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Methods Protoc. 2018, 1(1), 3; https://doi.org/10.3390/mps1010003

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

1,2,3,* , 1,* , 1,2
and
1,*
1
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
2
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
3
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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Abstract

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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