Development of a fluorescent high throughput assay for tau aggregation.
A high throughput assay for measuring tau aggregation using fluorescent resonance energy transfer (FRET) is described. Full-length recombinant tau labeled with Cy3 or Cy5 dye is used as ligand, and the induction of aggregation is accomplished by the addition of arachidonic acid. In the presence of this fatty acid, tau aggregation is measured by FRET in a 384-well format. The nature of tau aggregation is further characterized by competition with unlabeled tau and cross-linking experiments. It is concluded that the FRET observed under the experimental condition is due to the accumulation of tau dimers and tetramers. A model for tau aggregation is presented. The performance of this assay in a high throughput format is demonstrated and can be used to identify inhibitors of tau aggregation.
Liu
Min
M
Laboratory for Drug Discovery in Neurodegeneration, Brigham and Women's Hospital and Harvard Medical School, Cambridge, MA, USA.
Ni
Jake
J
Kosik
Kenneth S
KS
Yeh
Li-An
LA
D016718:Arachidonic Acid / Q000494:pharmacology; D001667:Binding, Competitive / Q000187:drug effects; D003432:Cross-Linking Reagents; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004353:Drug Evaluation, Preclinical; D004926:Escherichia coli / Q000378:metabolism; D005227:Fatty Acids / Q000737:chemistry; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D016875:tau Proteins / Q000737:chemistry* / Q000187:drug effects* / Q000302:isolation & purification
D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D005227:Fatty Acids; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D016875:tau Proteins; D016718:Arachidonic Acid; D004121:Dimethyl Sulfoxide