Journal
TALANTA
Volume 128, Issue -, Pages 360-365Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.talanta.2014.04.061
Keywords
Protein kinase; Phosphorylation; Positively charged gold nanoparticles; Fluorometric method
Categories
Funding
- National Natural Science Foundation of China [21190044, 21222507, 21175036, 21075031]
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Herein, we report a fluorometric method for monitoring the activity and inhibition of protein kinase based on positively charged gold nanoparticles, (+)AuNPs. In this assay, when the cationic substrate peptide (S-peptide) is phosphorylated by protein kinase, the resulting negatively charged product peptide (P-peptide) will be adsorbed onto (+)AuNPs through electrostatic interaction, and the fluorescence of fluorescein isothiocyanate (FITC) on the peptide will be quenched by (+)AuNPs. Thus, the fluorescence of solution can respond to the activity of protein kinase. The feasibility of this (+) AuNPs-based method has been demonstrated by sensitive measurement of the activity of cAMP-dependent protein kinase (PKA) with a low detection limit (0.5 mU mu L-1). Furthermore, the system is successfully applied to estimate the IC50 value of PKA inhibitor H-89. The fast mix-and-readout detection process as well as the simple synthesis of the unmodified (+)AuNPs makes this proposed method a promising candidate for simple and cost-effective kinase activity detection and a good potential in high-throughput screening of kinase-related drugs. (C) 2014 Elsevier B.V. All rights reserved.
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