The analysis of macromolecular interactions by sedimentation equilibrium

The study of macromolecular interactions by sedimentation equilibrium is a highly technical method that requires great care in both the experimental design and data analysis. The complexity of the interacting system that can be analyzed is only limited by the ability to deconvolute the exponential contributions of each of the species to the overall concentration gradient. This is achieved in part through the use of multi-signal data collection and the implementation of soft mass conservation. We illustrate the use of these constraints in SEDPHAT through the study of an A+B+B⇌AB+B⇌ABBA+B+B⇌AB+B⇌ABB system and highlight some of the technical challenges that arise. We show that both the multi-signal analysis and mass conservation result in a precise and robust data analysis and discuss improvements that can be obtained through the inclusion of data from other methods such as sedimentation velocity and isothermal titration calorimetry.