AAT Bioquest

How do I measure kinase activity?

Posted May 10, 2023


Kinase activity is measured through several methods such as radioactive assays, non-radioactive assays, mass spectrometry, and western blot analysis. Radioactive assays are used for measuring kinase activity, where radiolabeled ATP is utilized as a phosphate donor. The substrate molecule is a protein, lipid, or peptide that is added to the reaction mixture. After the kinase reaction, the phosphorylated substrate is separated from the non phosphorylated substrate. The radioactivity is then measured using scintillation counting. In western blot analysis, levels of phosphorylated substrate or downstream targets are indirectly measured by observation. Phosphorylation of a substrate can be detected using a phospho-specific antibody. Mass spectrometry is used to identify the phosphorylation sites of a substrate proceeding a kinase reaction. This technique involves purifying the phosphorylated substrate, digesting it into small peptides, and analyzing the resulting peptides using mass spectrometry. Non-radioactive assays can also be used and can either utilize fluorescent, colorimetric, and luminescent detection of phosphate transfer of ATP to the substrate. Some examples of these assays include ADP-Glo kinase assay and LanthaScreen kinase assay.  

Additional resources

Enzyme Activity Assays for Protein Kinases: Strategies to Identify Active Substrates


Amplite® Universal Fluorimetric Kinase Assay Kit *Red Fluorescence*