Analytical ultracentrifugation (AUC) is a first principle biophysical technique which has
been used for over a century to investigate the behaviour of biomolecules. AUC encompasses
a range of experimental approaches which enable the investigation of the size, shape, and
interactions of macromolecules in solution. While the technique is well established within the
annals of biochemistry, improvements to both the physical instrumentation and the data
processing software have provided new opportunities for the use of AUC. We highlight key
aspects of experimental design and showcase several modern applications of AUC,
encouraging its integration with other structural biology techniques or its use when such
methods fall short. Specific examples include: 1. Quantification of protein-bound detergent
during membrane protein sample preparation, 2. Vaccine quality control through
characterization of mRNA-loaded lipid nanoparticles, and 3. Multiwavelength-AUC for
deconvolution of spectrally dissimilar biomolecules, such as nucleic acids and proteins.