Journal
ANALYTICAL BIOCHEMISTRY
Volume 557, Issue -, Pages 120-122Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2018.07.018
Keywords
Protein kinase; Phosphorylation; Non-radioactive assay; Enzyme kinetics
Funding
- Agencia Nacional de Promocion Cientifica y Tecnologica [PICT 2015 0642, PICT 2015 1767]
- Universidad Nacional del Litoral (CAI + D 2016)
- Max Planck Gesellschaft (Partner Group for Plant Biochemistry)
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Protein kinases constitute one of the largest protein families in nature. Current methods to assay their activity involve the use of radioactive ATP or very expensive reagents. In this work, we developed a highly sensitive, cost-effective and straightforward protocol to measure protein kinase activity using a microplate layout. Released ADP is converted into NAD(+), which is quantified by its fluorescent properties after alkaline treatment (linear range 0-10 nmol ADP). To validate our protocol, we characterized a recombinant calcium-dependent protein kinase from potato. Overall, this tool represents a critical step forward in the functional characterization of protein kinases.
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