A fluorometric method for the assay of protein kinase activity

Abstract

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.

Keywords: Enzyme kinetics; Non-radioactive assay; Phosphorylation; Protein kinase.