一种用于AMP激活蛋白激酶（AMPK）的高灵敏度非放射性活性检测方法。
A Highly Sensitive Non-Radioactive Activity Assay for AMP-Activated Protein Kinase (AMPK).
作者信息
Yan Yan, Gu Xin, Xu H Eric, Melcher Karsten
机构信息
Laboratory of Structural Sciences and Laboratory of Structural Biology and Biochemistry, Center of Cancer and Cell Biology, Van Andel Research Institute, 333 Bostwick Avenue Northeast, Grand Rapids, MI 49503, USA;
VARI-SIMM Center, Center for Structure and Function of Drug Targets, The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), Shanghai 201203, China.
出版信息
Methods Protoc. 2018 Mar;1(1). doi: 10.3390/mps1010003. Epub 2017 Oct 13.
While many methods exist to quantitatively determine protein kinase activities, P-based radioactive assays remain the workhorse of many laboratories due to their high sensitivity, high signal to noise ratio, lack of interference by fluorescent and light-absorbing small molecules, and easy quantitation. Here, we demonstrate that the interaction between the yeast Rad53 Forkhead-associated (FHA) domain and a peptide optimized for phosphorylation by AMP-Activated Protein Kinase (AMPK), which has previously been exploited for the generation of intracellular phosphorylation sensors, can serve as a readout for a highly sensitive two-step AMPK AlphaScreen kinase assay with exceptional signal-to-noise ratio.
虽然存在多种定量测定蛋白激酶活性的方法，但基于磷的放射性测定法仍是许多实验室的主要方法，因为它们具有高灵敏度、高信噪比、不受荧光和吸光小分子干扰以及易于定量的特点。在这里，我们证明酵母Rad53叉头相关（FHA）结构域与经优化可被AMP激活的蛋白激酶（AMPK）磷酸化的肽之间的相互作用——该肽此前已被用于生成细胞内磷酸化传感器——可作为一种具有出色信噪比的高灵敏度两步AMPK AlphaScreen激酶测定法的读数。
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