Beckman Coulter Analytical Ultracentrifugation (AUC) is the most versatile, rigorous, and accurate technique for determining the molecular weight, hydrodynamic properties, stoichiometry, protein aggregation, ligand binding, conjugation efficiency, polydispersity, and thermodynamic properties of proteins and other macromolecules.

AUC is uniquely capable of analyzing molecules and particles across a wide range of concentrations and solvents, providing unmatched precision and accuracy. Unlike other macromolecular analysis techniques, AUC enables characterization of samples in their native state, under biologically relevant conditions, offering researchers two complementary insights into solution behavior:

Dual Modes of Analytical Ultracentrifugation

1. Sedimentation Velocity (SV): Provides first-principle, hydrodynamic data on the size, shape, oligomeric state, and interactions of proteins and protein complexes in solution.
2. Sedimentation Equilibrium (SE): Yields detailed information about solution molar masses, stoichiometries, association constants, and non-ideal solution behavior.

Density Gradient Equilibrium AUC (DGE-AUC)

A recent enhancement in AUC methodology, DGE-AUC combines traditional sedimentation equilibrium with density-based separation. Inspired by the landmark Meselson–Stahl experiment (1958), DGE-AUC separates analytes based on buoyant density:

1. The analyte is mixed with a gradient-forming material (GFM) such as iodixanol or CsCl.
2. Upon ultracentrifugation, the GFM establishes a stable density gradient through the sample cell.
3. Analyte particles (e.g., proteins, nucleic acids, viruses, or nanoparticles) migrate to a radial position where their buoyant density matches the local GFM density.

For instance, in a mixture of Adeno-Associated Virus (AAV) capsids, fully loaded capsids (higher density) migrate further down the cell than empty capsids (lower density). Similarly, drug-loaded nanoparticles can be distinguished from empty carriers due to differences in density.

Key Analytical Insights from AUC

✔ Molecular Weight Determination: Estimated from sedimentation coefficients and protein shape analysis in solution.
✔ Hydrodynamic Radius & Protein Shape: Derived from sedimentation coefficient and solution viscosity, revealing structural details.
✔ Thermodynamic Properties & Binding Affinity: Determined by analyzing sedimentation data under varying conditions (e.g., different protein concentrations, ligand presence).
✔ Oligomeric State & Interactions: If the sedimentation coefficient is concentration-dependent, it indicates complex dissociation into subunits.
✔ Protein-Protein & Protein-Ligand Interactions: Estimated by analyzing sedimentation behavior with and without interacting partners, allowing determination of association constants and stoichiometry.

Applications of AUC Across Industries

Pharmaceutical & Drug Development

AUC is widely used in drug discovery and development, including:
✔ Drug Binding Affinity Determination
✔ Study of Drug-Target Interactions
✔ Characterization of Drug Delivery Systems
✔ Assessment of Drug Formulation Stability
✔ Determination of Pharmacokinetic Parameters

Peptide & Polymer Research

AUC provides crucial insights into peptides, polymers, and biomolecules, helping researchers understand:
✔ Molecular weight, size, and conformational changes
✔ Self-assembly and aggregation behavior (critical for drug delivery & nanotechnology applications)
✔ Interactions with biomolecules (proteins, nucleic acids) and solvents/surfactants
✔ Structure-function relationships for optimization in biomedical and material science applications

Food Science & Industry Applications

AUC plays a critical role in food research, aiding in:
✔ Development & optimization of emulsion-based food products (e.g., dressings, sauces)
✔ Characterization of food proteins and emulsions (molecular weight, size distribution, interactions)
✔ Analysis of food additives and ingredient interactions
✔ Detection of food adulteration by identifying changes in protein or lipid profiles