Abstract
Recent advances in proteomic screening approaches have led to the isolation of a wide variety of binding partners to interacting proteins and opened an avenue to analyze and understand signaling pathways. The study of protein-protein interactions is a key component in elucidating and understanding signaling pathways. Despite the importance of these interactions, very few studies are quantitative or report binding affinities. Here we present a simple method for examination and analysis of direct protein-protein binding interactions between two purified proteins. In the quantitative pull-down assay, one protein (the bait protein) is immobilized on beads whereas a second protein (the prey) is kept in solution. The concentration of the bait protein is kept constant, whereas the concentration of the prey protein is increased until binding saturation is achieved. After incubation, the beads are precipitated to separate unbound prey protein in solution from prey protein bound to the bait. The fraction of bound prey protein can then be loaded on a protein gel and the resulting bands can be analyzed with standard software. The quantitative pull-down assay with purified recombinant proteins provides a simple method to obtain dissociation constants (K d). These quantifications are invaluable to compare relative binding of proteins, to map binding sites, and to show that binding is direct. This assay presents a powerful method to quantitatively analyze protein-protein interactions with tools that are available in most biochemistry laboratories and does not require the use of specialized or expensive equipment.