用于以定量高通量筛选（qHTS）形式检测激酶的生物发光方法，应用于Yes1酪氨酸激酶、葡萄糖激酶和PI5P4Kα脂质激酶
Bioluminescence Methods for Assaying Kinases in Quantitative High-Throughput Screening (qHTS) Format Applied to Yes1 Tyrosine Kinase, Glucokinase, and PI5P4Kα Lipid Kinase.
作者信息
Davis Mindy I, Auld Douglas S, Inglese James
机构信息
Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA.
Center for Proteomic Chemistry, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
出版信息
Methods Mol Biol. 2016;1360:47-58. doi: 10.1007/978-1-4939-3073-9_4.
Abstract
Assays in which the detection of a biological phenomenon is coupled to the production of bioluminescence by luciferase have gained widespread use. As firefly luciferases (FLuc) and kinases share a common substrate (ATP), coupling of a kinase to FLuc allows for the amount of ATP remaining following a kinase reaction to be assessed by quantitating the amount of luminescence produced. Alternatively, the amount of ADP produced by the kinase reaction can be coupled to FLuc through a two-step process. This chapter describes the bioluminescent assays that were developed for three classes of kinases (lipid, protein, and metabolic kinases) and miniaturized to 1536-well format, enabling their use for quantitative high-throughput (qHTS) of small-molecule libraries.
摘要
将生物现象的检测与荧光素酶产生生物发光相结合的检测方法已得到广泛应用。由于萤火虫荧光素酶（FLuc）和激酶共享一种共同底物（ATP），将激酶与FLuc偶联可通过定量产生的发光量来评估激酶反应后剩余的ATP量。或者，激酶反应产生的ADP量可通过两步过程与FLuc偶联。本章描述了为三类激酶（脂质激酶、蛋白激酶和代谢激酶）开发并小型化为1536孔板形式的生物发光检测方法，使其能够用于小分子文库的定量高通量（qHTS）筛选。
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