Institutes of Health
The study of high-affinity protein interactions with equilibrium dissociation constants (K-D) in the picomolar range is of significant interest in many fields, but the characterization of stoichiometry and free energy of such high-affinity binding can be far from trivial. Analytical ultracentrifugation has long been considered a gold standard in the study of protein interactions but is typically applied to systems with micromolar K-D. Here we present a new approach for the study of high-affinity interactions using fluorescence detected sedimentation velocity analytical ultracentrifugation (FDS-SV). Taking full advantage of the large data sets in FDS-SV by direct boundary modeling with sedimentation coefficient distributions c(s), we demonstrate detection and hydrodynamic resolution of protein complexes at low picomolar concentrations. We show how this permits the characterization of the antibody antigen interactions with low picomolar binding constants, 2 orders of magnitude lower than previously achieved. The strongly size-dependent separation and quantitation by concentration, size, and shape of free and complex species in free solution by FDS-SV has significant potential for studying high-affinity multistep and multicomponent protein assemblies.
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