State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, China.
Anal Chem. 2010 Apr 1;82(7):3067-72. doi: 10.1021/ac902804h.
In this technical note, a microarray-based spectroscopic assay with two readout principles, fluorescence and resonance light scattering (RLS), for screening kinase inhibitors has been reported. In this assay, the phosphorylation and inhibition events are marked by biotinylated antiphosphoserinen/antiphosphotyrosine antibodies, and gold nanoparticles are attached to the antibodies by standard avidin-biotin chemistry followed by silver deposition for RLS signal enhancement. The avidin conjugated fluorescein is used as a fluorescent probe. Assays for both serine kinase, the alpha-catalytic subunit of cyclic adenosine 5'-monophosphate (cAMP) dependent protein kinase (PKA), and tyrosine kinase, leukocyte-specific protein tyrosine kinase (LCK), have been developed. The utility of this assay to high-throughput screening was demonstrated with a commercial inhibitor library, a collection of 80 kinase inhibitors, and satisfactory results were obtained. In addition, quantitative determination of binding strength and the inhibiting type (type I) of these inhibitors are also demonstrated by the adenosine 5'-triphosphate (ATP) competing assays.
在本技术说明中，报道了一种基于微阵列的光谱分析测定法，具有两种读取原理，即荧光和共振光散射（RLS），用于筛选激酶抑制剂。在该测定法中，磷酸化和抑制事件由生物素化抗磷酸丝氨酸/抗磷酸酪氨酸抗体标记，并且通过标准的亲和素-生物素化学将金纳米颗粒附着到抗体上，随后进行银沉积以增强 RLS 信号。连接有亲和素的荧光素被用作荧光探针。已经开发了用于丝氨酸激酶（环腺苷酸 5'-单磷酸（cAMP）依赖性蛋白激酶（PKA）的α-催化亚基）和酪氨酸激酶（白细胞特异性蛋白酪氨酸激酶（LCK）的测定法。通过使用商业抑制剂文库（包含 80 种激酶抑制剂）证明了该测定法在高通量筛选中的实用性，并获得了令人满意的结果。此外，通过三磷酸腺苷（ATP）竞争测定法还证明了这些抑制剂的结合强度和抑制类型（I 型）的定量测定。
