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Background <p>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.</p> Methods <p>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.</p> Results <p>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&#xa0;h.</p> Conclusions <p>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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月13日

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  • Maria Alessandra Martini,
  • Astrid Winterhalter,
  • Annika J. Weber,
  • Jennifer Kühne,
  • Julian S. Hertel,
  • Kersten S. Rabe,
  • Christof M. Niemeyer

摘要

Background

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.

Methods

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.

Results

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.

Conclusions

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.