Electrokinetic Detection of Single-Molecule Phosphorylation

Current single-molecule fluorescence experiments lack direct sensitivity to molecular phosphorylation states. We show that electrokinetic properties measured with an anti-Brownian trap resolve the number of phosphorylated sites on individual biomolecules and can monitor phosphorylation cycles with single kinase turnovers. As an application, we directly visualize the rate-limiting step in the kinase catalytic cycle.