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

While many methods exist to quantitatively determine protein kinase activities, (32)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 quanti...

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Detalles Bibliográficos
Autores principales: Yan, Yan, Gu, Xin, Xu, H. Eric, Melcher, Karsten
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2017
Materias:
Benchmark
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809138/
https://www.ncbi.nlm.nih.gov/pubmed/29451563
http://dx.doi.org/10.3390/mps1010003
Descripción
Sumario:While many methods exist to quantitatively determine protein kinase activities, (32)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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