Monitoring Protein Kinase Activity in Cell Lysates Using a High-Density Peptide Microarray
Monitoring Protein Kinase Activity in Cell Lysates Using a High-Density Peptide Microarray
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DOI：
10.1177/1087057108329348
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发表时间：
2009-03-01
影响因子：
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通讯作者：
Katayama, Yoshiki
中科院分区：
文献类型：
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作者：
Han, Xiaoming;Yamanouchi, Go;Katayama, Yoshiki
监测和靶向蛋白激酶被广泛认为是疾病诊断和药物发现的一种很有前途的方法。为此，作者开发了一种原始类型的多肽阵列，作为定量评估激酶活性的高通量筛选试验。通过使用与蛋白质芯片技术一起设计的阵列，将体积为2nL的多肽溶液斑点到甲酰基修饰的玻璃片上。用荧光标记抗体识别磷酸化，并用DNA芯片技术中广泛使用的自动芯片扫描仪进行检测。该系统所需样品体积小，提供了高密度的多肽阵列，并具有高重复性。即使使用相对较低浓度的纯化的激酶，它也为抑制剂的筛选提供了足够的灵敏度。该方法还可用于检测细胞内蛋白激酶活性，以及测量细胞内蛋白激酶活性随药物刺激的变化。因此，这种多肽阵列将适用于激酶靶向诊断、基于细胞的药物筛选和信号通路研究。(生物分子筛选杂志2009：256-262)
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)