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

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.

SUBMITTER: Yan Y

PROVIDER: S-EPMC5809138 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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

Yan Yan Y Gu Xin X Xu H Eric HE Melcher Karsten K

Methods and protocols 20171013 1

While many methods exist to quantitatively determine protein kinase activities, <sup>32</sup>P-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), ...[more]

PMID: 29451563

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