An Aggregate Weight-Normalized Thioflavin-T Measurement Scale for Characterizing Polymorphic Amyloids and Assembly Intermediates

  • Ronald Wetzel
  • Saketh Chemuru
  • Pinaki Misra
  • Ravi Kodali
  • Smita Mukherjee
  • Karunakar Kar
Part of the Methods in Molecular Biology book series (MIMB, volume 1777)


The red shift in the fluorescence excitation spectra of thioflavin dyes upon binding to fibrils has been a boon to the amyloid field, offering simple and effective methods for the qualitative detection of amyloid in tissue samples and for quantitation of particular fibril preparations with gravimetric linearity. The quantitative aspect of the thioflavin T (ThT) response, however, comes with an important caveat that bestows both significant limitations and great untapped power. It is now well established that amyloid fibrils of different proteins, as well as polymorphic fibrils of the same protein, can exhibit vastly different ThT fluorescence intensities for the same weight concentration of aggregates. Furthermore, the aggregated intermediates commonly observed in amyloid assembly reactions can exhibit aggregate weight-normalized (AWN) ThT fluorescence intensities that vary from essentially zero through a wide range of intermediate values before reaching the intensity of homogeneous, mature amyloid. These features make it very difficult to quantitatively interpret, without additional data, the time-dependent development of ThT fluorescence intensity in an assembly reaction. In this chapter, we describe a method for coupling ex situ ThT fluorescence determinations with an analytical HPLC supported sedimentation assay (also described in detail) that can provide significant new insights into amyloid assembly reactions. The time dependent aggregation data provided by the sedimentation assay reveals a time course of aggregation that is largely independent of aggregate properties. In addition, the combination of these data with ThT measurements of the same reaction time points reveals important aspects of average aggregate structure at each time point. Examples of the use and potential value of AWN-ThT measurements during amyloid assembly Aβ and polyglutamine peptides are provided.

Key words

Amyloid Oligomer Thioflavin T Centrifugation HPLC Assembly intermediates Polymorphic Fibrils Nucleation Elongation 



We acknowledge financial support from N.I.H. grant R01 GM099718.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Ronald Wetzel
    • 1
  • Saketh Chemuru
    • 1
    • 2
  • Pinaki Misra
    • 1
    • 3
  • Ravi Kodali
    • 1
    • 4
  • Smita Mukherjee
    • 1
    • 5
  • Karunakar Kar
    • 1
    • 6
  1. 1.Department of Structural Biology and Pittsburgh Institute for Neurodegenerative DiseasesUniversity of Pittsburgh School of MedicinePittsburghUSA
  2. 2.Department of Physiology and BiophysicsCase Western Reserve UniversityClevelandUSA
  3. 3.Department of Biochemistry and Molecular BiologyMayo Clinic College of MedicineRochesterUSA
  4. 4.Department of Chemistry and BiochemistryDuquesne UniversityPittsburghUSA
  5. 5.The Wharton SchoolUniversity of PennsylvaniaPhiladelphiaUSA
  6. 6.School of Life SciencesJawaharlal Nehru UniversityNew DelhiIndia

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