Kinase activity and specificity assay using synthetic peptides

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 specifi city of protein kinases are important questions in characterization of kinase functions. Here, we describe a procedure to study kinase activity and specifi city using kinase-GFP complexes purifi ed 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 identifi ed and quantifi ed by mass spectrometry.