Dynamic light scattering is a non-invasive technique which allows determination of particle sizes of samples containing only a single particle species as well as particle mixtures. If particle populations in a mixture can be resolved - meaning they are visible as baseline separated peaks in the size distribution - depends on their hydrodynamic radius difference and their mixing ratio.

Prometheus Panta can resolve particles of approximately 5x difference in radius. For particles with a smaller difference in radius there is likely no baseline separation between the peaks of the size distribution. However, shifts in the average size and an increase in the polydispersity index may indicate the presence of multiple particle populations. Another important factor that determines DLS resolution is the mixing ratio of particles with different sizes: Larger particles will scatter more light, which is why DLS is very sensitive to even trace amounts of large aggregates. However, when measuring particle mixtures, the scattering signal from larger particles may mask the signal of smaller particles, if the smaller particles are not present in large excess. Whether two populations can be resolved by Prometheus Panta is best tested experimentally. 

Monomer-dimer transitions

DLS measurements carried out on Prometheus Panta are sensitive to the size difference between monomeric and dimeric proteins. However, if there is a mixture of monomer and dimers in solution, Prometheus Panta will not be able to resolve baseline separated peaks for the two populations. Note that a dimeric protein will most likely not yield a radius of twice the size of the monomer. The changes in the measured radius due to dimer formation will be smaller. The formation of dimer/oligomer should always be confirmed by orthogonal techniques, such as size exclusion chromatography.

Conformational changes

Changes to the shape of a particle, such as conformational changes, will alter its diffusion coefficient and thereby also the rH. However, not all conformational changes will be detectable with Prometheus Panta – this sensitivity will be specific to the nature of the protein in question and the type/magnitude of its conformational change. Like the analysis of monomer-dimer transitions, the change in the observed rH might be small and orthogonal techniques are required to confirm the results.