Real-time monitoring of protein self-assembly by dynamic light scattering

Dynamic light scattering (DLS) is a widely accessible technique for determining particle size distributions in solution. Although most commonly used to characterize samples at equilibrium, DLS can also provide insight into dynamic processes. Here, we present a protocol that employs DLS for real-time monitoring of protein self-assembly, enabling quantitative characterization of assembly kinetics. We have applied this approach to enzyme-based nanogels and hydrogels for biocatalytic applications, and it can be readily extended to other self-assembling biomolecular systems. The protocol comprises four main components: (1) preparation and quality control of individual proteins; (2) initiation of the self-assembly reaction coupled with continuous DLS measurements; (3) time-resolved control measurements of the individual proteins; and (4) evaluation and analysis of the resulting data to extract growth profiles and assess assembly dynamics. All measurements and metadata are organized in a FAIR-compliant research data management workflow, enabling transparent analysis and long-term reuse of time-resolved DLS datasets. The protocol provides time-resolved hydrodynamic diameters with a temporal resolution on the order of minutes. It requires only small sample volumes and a standard benchtop DLS instrument. From sample preparation to data visualization and deposition, the complete workflow can be completed within approximately 8 h. This protocol enables robust, real-time monitoring of protein self-assembly and provides quantitative insights into the kinetics and performance of self-assembling systems. The integration of FAIR research data management facilitates reproducibility and comparative analysis across proteins, variants, and experimental conditions.