Prometheus Panta enables analysis of samples over a broad concentration range. Most nanoDSF experiments will produce high quality data with a few µg/mL of protein and the limit of detection for a given protein will depend on its amino acid composition, i.e. the number of fluorescent amino acids, and the position of these amino acids within the protein. The upper concentration limit for nanoDSF is determined by the viscosity and fluorescence of the sample, but measurements of protein samples with concentrations above 200 mg/mL are possible.

The lower concentration limit for the DLS optics is dependent upon the amount of scattered light from the particles, which in turn depends on particle size and refractive index. For small and weakly scattering proteins such as lysozyme, the lowest concentration that can be precisely and accurately characterized with Prometheus Panta is 0.5 mg/mL. For larger proteins (e.g. mAbs), the limit is lower. For instance, Herceptin can be characterized at 0.08 mg/mL. When working with low concentrations of very large and strongly scattering particles (e.g. polysterene beads with several hundred nm), number fluctuations can occur.

The upper concentration limit for DLS measurements is determined by secondary effects, such as restricted diffusion, particle interactions, inner filter effect (absorbance around 405 nm), and multiple scattering. When multiple scattering is present in your sample, the reported particle sizes will be too small. Restricted diffusion relates to the phenomenon that particles require more time to travel a certain distance when hindered in free diffusion by other particles. This leads to artificially high sizes with increasing sample concentration but can, to a certain extent, be corrected for by using the bulk viscosity instead of the solvent viscosity. It is impossible to remove the effect of particle interactions occurring at high sample concentration, but even if the resulting particle sizes will be incorrect, it may be helpful to know that particles in the sample repel or attract each other. Particle interactions at high concentration might even cause aggregation, which will be visible as an increased polydispersity index, additional peaks at higher sizes appearing in the size distribution, and/or increased turbidity.

To investigate if these effects influence the output particle sizes, record DLS data from a concentration series of your sample. When reporting particle sizes, it is recommended to use data which is devoid of sample concentration effects.

A good starting concentration for an experiment on Prometheus Panta is 1-2 mg/mL protein.