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Updated: Feb 14, 2026

Assaying Protein Kinase Activity with Radiolabeled ATP
Published on: May 26, 2017
A Highly Sensitive Non-Radioactive Activity Assay for AMP-Activated Protein Kinase (AMPK)
Yan Yan1,2,3, Xin Gu1, H Eric Xu1,2
1Laboratory 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; eric.xu@vai.org.
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.
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