What type of assay would be useful for determining protein-protein binding affinities?

Determining protein-protein binding affinities often requires methods that allow for the direct measurement of interactions between two proteins. Pull down assays are particularly effective for this purpose. In a pull down assay, a bait protein (usually tagged for easy identification) is expressed and allowed to interact with a complex mixture that may contain a prey protein. The bait protein is then isolated using affinity purification methods, typically involving beads that bind the tag, and any proteins that bind to it can be recovered and analyzed.

This method is advantageous for measuring binding affinities because it allows researchers to manipulate conditions (like protein concentrations) and to directly observe the strength of the interaction through changes in the amount of prey protein that can be recovered. By assessing the concentration at which binding occurs, and using techniques like SDS-PAGE to quantify the bound proteins, researchers can derive binding constants and other important parameters of the interaction.

In contrast, other assay types are either not designed specifically for quantifying affinity or may not provide enough specificity or sensitivity for protein-protein interactions. For example, enzymatic assays are more suited to measuring reaction rates catalyzed by enzymes rather than specific binding interactions. Co-immunoprecipitation is a related technique that can also help identify interacting partners, but it