Development of an assay to screen for inhibitors of tau phosphorylation by Cdk5

Jae Suk Ahn, Andrea Musacchio, Marina Mapelli, Jake Ni, Leonard Scinto, Ross Stein, Kenneth S. Kosik, Li An Yeh

Research output: Contribution to journalArticle

15 Citations (Scopus)

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 languageEnglish
Pages (from-to)122-131
Number of pages10
JournalJournal of Biomolecular Screening
Volume9
Issue number2
DOIs
Publication statusPublished - Mar 2004

Fingerprint

Phosphorylation
Assays
Epitopes
Serine
Throughput
Immunosorbents
Catalysis
Kinetic parameters
Rate constants
Adenosine Triphosphate
Enzyme-Linked Immunosorbent Assay
Antibodies
Substrates
Enzymes

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 journalArticle

Ahn, JS, Musacchio, A, Mapelli, M, Ni, J, Scinto, L, Stein, R, Kosik, KS & Yeh, LA 2004, 'Development of an assay to screen for inhibitors of tau phosphorylation by Cdk5', Journal of Biomolecular Screening, vol. 9, no. 2, pp. 122-131. https://doi.org/10.1177/1087057103260594
Ahn, Jae Suk ; Musacchio, Andrea ; Mapelli, Marina ; Ni, Jake ; Scinto, Leonard ; Stein, Ross ; Kosik, Kenneth S. ; Yeh, Li An. / Development of an assay to screen for inhibitors of tau phosphorylation by Cdk5. In: Journal of Biomolecular Screening. 2004 ; Vol. 9, No. 2. pp. 122-131.
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