The analytical ultracentrifuge (AUC) remains the most versatile, rigorous and accurate means for determining the molecular weight and hydrodynamic/thermodynamic properties of a protein or other macromolecule. No other technique is capable of providing the same range of information with a comparable level of precision and accuracy. Learn about AUC in our Introduction to Analytical Ultracentrifugation handbook.
Described below are some fundamental applications for the analytical centrifuge for which it is either the preferred or only method of analysis. For more detail, refer to our handbook Self-Associating Systems in the Analytical Ultracentrifuge.
Sedimentation analysis has a long history in examining solution heterogeneity or polydispersity. Determining average molecular weights by AUC experiments can provide sensitive and rigorous analysis to quantify minor species and allow quantitation of the size distributions in polydisperse samples.
AUC velocity experiments also allow for rapid rigorous quantitative assessment of sample heterogeneity and analysis of purity, integrity of native structure and degree of aggregation unaffected by interactions of the macromolecules with gel matrix or support.
Molecular weight determination
The analytical ultracentrifuge is unsurpassed for directly measuring molecular weights of solutes in their native state and as they exist in solution without relying on calibration or making assumptions concerning shape. The method is applicable to molecules with molecular weights ranging from several hundreds (such as sucrose) up to many millions (for virus particles and organelles).
This method is applicable to proteins, nucleic acids, carbohydrates and other substances with absorbance (or refractive index) that differs from that of the solvent. AUC experiment methods allow for a wide variety of sample sizes (15 μL-400 μL) and a wide range of concentrations can also be used to explore macromolecules in concentrated solutions (i.e., studies of very weak interactions).
Sedimentation analysis is even more valuable when analyzing molecular changes in cases where molecules associate to form more complex structures.
Detecting conformation changes
Experiments in the analytical ultracentrifuge provide sedimentation and diffusion coefficients that contain information concerning the size and shape of macromolecules and the interactions between them.
Sedimentation coefficients are particularly useful for monitoring changes in conformation in proteins and nucleic acids. Bending in nucleic acids induced by protein binding may also be amenable to study. Through the combination of several different hydrodynamic or thermodynamic measurements, it is now possible to discriminate more clearly between different idealized shapes used to model the overall shape of a macromolecule in solution.
Absorbance optics are particularly well suited to studies of ligand binding. Ligands and acceptors may have different intrinsic absorbance or one of the species may be labeled with a chromophore, provided the modification does not alter the binding. Analysis can be simply done with sedimentation velocity methods when the ligand and acceptor differ greatly in sedimentation coefficient such as with small molecule-protein association, DNA-protein binding or the binding of relatively large proteins to filaments such as F-actin.