A1 Journal article – refereed
Homogeneous single-label tyrosine kinase activity assay for high throughput screening




List of Authors: Natalia Tong-Ochoa, Kari Kopra, Markku Syrjänpää, Nicolas Legrand, Harri Härmä
Publication year: 2015
Journal: Analytica Chimica Acta
Volume number: 897
Number of pages: 6

Abstract

Protein post-translational modifications (PTMs) are regulatory mechanisms carried out by different enzymes in a cell. Kinase catalyzed phosphorylation is one of the most important PTM affecting the protein activity and function. We have developed a single-label quenching resonance energy transfer (QRET) assay to monitor tyrosine phosphorylation in a homogeneous high throughput compatible format. Epidermal growth factor receptor (EGFR) induced phosphorylation was monitored using Eu 3+-chelate labeled peptide and label-free phosphotyrosine specific antibody in presence of a soluble quencher molecule. In the QRET


kinase assay, antibody binding to phosphorylated Eu 3+-peptide protects the Eu3+-chelate from luminescence quenching, monitoring high time-resolved luminescence (TRL) signals. In the presence of specific kinase inhibitor, antibody recognition and Eu3+-chelate protection is prevented, allowing an efficient  luminescence quenching. The assay functionality was demonstrated with a panel of EGFR inhibitors (AG-1478, compound 56, erlotinib, PD174265, and staurosporine). The monitored IC50values ranged from 0.08 to 155.3 nM and were comparable to those found in the literature. EGFR activity and inhibition assays were performed using low nanomolar enzyme and antibody concentration in a 384-well plate format, demonstrating its compatibility for high throughput screening (HTS).


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