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

Overview

Journal Methods Protoc

Specialty General Medicine

Date 2018 Feb 17

PMID 29451563

Citations 4

Authors

Yan Yan

Xin Gu

H Eric Xu

Karsten Melcher

Affiliations

Soon will be listed here.

Abstract

While many methods exist to quantitatively determine protein kinase activities, 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), 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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