A fluorometric method for the assay of protein kinase activity

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

id	todo:paper_00032697_v557_n_p120_Rojas
record_format	dspace
spelling	todo:paper_00032697_v557_n_p120_Rojas2023-10-03T13:55:55Z A fluorometric method for the assay of protein kinase activity Rojas, B.E. Santin, F. Ulloa, R.M. Iglesias, A.A. Figueroa, C.M. 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 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. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_00032697_v557_n_p120_Rojas
institution	Universidad de Buenos Aires
institution_str	I-28
repository_str	R-134
collection	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA)
topic	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
spellingShingle	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 Rojas, B.E. Santin, F. Ulloa, R.M. Iglesias, A.A. Figueroa, C.M. A fluorometric method for the assay of protein kinase activity
topic_facet	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
description	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.
format	JOUR
author	Rojas, B.E. Santin, F. Ulloa, R.M. Iglesias, A.A. Figueroa, C.M.
author_facet	Rojas, B.E. Santin, F. Ulloa, R.M. Iglesias, A.A. Figueroa, C.M.
author_sort	Rojas, B.E.
title	A fluorometric method for the assay of protein kinase activity
title_short	A fluorometric method for the assay of protein kinase activity
title_full	A fluorometric method for the assay of protein kinase activity
title_fullStr	A fluorometric method for the assay of protein kinase activity
title_full_unstemmed	A fluorometric method for the assay of protein kinase activity
title_sort	fluorometric method for the assay of protein kinase activity
url	http://hdl.handle.net/20.500.12110/paper_00032697_v557_n_p120_Rojas
work_keys_str_mv	AT rojasbe afluorometricmethodfortheassayofproteinkinaseactivity AT santinf afluorometricmethodfortheassayofproteinkinaseactivity AT ulloarm afluorometricmethodfortheassayofproteinkinaseactivity AT iglesiasaa afluorometricmethodfortheassayofproteinkinaseactivity AT figueroacm afluorometricmethodfortheassayofproteinkinaseactivity AT rojasbe fluorometricmethodfortheassayofproteinkinaseactivity AT santinf fluorometricmethodfortheassayofproteinkinaseactivity AT ulloarm fluorometricmethodfortheassayofproteinkinaseactivity AT iglesiasaa fluorometricmethodfortheassayofproteinkinaseactivity AT figueroacm fluorometricmethodfortheassayofproteinkinaseactivity
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A fluorometric method for the assay of protein kinase activity

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