Homogeneous single-label tyrosine kinase activity assay for high throughput screening
| Autor: | Markku Syrjänpää, Natalia Tong-Ochoa, Nicolas Legrand, Kari Kopra, Harri Härmä |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
Luminescence
ta3111 Biochemistry Analytical Chemistry chemistry.chemical_compound Europium Fluorescence Resonance Energy Transfer medicine Environmental Chemistry Staurosporine Phosphorylation Tyrosine ta116 Spectroscopy Enzyme Assays EGFR inhibitors Quenching (fluorescence) Kinase Tyrosine phosphorylation Molecular biology High-Throughput Screening Assays Enzyme Activation ErbB Receptors chemistry Peptides Tyrosine kinase medicine.drug |
| Zdroj: | Analytica Chimica Acta. 897:96-101 |
| ISSN: | 0003-2670 |
| Popis: | 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 Eu(3+)-chelate from luminescence quenching, monitoring high time-resolved luminescence (TRL) signals. In the presence of specific kinase inhibitor, antibody recognition and Eu(3+)-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 IC50 values 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). |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect