A Highly Sensitive Non-Radioactive Activity Assay for AMP-Activated Protein Kinase (AMPK)
2017
Yan Yan | Xin Gu | H. Eric Xu | Karsten Melcher
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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