Monitoring protein kinase activity in cell lysates using a high-density peptide microarray.
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.
Han
Xiaoming
X
Graduate School of System Life Sciences, Department of Applied Chemistry, Faculty of Engineering, Center of Future Chemistry, Kyushu University, Japan.
Yamanouchi
Go
G
Mori
Takeshi
T
Kang
Jeong-Hun
JH
Niidome
Takuro
T
Katayama
Yoshiki
Y
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D016466:CHO Cells; D002232:Carbocyanines / Q000378:metabolism; D002457:Cell Extracts / Q000737:chemistry*; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D010455:Peptides / Q000378:metabolism*; D010766:Phosphorylation; D040081:Protein Array Analysis / Q000379:methods*; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000906:Antibodies; D002232:Carbocyanines; D002457:Cell Extracts; D005456:Fluorescent Dyes; D010455:Peptides; C085321:cyanine dye 5; D011494:Protein Kinases