Abstract
A high-throughput assay for tau phosphorylation by cdk5/p25 is described. Full-length recombinant tau was used as a substrate in the presence of saturating adenosine triphosphate (ATP). Using PHF-1, an antibody directed specifically against 2 tau phosphorylation epitopes (serine 396 and serine 404), an enzyme-linked immunosorbent assay (ELISA)-based colorimetric assay was formatted in 384-well plates. The assay was validated by measuring kinetic parameters for cdk5/p25 catalysis and known inhibitors. Rate constants for the site-specific phosphorylations at the PHF-1 epitopes were determined and suggested preferential phosphorylation at these sites. The performance of this assay in a high-throughput format was demonstrated and used to identify inhibitors of tau phosphorylation at specific epitopes phosphorylated by cdk5/p25.
Original language | English |
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Pages (from-to) | 122-131 |
Number of pages | 10 |
Journal | Journal of Biomolecular Screening |
Volume | 9 |
Issue number | 2 |
DOIs | |
Publication status | Published - Mar 2004 |
Keywords
- Alzheimer's disease
- Cdk5
- HTS
- Tau phosphorylation
ASJC Scopus subject areas
- Analytical Chemistry
- Clinical Biochemistry
- Biotechnology
- Biochemistry
- Molecular Biology