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 |
|---|---|
| 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 |
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Keywords
- Alzheimer's disease
- Cdk5
- HTS
- Tau phosphorylation
ASJC Scopus subject areas
- Analytical Chemistry
- Clinical Biochemistry
- Biotechnology
- Biochemistry
- Molecular Biology
Cite this
Development of an assay to screen for inhibitors of tau phosphorylation by Cdk5. / Ahn, Jae Suk; Musacchio, Andrea; Mapelli, Marina; Ni, Jake; Scinto, Leonard; Stein, Ross; Kosik, Kenneth S.; Yeh, Li An.
In: Journal of Biomolecular Screening, Vol. 9, No. 2, 03.2004, p. 122-131.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Development of an assay to screen for inhibitors of tau phosphorylation by Cdk5
AU - Ahn, Jae Suk
AU - Musacchio, Andrea
AU - Mapelli, Marina
AU - Ni, Jake
AU - Scinto, Leonard
AU - Stein, Ross
AU - Kosik, Kenneth S.
AU - Yeh, Li An
PY - 2004/3
Y1 - 2004/3
N2 - 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.
AB - 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.
KW - Alzheimer's disease
KW - Cdk5
KW - HTS
KW - Tau phosphorylation
UR - http://www.scopus.com/inward/record.url?scp=14644434917&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=14644434917&partnerID=8YFLogxK
U2 - 10.1177/1087057103260594
DO - 10.1177/1087057103260594
M3 - Article
VL - 9
SP - 122
EP - 131
JO - Journal of Biomolecular Screening
JF - Journal of Biomolecular Screening
SN - 1087-0571
IS - 2
ER -