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Updated: Feb 28, 2026
In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein
Published on: January 2, 2015
Thioflavin T fluorescence to analyse amyloid formation kinetics: Measurement frequency as a factor explaining
Mathew Sebastiao1, Noe Quittot1, Steve Bourgault1
1Department of Chemistry, University of Québec in Montreal, Montreal, C.P. 8888, Succursale Centre-Ville, Montreal, H3C 3P8, Canada; Quebec Network for Research on Protein Function, Engineering, and Applications, PROTEO, Canada.
Abstract:
The most frequent method to monitor amyloid formation relies on the fluorescence of thioflavin T (ThT). The present study reports a novel factor of irreproducibility in ThT kinetic assays performed in microplate. Discrepancies among kinetics of amyloid assembly, performed under quiescent conditions, were associated with the frequency of fluorescence measurement. Evaluating self-assembly of the islet amyloid polypeptide at short intervals hastened its fibrillization. This observation was confirmed by transmission electron microscopy, circular dichroism spectroscopy and 8-anilino-1-naphthalenesulfonic acid fluorescence. This effect, attributed to agitation during microplate displacements between fluorescence measurements, reinforces the importance of a better standardization in amyloid formation assays.
Insights
The frequency of fluorescence measurement in thioflavin T (ThT) assays can alter amyloid formation kinetics. This agitation effect highlights the need for improved standardization in amyloid detection methods.
Area of Science:
- Biochemistry
- Biophysics
- Materials Science
Background:
- Amyloid formation is crucial in diseases like Alzheimer's and Type 2 diabetes.
- Thioflavin T (ThT) fluorescence assays are standard for monitoring amyloid aggregation kinetics.
- Reproducibility issues in ThT assays can hinder accurate amyloidosis research.
Purpose of the Study:
- To identify a novel source of irreproducibility in microplate-based ThT kinetic assays.
- To investigate the impact of fluorescence measurement frequency on amyloid self-assembly.
- To propose enhanced standardization for amyloid formation assays.
Main Methods:
- Kinetic monitoring of amyloid formation using thioflavin T (ThT) fluorescence.
- Transmission electron microscopy (TEM) for structural analysis.
- Circular dichroism (CD) spectroscopy for conformational changes.
- 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence for hydrophobic patch exposure.
Main Results:
- The frequency of fluorescence measurements in ThT assays significantly influenced amyloid assembly kinetics.
- Shorter measurement intervals, causing increased microplate agitation, accelerated islet amyloid polypeptide (IAPP) fibrillization.
- Confirmatory evidence from TEM, CD, and ANS fluorescence supported the agitation-induced acceleration of amyloid formation.
Conclusions:
- Microplate agitation during frequent fluorescence measurements is a critical, previously unrecognized factor affecting amyloid formation.
- Standardization protocols for ThT kinetic assays must account for mechanical agitation to ensure reliable results.
- This finding is vital for accurate amyloidosis research and therapeutic development.
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