Kinase Activity Assay Using Unspecific Substrate or Specific Synthetic Peptides

Phosphorylation of a substrate by protein kinases leads to the activation or inactivation of numerous signaling pathways and metabolic processes. The assessment of kinase activity by using a specific or generic substrate plays a crucial role in characterization of kinase specificity and activity. Here we describe a protocol using either a synthetic peptide as a specific substrate or using myelin basic protein (MBP) as a generic substrate for the kinase activity assay. The kinase of interest is fused with a GFP (green fluorescent protein) tag and can be purified by GFP magnetic beads. Kinase-GFP complexes are then incubated with ATP, substrate, and coordinated reaction reagent for the kinase reaction. The assay is then quantified through mass spectrometry or enzymatic luminescence.