Birk Jesper B, Wojtaszewski Jørgen F P
Section of Molecular Physiology, Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen, Copenhagen, Denmark.
Methods Mol Biol. 2018;1732:215-228. doi: 10.1007/978-1-4939-7598-3_14.
Measuring the kinase activity of the 5'-AMP-activated protein kinase (AMPK) is an essential part of understanding the regulation of this metabolic master switch. The AMPK heterotrimer can exist in 12 different constellations with potentially diverse activation patterns. It is therefore important to be able to measure heterotrimer-specific activity to discriminate between these patterns. In this chapter we describe how to measure the AMPK activity of specific heterotrimeric complexes by consecutive immunoprecipitations and how the assay can be performed in a medium throughput fashion using 96-well plates.
测量5'-AMP激活蛋白激酶（AMPK）的激酶活性是理解这一代谢主开关调控机制的重要组成部分。AMPK异源三聚体可以以12种不同的组合形式存在，具有潜在的多种激活模式。因此，能够测量异源三聚体特异性活性以区分这些模式非常重要。在本章中，我们描述了如何通过连续免疫沉淀来测量特定异源三聚体复合物的AMPK活性，以及如何使用96孔板以中等通量的方式进行该测定。
