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Methods Protoc. 2018, 1(1), 3;

A Highly Sensitive Non-Radioactive Activity Assay for AMP-Activated Protein Kinase (AMPK)

1,2,3,* , 1,* , 1,2
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
University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China
Authors to whom correspondence should be addressed.
Received: 15 August 2017 / Revised: 26 September 2017 / Accepted: 6 October 2017 / Published: 13 October 2017
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While many methods exist to quantitatively determine protein kinase activities, 32P-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. View Full-Text
Keywords: AMP-activated protein kinase (AMPK); Forkhead-associated (FHA) domain; kinase assay; AlphaScreen AMP-activated protein kinase (AMPK); Forkhead-associated (FHA) domain; kinase assay; AlphaScreen

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    Description: Size exclusion chromatography and SDS PAGE profiles of purified proteins. (A) H6GST-FHA[Rad53(22-162)], (B) H6-α1(13-550)-β1(68-270; S108D)-γ1-AMPK, and (C) MBP-α1(13-550)-β1(68-270; S108D)-γ1(24-327)-AMPK. Black line: non-phosphorylated (non-P) AMPK; red line: phosphorylated AMPK (P).

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Yan, Y.; Gu, X.; Xu, H.E.; Melcher, K. A Highly Sensitive Non-Radioactive Activity Assay for AMP-Activated Protein Kinase (AMPK). Methods Protoc. 2018, 1, 3.

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