Wu Meng, Li Min
High Throughput Screening Facility at Univ. of Iowa (UIHTS), Division of Medicinal and Natural Products Chemistry, Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, 115 S. Grand Avenue, 316 PHAR, Iowa City, IA, 52242, USA,
Methods Mol Biol. 2015;1278:139-52. doi: 10.1007/978-1-4939-2425-7_8.
Label-free detection technologies have been widely used to characterize biomolecular interactions without having to label the target molecules. These technologies exhibit considerable potential in facilitating assay development and enabling new integrated readouts. When combined with high-throughput capability, label-free detection may be applied to small molecule screens for drug candidates. Based on the resonant waveguide grating biosensors, a label-free high-throughput detection system, the Epic(®) System, has been applied to monitor molecular interactions. Here we describe a generic label-free assay to quantitatively measure phospho-specific interactions between a trafficking signal-phosphorylated SWTY peptide and 14-3-3 proteins or anti-phosphopeptide antibodies. Compared with the solution-based fluorescence anisotropy assay, our results support that the high-throughput resonant waveguide grating biosensor system has shown the capability not only for high-throughput characterization of binding rank and affinity but also for the exploration of potential interacting kinases for the substrates. Hence, it provides a new generic HTS platform for phospho-detection.
无标记检测技术已被广泛用于表征生物分子相互作用，而无需对目标分子进行标记。这些技术在促进检测方法开发和实现新的集成读数方面具有巨大潜力。当与高通量能力相结合时，无标记检测可应用于小分子筛选以寻找候选药物。基于共振波导光栅生物传感器的一种无标记高通量检测系统，即Epic(®)系统，已被用于监测分子相互作用。在此，我们描述了一种通用的无标记检测方法，用于定量测量携带运输信号磷酸化的SWTY肽与14-3-3蛋白或抗磷酸肽抗体之间的磷酸化特异性相互作用。与基于溶液的荧光各向异性检测相比，我们的结果表明，高通量共振波导光栅生物传感器系统不仅能够对结合等级和亲和力进行高通量表征，还能够探索底物潜在的相互作用激酶。因此，它为磷酸化检测提供了一个新的通用高通量筛选平台。
