High-Throughput Kinase Assay Based on Surface Plasmon Resonance
High-Throughput Kinase Assay Based on Surface Plasmon Resonance
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DOI：
10.1007/978-1-60761-670-2_8
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发表时间：
2010-01-01
期刊：
影响因子：
--
通讯作者：
Nomura, Nobuo
中科院分区：
文献类型：
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作者：
Takeda, Hiroyuki;Goshima, Naoki;Nomura, Nobuo
我们设计了一种新的高通量（HTP）激酶检测使用基于阵列的表面等离子体共振（SPR）装置。为了获得高灵活性和高性能，激酶测定程序分为体外磷酸化部分和传感器芯片上的磷酸检测部分。不仅生物素化的肽，而且与FLAG-GST串联标签融合的重组蛋白可以用作天然底物。通过固定在传感器芯片上的捕获抗体选择性地捕获底物，并且通过抗pTyr抗体检测磷酸酪氨酸（pTyr）残基。根据底物的捕获水平和SPR监测的抗pTyr抗体的结合水平计算酪氨酸磷酸化水平。SPR的宽动态范围和实时监测有助于提高数据可靠性，并且基于阵列的装置的程序的优化实现了多个样品处理（1，000个样品/天）。
We have designed a novel high-throughput (HTP) kinase assay using an array-based surface plasmon resonance (SPR) apparatus. For high flexibility and performance, the kinase assay procedure is divided into an in vitro phosphorylation part and a phospho-detection part on a sensor chip. Not only biotinylated peptides but also recombinant proteins fused with FLAG-GST tandem tag can be used as native substrates. The substrate is selectively captured by a capture antibody immobilized on a sensor chip, and phospho-tyrosine (pTyr) residues are detected by an anti-pTyr antibody. The level of tyrosine phosphorylation is calculated from the capture level of the substrates and the binding level of the anti-pTyr antibody monitored by SPR. A wide dynamic range and real-time monitoring of SPR contribute to improved data reliability, and optimization of the procedure for an array-based apparatus achieved multiple sample processing (1,000 samples/day).