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The thioflavin T fluorescence assay for amyloid fibril detection can be biased by the presence of exogenous compounds

Journal Article


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Abstract


  • Thioflavin T (ThT) dye fluorescence is used regularly to quantify the formation

    and inhibition of amyloid fibrils in the presence of anti-amyloidogenic

    compounds such as polyphenols. However, in this study, it was

    shown, using three polyphenolics (curcumin, quercetin and resveratrol),

    that ThT fluorescence should be used with caution in the presence of such

    exogenous compounds. The strong absorptive and fluorescent properties of

    quercetin and curcumin were found to significantly bias the ThT fluorescence

    readings in both in situ real-time ThT assays and single time-point

    dilution ThT-type assays. The presence of curcumin at concentrations as

    low as 0.01 and 1 uM was sufficient to interfere with the ThT fluorescence

    associated with fibrillar amyloid-b(1-42) (0.5 uM) and fibrillar reduced and

    carboxymethylated kappa-casein (50 lm), respectively. The ThT fluorescence

    associated with fibrillar amyloid-b(1-42) was also biased using higher concentrations

    of resveratrol, a polyphenol that is not spectroscopically active

    at the wavelengths of ThT fluorescence, implying that there can be direct

    interactions between ThT and the exogenous compound and/or competitive

    binding with ThT for the fibrils. Thus, in all cases where ThT is used

    in the presence of an exogenous compound, biases for amyloid-associated

    ThT fluorescence should be tested, regardless of whether the additive is

    spectroscopically active. Simple methods to conduct these tests were

    described. The Congo red spectral shift assay is demonstrated as a more

    viable spectrophotometric alternative to ThT, but allied methods, such as

    transmission electron microscopy, should also be used to assess fibril

    formation independently of dye-based assays.

Authors


  •   Hudson, Sean A. (external author)
  •   Ecroyd, Heath
  •   Kee, Tak W. (external author)
  •   Carver, John A. (external author)

Publication Date


  • 2009

Citation


  • Hudson, S. A., Ecroyd, H., Kee, T. W. & Carver, J. A. (2009). The thioflavin T fluorescence assay for amyloid fibril detection can be biased by the presence of exogenous compounds. Febs Journal, 276 (20), 5960-5972.

Scopus Eid


  • 2-s2.0-70349481513

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=1984&context=scipapers

Ro Metadata Url


  • http://ro.uow.edu.au/scipapers/945

Has Global Citation Frequency


Number Of Pages


  • 12

Start Page


  • 5960

End Page


  • 5972

Volume


  • 276

Issue


  • 20

Abstract


  • Thioflavin T (ThT) dye fluorescence is used regularly to quantify the formation

    and inhibition of amyloid fibrils in the presence of anti-amyloidogenic

    compounds such as polyphenols. However, in this study, it was

    shown, using three polyphenolics (curcumin, quercetin and resveratrol),

    that ThT fluorescence should be used with caution in the presence of such

    exogenous compounds. The strong absorptive and fluorescent properties of

    quercetin and curcumin were found to significantly bias the ThT fluorescence

    readings in both in situ real-time ThT assays and single time-point

    dilution ThT-type assays. The presence of curcumin at concentrations as

    low as 0.01 and 1 uM was sufficient to interfere with the ThT fluorescence

    associated with fibrillar amyloid-b(1-42) (0.5 uM) and fibrillar reduced and

    carboxymethylated kappa-casein (50 lm), respectively. The ThT fluorescence

    associated with fibrillar amyloid-b(1-42) was also biased using higher concentrations

    of resveratrol, a polyphenol that is not spectroscopically active

    at the wavelengths of ThT fluorescence, implying that there can be direct

    interactions between ThT and the exogenous compound and/or competitive

    binding with ThT for the fibrils. Thus, in all cases where ThT is used

    in the presence of an exogenous compound, biases for amyloid-associated

    ThT fluorescence should be tested, regardless of whether the additive is

    spectroscopically active. Simple methods to conduct these tests were

    described. The Congo red spectral shift assay is demonstrated as a more

    viable spectrophotometric alternative to ThT, but allied methods, such as

    transmission electron microscopy, should also be used to assess fibril

    formation independently of dye-based assays.

Authors


  •   Hudson, Sean A. (external author)
  •   Ecroyd, Heath
  •   Kee, Tak W. (external author)
  •   Carver, John A. (external author)

Publication Date


  • 2009

Citation


  • Hudson, S. A., Ecroyd, H., Kee, T. W. & Carver, J. A. (2009). The thioflavin T fluorescence assay for amyloid fibril detection can be biased by the presence of exogenous compounds. Febs Journal, 276 (20), 5960-5972.

Scopus Eid


  • 2-s2.0-70349481513

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=1984&context=scipapers

Ro Metadata Url


  • http://ro.uow.edu.au/scipapers/945

Has Global Citation Frequency


Number Of Pages


  • 12

Start Page


  • 5960

End Page


  • 5972

Volume


  • 276

Issue


  • 20