A fundamental aspect of the thermodynamic characterization of macromolecular complexes is the determination of the component stoichiometry. There are a number of approaches that may be employed in this effort, including mass spectrometry, light scattering, and sedimentation equilibrium analytical ultracentrifugation (AUC). In recent years, an AUC method called multi-signal sedimentation velocity (MSSV) has emerged as an excellent means to elucidate stoichiometry. This technique, pioneered in the laboratory of Peter Schuck, capitalizes on the Beckman XL-A/I centrifuge’s ability to acquire sedimentation velocity (SV) data using multiple wavelengths and, in the case of the XL-I, multiple optical systems. Using the popular c(s) formalism, MSSV can supply the molar ratio of 2 – 4 species in a stable, co-sedimenting complex. In conjunction with the hydrodynamic information inherently available from a SV experiment, this often allows the unambiguous determination of stoichiometry. Basic theory and experimental design for MSSV will be featured in this webinar, along with the analytical strategies necessary to produce reliable results. Also, advanced topics, including measures that may be taken in difficult cases, will be covered. Throughout, concrete, real-world examples of MSSV will be presented.

Learning Objectives:

• Learn why an analytical ultracentrifugation (AUC) method called multi-signal sedimentation velocity (MSSV) has emerged as an excellent means to elucidate stoichiometry in recent years 
• Learn the basic theory and experimental design for MSSV, along with the analytical strategies necessary to produce reliable results.
• See real-world examples of MSSV application