Monitoring the Kinase Activity of Heme-Based Oxygen Sensors and Its Dependence on O 2 and Other Ligands Using Phos-Tag Electrophoresis

The nonradioactive method, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of Phos-tag (Phos-tag electrophoresis), is used to evaluate a kinase autophosphorylation and/or phosphotransfer reaction from a kinase/ATP to its protein substrate. This method outperforms...

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Bibliographic Details
Published inMethods in molecular biology (Clifton, N.J.) Vol. 2648; p. 63
Main Authors Vávra, Jakub, Sergunin, Artur, Shimizu, Toru, Martínková, Markéta
Format Journal Article
LanguageEnglish
Published United States 2023
Subjects
Adenosine Triphosphate - metabolism
Bacteria - metabolism
Electrophoresis, Polyacrylamide Gel
Heme - metabolism
Ligands
Oxygen - metabolism
Proteins
Kinase activity
Ligand binding
Phos-tag electrophoresis
Phosphotransfer reaction
Autophosphorylation reaction
Ligand-dependent kinase activity
Heme-based oxygen sensors
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Summary:The nonradioactive method, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of Phos-tag (Phos-tag electrophoresis), is used to evaluate a kinase autophosphorylation and/or phosphotransfer reaction from a kinase/ATP to its protein substrate. This method outperforms radioisotope methods using [ P]ATP for detecting trace amounts of phosphorylated protein in fresh protein preparations. Phos-tag electrophoresis has been used to perform detailed analyses of the kinase activity of a heme-based oxygen sensor-specifically, a globin-coupled histidine kinase from the soil bacterium Anaeromyxobacter sp. Fw109-5 (AfGcHK).
ISSN:1940-6029