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An Aggregate Weight-Normalized Thioflavin-T Measurement Scale for Characterizing Polymorphic Amyloids and Assembly
Ronald Wetzel1, Saketh Chemuru2,3, Pinaki Misra2,4
1Department of Structural Biology and Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. rwetzel@pitt.edu.
Methods in Molecular Biology (Clifton, N.J.)
|May 11, 2018
Summary
Thioflavin T (ThT) fluorescence is useful for amyloid detection but challenging for quantitation. Combining ThT assays with HPLC-supported sedimentation reveals detailed insights into amyloid assembly dynamics and aggregate structure.
Area of Science:
- Biochemistry
- Molecular Biology
- Neuroscience
Background:
- Thioflavin T (ThT) fluorescence is widely used for amyloid detection and quantitation.
- However, ThT fluorescence intensity varies significantly between different amyloid fibrils and intermediates, complicating quantitative analysis.
- Aggregate weight-normalized (AWN) ThT fluorescence can be misleading without additional data.
Purpose of the Study:
- To develop a method for more accurate quantitative analysis of amyloid assembly reactions.
- To gain deeper insights into the time-dependent aggregation process and average aggregate structure.
- To overcome the limitations of traditional ThT fluorescence assays.
Main Methods:
- Coupling ex situ ThT fluorescence measurements with an analytical HPLC-supported sedimentation assay.
- Analyzing time-dependent aggregation data from the sedimentation assay.
- Correlating sedimentation data with ThT measurements at identical time points.
Main Results:
- The sedimentation assay provides a time course of aggregation largely independent of aggregate properties.
- Combining sedimentation and ThT data reveals insights into average aggregate structure over time.
- Aggregate weight-normalized (AWN) ThT measurements, when combined with sedimentation data, offer a more comprehensive understanding of amyloid assembly.
Conclusions:
- The integrated method overcomes limitations of standalone ThT assays for amyloid research.
- This approach provides significant new insights into the kinetics and structural changes during amyloid assembly.
- The method is valuable for studying amyloid-beta (Aβ) and polyglutamine peptide aggregation.
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