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

[摘要] 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.

[学科分类] 生物技术

[关键词] AMP-activated protein kinase (AMPK);Forkhead-associated (FHA) domain;kinase assay;AlphaScreen