Real-time observation of temperature-dependent protein–protein interactions using real-time dual-color detection system Original Research Article

This study examined the ability of a real-time dual-color detection system to allow direct observations of the kinetics of temperature-dependent protein–protein interaction at a single-molecule level. The primary target protein was an Alexa Fluor® 488-labeled actin conjugate, which had been pre-incubated with an unlabeled rabbit anti-actin antibody (IgG). The complementary fluorescent protein was Alexa Fluor® 633-labeled goat anti-rabbit IgG antibody, which interacts with the rabbit anti-actin antibody (IgG) bound to the Alexa Fluor® 488-labeled actin conjugate. The individual protein molecules labeled with different fluorescent dyes in solution were effectively focused, interacted with the other protein molecules at 500 aM, and detected directly in real-time using the dual-wavelength (λex = 488 and 635 nm) laser-induced fluorescence detection system. The kinetics of the protein–protein interactions were examined at different temperatures (12–32 °C). At concentrations in the aM range, the number of bound complex molecules through the protein–protein interaction decreased gradually with time at a given temperature, and increased with decreasing temperature at a set time. A high concentration (above 500 pM) of the protein sample caused aggregation and nonspecific binding of the protein molecules, even though the protein molecules were not an example of complementary binding. The results demonstrated that the real-time kinetics of a protein–protein interaction could be analyzed effectively at the single-molecule level without any time delay using the real-time dual-color detection system.