A fluorometric method for the assay of protein kinase activity

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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 activi...

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Detalles Bibliográficos
Autores principales: Rojas, B.E., Santin, F., Ulloa, R.M., Iglesias, A.A., Figueroa, C.M.
Formato: JOUR
Materias:
Enzyme kinetics
Non-radioactive assay
Phosphorylation
Protein kinase
lactate dehydrogenase
protein kinase
pyruvate kinase
Article
enzyme activity
fluorometry
kinetic parameters
limit of detection
potato
priority journal
substrate concentration
Acceso en línea:http://hdl.handle.net/20.500.12110/paper_00032697_v557_n_p120_Rojas
Aporte de:
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) de Universidad de Buenos Aires
Descripción
Sumario: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. © 2018 Elsevier Inc.

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