Simultaneous Imaging of Single Protein Size, Charge, and Binding Using A Protein Oscillation Approach

Electrophoresis and Western blot are important tools in protein research for detection and identification of proteins. These traditional techniques separate the proteins based on size and charge differences and identify the proteins by antibody binding. Over the past decade, the emergence of single-molecule techniques has shown great potential in improving the resolution of the traditional protein analysis methods to the single-molecule level. However, such single-molecule techniques measure either size or charge, and it is challenging to measure both at the same time. Recently, we have developed a single-molecule approach to address this problem. We tether the single proteins to a surface with a polymer linker and drive them into oscillation with an electric field. By tracking the electromechanical response of the proteins to the field using an optical imaging method, the size and charge can be obtained simultaneously. Binding of antibodies or ions to the tethered protein also changes the size and charge, which allows us to probe the interactions. This protocol includes fabrication of protein oscillators, configuration of the optical detection system, and analysis of the oscillation signal for quantification of protein size and charge. We wish this protocol will enable researchers to perform comprehensive single-protein analysis on a single platform.