Kinase activity and specificity assay using synthetic peptides.
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.
Department of Plant Systems Biology, Universität Hohenheim, Fruwirthstraße 12, Stuttgart, 70593, Germany.
Xu Na
XN
Wu
Waltraud X
WX
Schulze
D001665:Binding Sites; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry*; D010766:Phosphorylation; D010940:Plant Proteins / Q000378:metabolism; D010944:Plants / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D015398:Signal Transduction; D013379:Substrate Specificity
D010455:Peptides; D010940:Plant Proteins; D011494:Protein Kinases