ThT 101: a primer on the use of thioflavin T to investigate amyloid formation

Abstract

Thioflavin T (ThT) has been widely used to investigate amyloid formation since 1989. While concerns have recently been raised about its use as a probe specific for amyloid, ThT still continues to be a very valuable tool for studying kinetic aspects of fibrillation and associated inhibition mechanisms. This review aims to provide a conceptual instruction manual, covering appropriate considerations and pitfalls related to the use of ThT. We start by giving a brief introduction to amyloid formation with focus on the morphology of different aggregate species, followed by a discussion of the quality of protein needed to obtain reliable fibrillation data. After an overview of the photochemical basis for ThT’s amyloid binding properties and artifacts that may arise from this, we describe how to plan and analyze ThT assays. We conclude with recommendations for complementary techniques to address shortcomings in the ThT assay.

Keywords:

• Amyloid
• thioflavin T
• aggregation mechanisms
• photochemistry

Disclosure statement

The authors report no conflicts of interest. LBM and MRA have been supported by the National Institutes of Health funding in the US, the Mayo Foundation, and the generous support from amyloidosis patients and their families. BW and JB have been supported by a grant from the Deutsche Forschungsgemeinschaft (DFG). HN is supported in part by grants-in-aid for scientific research (B) 15390123, 18390120, 22390075, 25293094, and 16H05170 from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and for research on specific diseases “amyloidosis” from the Ministry of Health, Labor and Welfare, Japan. KGM and DO are funded by the Danish Research Foundation in SPIN Grant DNRF59.

Additional information

Funding

LBM and MRA have been supported by the National Institutes of Health funding in the US [Grant number R01 GM 071514], the Mayo Foundation, and the generous support from amyloidosis patients and their families. BW and JB have been supported by a grant from the Deutsche Forschungsgemeinschaft (DFG). HN is supported in part by grants-in-aid for scientific research (B) 15390123, 18390120, 22390075, 25293094, and 16H05170 from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and for research on specific diseases “amyloidosis” from the Ministry of Health, Labor and Welfare, Japan. KGM and DO are funded by the Danish Research Foundation in SPIN Grant DNRF59.