Abstract
Phosphorylation of substrate proteins by protein kinases can lead to activation or inactivation of signaling pathways or metabolic processes. Precise understanding of activity and specificity of protein kinases are important questions in characterization of kinase functions. Here, we describe a procedure to study kinase activity and specificity using kinase-GFP complexes purified from plant material and synthetic peptides as substrates. Magnetic GFP beads allow purifying receptor-like kinase-GFP complexes from microsomal fractions. Kinase-GFP complexes are then incubated with ATP and the synthetic peptides for kinase reaction. Phosphorylation of substrate peptides is then identified and quantified by mass spectrometry.
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Acknowledgement
Thanks to Dr. Heidi Pertl-Obermeyer for optimization of the microsome preparation protocol.
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Wu, X.N., Schulze, W.X. (2015). Kinase Activity and Specificity Assay Using Synthetic Peptides. In: Schulze, W. (eds) Plant Phosphoproteomics. Methods in Molecular Biology, vol 1306. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2648-0_7
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DOI: https://doi.org/10.1007/978-1-4939-2648-0_7
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2647-3
Online ISBN: 978-1-4939-2648-0
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