title: Agitation of amyloid proteins to speed aggregation measured by ThT fluorescence: a call for standardization.
abstract: 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.
authors.affiliation: Department of Materials Science and Engineering, University of Michigan, Ann Arbor, MI 48109, USA. Electronic address: kbatzli@umich.edu.
authors.forename: Kiersten M
authors.initials: KM
authors.lastname: Batzli
authors.affiliation: Department of Materials Science and Engineering, University of Michigan, Ann Arbor, MI 48109, USA; Macromolecular Science and Engineering Research Center, University of Michigan, Ann Arbor, MI 48109, USA; Department of Biomedical Engineering and Biologic and Materials Sciences (Dentistry), University of Michigan, Ann Arbor, MI 48109, USA. Electronic address: bjlove@umich.edu.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Love
mesh_terms: D000682:Amyloid / Q000737:chemistry* / Q000592:standards; D052160:Benzothiazoles; D005453:Fluorescence; D006801:Humans; D066329:Protein Aggregates; D011489:Protein Denaturation; D013844:Thiazoles / Q000737:chemistry*
chemical_list: D000682:Amyloid; D052160:Benzothiazoles; D066329:Protein Aggregates; D013844:Thiazoles; C009462:thioflavin T
keywords: Aggregation; Agitation; Kinetics; Sigmoidal modeling; Thioflavin T fluorescence