A Highly Sensitive Non-Radioactive Activity Assay for AMP-Activated Protein Kinase (AMPK)
AbstractWhile 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. View Full-Text
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Description: Size exclusion chromatography and SDS PAGE profiles of purified proteins. (A) H6GST-FHA[Rad53(22-162)], (B) H6-α1(13-550)-β1(68-270; S108D)-γ1-AMPK, and (C) MBP-α1(13-550)-β1(68-270; S108D)-γ1(24-327)-AMPK. Black line: non-phosphorylated (non-P) AMPK; red line: phosphorylated AMPK (P).
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Yan, Y.; Gu, X.; Xu, H.E.; Melcher, K. A Highly Sensitive Non-Radioactive Activity Assay for AMP-Activated Protein Kinase (AMPK). Methods Protoc. 2018, 1, 3.
Yan Y, Gu X, Xu HE, Melcher K. A Highly Sensitive Non-Radioactive Activity Assay for AMP-Activated Protein Kinase (AMPK). Methods and Protocols. 2018; 1(1):3.Chicago/Turabian Style
Yan, Yan; Gu, Xin; Xu, H. Eric; Melcher, Karsten. 2018. "A Highly Sensitive Non-Radioactive Activity Assay for AMP-Activated Protein Kinase (AMPK)." Methods Protoc. 1, no. 1: 3.
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