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Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis
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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
PubMed
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.

Keywords:
AmyloidAssembly intermediatesCentrifugationElongationFibrilsHPLCNucleationOligomerPolymorphicThioflavin T

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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.