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Monitoring Protein Kinase Activity in Cell Lysates Using a High-Density Peptide Microarray

Graduate School of System Life Sciences, Kyushu University, Japan

Go Yamanouchi

Graduate School of System Life Sciences, Kyushu University, Japan

Takeshi Mori

Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Japan, Center for Future Chemistry, Kyushu University, Japan

Jeong-Hun Kang

CREST, JST, Japan

Takuro Niidome

Graduate School of System Life Sciences, Kyushu University, Japan, Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Japan, Center for Future Chemistry, Kyushu University, Japan

Yoshiki Katayama

Graduate School of System Life Sciences, Kyushu University, Japan, CREST, JST, Japan, Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Japan, Center for Future Chemistry, Kyushu University, Japan, ykatatcm{at}mbox.nc.kyushu-u.ac.jp

Monitoring and targeting protein kinases is widely accepted as a promising approach for disease diagnosis and drug discovery. For this purpose, the authors have developed an original type of peptide array as a high-throughput screening assay for quantitatively evaluating kinase activity. A volume of 2 nL of peptide solution was spotted onto a formyl group-modified glass slide by using an arrayer, which was designed for use with protein chip technology. The phosphorylation was recognized by fluorescence-label antibody and detected with an automatic microarray scanner widely used in DNA chip technology. The system needs low sample volume, provides a high-density peptide array, and supplies high reproducibility. It provided enough sensitivity for inhibitor screening, even though a relatively low concentration of purified kinase was employed. The assay also proved useful for the detection of intracellular kinase activity as well as for the measurement of the fluctuations of intracellular protein kinase activity with drug stimulation. Thus, this peptide array would be applicable for kinase-targeted diagnosis, cell-based drug screening, and signal pathway investigation. (Journal of Biomolecular Screening 2009:256-262)

Key Words: protein kinase • peptide • microarray • high throughput • cell lysate

This version was published on March 1, 2009

Journal of Biomolecular Screening, Vol. 14, No. 3, 256-262 (2009)
DOI: 10.1177/1087057108329348


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