The last two decades have led to significant progress in the field of analytical ultracentrifugation driven by instrumental theoretical and computational methods. implicit mass conservation. For SV this includes the development and applications of numerical solutions of the Lamm equation noise decomposition techniques enabling direct boundary fitting diffusion deconvoluted sedimentation coefficient distributions and multi-signal sedimentation coefficient distributions. Recently effective particle theory has uncovered simple physical rules for the co-migration of rapidly exchanging systems of interacting components in SV. This has opened new possibilities for the robust interpretation of the boundary patterns of heterogeneous interacting systems. Together these SE and SV techniques have led to new approaches to study macromolecular interactions across the entire the spectrum of affinities including both attractive and repulsive interactions in both dilute and highly concentrated solutions which can be applied to single-component solutions of self-associating proteins as well as the study of multi-protein complex formation in multi-component solutions. in the centrifugal field can be described as and are the chemical potential and activity respectively as a function of the distance from the center of rotation a reference radius and ω the angular velocity the gas constant and the absolute temperature. On the right-hand-side the mechanical potential energy in the centrifugal field is governed by and the buoyant molar mass is the species molar mass and (is the density increment at constant chemical potential of all other species. In aqueous solvents and in the absence of preferential solvation the latter is often approximated as the buoyancy term (1-denoting the protein partial specific volume and ρ the solvent density. Dependent on the LY2090314 strength and type of the interaction to be studied and correspondingly the macromolecular concentration range used in the SE experiment the description branches into that of thermodynamically ideal sedimentation at low concentrations typically applied to study species molecular weights and specific biochemical interactions in a traditional analytical ultracentrifuge with real-time optical detection and into non-ideal sedimentation at high concentrations typically applied to study weakly attractive or repulsive interactions and conducted in analytical ultracentrifugation experiments with post-centrifugal fractionation and quantitation. Sedimentation equilibrium at high concentrations: Repulsive and weakly attractive interactions The study of SE at high concentrations of macromolecules of interest can reveal both attractive interactions from weak self-association or hetero-association as well as repulsive interactions from steric or electrostatic forces. In view of mimicking the intracellular LY2090314 environment or other milieus crowded by a high concentration of unrelated molecules which can have profound effects on the thermodynamics of protein interactions (Zhou et al. 2008 SE may also be conducted in the presence of a high concentration of unrelated macromolecules that produce a solution with a background of volume-excluding but otherwise inert macromolecules as pioneered by Minton and coworkers (Rivas et al. 1999 In both cases due to the potential of optical aberrations caused by refractive index gradients (González et al. 2003 experiments at very high total macromolecular concentrations are often conducted in preparative LY2090314 ultracentrifuge followed by post-centrifugal fractionation and protein quantitation by various methods (Darawshe & Minton 1994 Commensurate with the radial resolution of this approach it is convenient to phrase this type of SE in terms of apparent molar mass = between concentration chemical activity and the activity coefficient ln may be expanded Rabbit Polyclonal to PHCA. into a power series for two-body interactions for LY2090314 three-body interactions etc. (Hall & Minton 2003 Zimmerman & Minton 1993 If the solution is sufficiently dilute such that only two-body interactions are significant then the second virial coefficient may be extracted from experimental data and interpreted for example in the context of effective rigid particles.