Analysis of high affinity self-association by fluorescence optical sedimentation velocity analytical ultracentrifugation of labeled proteins: opportunities and limitations.

ABSTRACT: Sedimentation velocity analytical ultracentrifugation (SV) is a powerful first-principle technique for the study of protein interactions, and allows a rigorous characterization of binding stoichiometry and affinities. A recently introduced commercial fluorescence optical detection system (FDS) permits analysis of high-affinity interactions by SV. However, for most proteins the attachment of an extrinsic fluorophore is an essential prerequisite for analysis by FDS-SV. Using the glutamate receptor GluA2 amino terminal domain as a model system for high-affinity homo-dimerization, we demonstrate how the experimental design and choice of fluorescent label can impact both the observed binding constants as well as the derived hydrodynamic parameter estimates for the monomer and dimer species. Spe

SUBMITTER: Zhao H 

PROVIDER: S-EPMC3866193 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature