Agitation of amyloid proteins to speed aggregation measured by ThT fluorescence: A call for standardization

• The breadth of ThT fluorescence protocols tracking amyloid aggregation is shown.
• Kinetic constants from published ThT fluorescence assays are determined and compared.
• Agitation methods of shaking, stirring and shearing are often inadequately quantified.
• A standard protocol with known denaturing conditions could help draw comparisons.

This retrospective study of protein aggregation measured by Thioflavin T (ThT) fluorescence assay in published literature has assessed protein sensitivity to denaturing conditions that include elevated temperatures, fluctuations in pH, and concentration and, in particular, agitation to induce amyloid structure formation. The dynamic tracking of fluorescence shows a sigmoidal evolution as aggregates form; the resulting kinetics of association have been analyzed to explore the range of aggregation behavior which occurs based on environmental parameters. Comparisons between the experimental results of different groups have been historically difficult due to subtleties of experimental procedures including denaturing temperature, protein type and concentration, formulation differences, and how agitation is achieved. While it is clear that agitation has a strong influence on the driving force for aggregation, the use of magnetic stirring bar or shaker table rotational speed is insufficient to characterize the degree of turbulence produced during shear. The pathway forward in resolving dependence of aggregate formation on shear may require alternative methodologies or better standardization of the experimental protocols.

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