Thioflavin T as an amyloid dye: fibril quantification, optimal concentration and effect on aggregation

Area of Science:

• Biochemistry
• Molecular Biology
• Neuroscience

Background:

• Amyloid fibril formation is implicated in neurodegenerative diseases like Alzheimer's and prion diseases.
• Thioflavin T (ThT) is a fluorescent dye commonly used to detect and quantify amyloid fibrils.

Purpose of the Study:

• To investigate the optimal ThT concentration for amyloid quantification.
• To determine the effect of ThT concentration on amyloid aggregation kinetics.
• To establish the relationship between ThT fluorescence and amyloid concentration.

Main Methods:

• Utilized amyloid fibrils from Aβ40, Aβ42 (Alzheimer's disease-related), and yeast prion protein Ure2.
• Measured ThT fluorescence across a range of ThT concentrations (0.2–500 µM).
• Performed aggregation kinetics experiments with varying ThT concentrations.

Main Results:

• ThT fluorescence showed a linear correlation with amyloid concentration from 0.2 to 500 µM ThT.
• Maximal ThT fluorescence occurred at 20–50 µM ThT, independent of the amyloid-to-ThT ratio.
• ThT concentrations ≤ 20 µM had minimal impact on aggregation kinetics; higher concentrations showed protein-dependent effects.

Conclusions:

• ThT is a reliable tool for amyloid quantification, with optimal performance at 20–50 µM.
• ThT concentrations below 20 µM are recommended to avoid influencing amyloid aggregation dynamics.
• Understanding ThT binding characteristics is crucial for accurate amyloidosis research.