Experimental Protocol for Sedimentation Equilibrium Analytical Ultracentrifugation

This step-by-step protocol illustrates a sedimentation equilibrium (SE) study of a heterogeneous interaction between two proteins ‘A’ and ‘B’ forming a reversible complex. The goal of the experiment is the determination of the binding constant and the binding stoichiometry. It includes the determination of the molar mass of a non-interacting protein (or stable protein complex), and can be easily adapted to the study of protein self-association. Again, the details of the practical steps are given in conjunction with the workflow of the data analysis, which is closely tied to the experimental configuration. It will be assumed that a sedimentation velocity (SV) study of the same proteins and their mixture has taken place before, for example, using the SV protocol provided by us.