A3 Refereed book chapter or chapter in a compilation book
In Vitro Kinase Assay with Recombinant SnRK2s : An Example for Assaying Stress-Responsive Kinases in Plants
Authors: Fujii, Hiroaki
Editors: Sunkar, Ramanjulu
Edition: 3rd ed.
Publication year: 2024
Journal: Methods in Molecular Biology
Book title : Plant Stress Tolerance : Methods and Protocols
Journal name in source: Methods in molecular biology (Clifton, N.J.)
Journal acronym: Methods Mol Biol
Series title: Methods in Molecular Biology
Number in series: 2832
Volume: 2832
First page : 163
Last page: 170
ISBN: 978-1-0716-3972-6
eISBN: 978-1-0716-3973-3
ISSN: 1064-3745
eISSN: 1940-6029
DOI: https://doi.org/10.1007/978-1-0716-3973-3_11
Web address : https://doi.org/10.1007/978-1-0716-3973-3_11
Abstract
Protein phosphorylation is one of the most important posttranslational modifications in cell signaling pathways. Kinases and phosphatases play essential roles in transferring information between sensors and effectors under stress conditions. Several methods have been developed to analyze the phosphorylation mechanisms. Each method has advantages and disadvantages. In vitro kinase assay using recombinant proteins is a method to analyze kinase activities under simplified conditions. It is a good strategy to understand each mechanism one by one, although it is not always suitable to estimate the feature of complex machinery in vivo. In this chapter, the purification of recombinant proteins produced in Escherichia coli followed by assaying a kinase activity using radioactivity is described.
Protein phosphorylation is one of the most important posttranslational modifications in cell signaling pathways. Kinases and phosphatases play essential roles in transferring information between sensors and effectors under stress conditions. Several methods have been developed to analyze the phosphorylation mechanisms. Each method has advantages and disadvantages. In vitro kinase assay using recombinant proteins is a method to analyze kinase activities under simplified conditions. It is a good strategy to understand each mechanism one by one, although it is not always suitable to estimate the feature of complex machinery in vivo. In this chapter, the purification of recombinant proteins produced in Escherichia coli followed by assaying a kinase activity using radioactivity is described.
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