AAT Bioquest

What are the methods used to detect phosphorylated proteins?

Posted June 15, 2023


The most common methods for detecting and quantifying protein phosphorylation are immunoprecipitation and western blotting. Both techniques require large quantities of cells of interest, which can be difficult for stem cells and tissue material. Cell lysis is also required for both methods. Immunoprecipitation involves the use of antibodies that recognize the phosphorylated target protein coupled to solid supports. After binding to the phosphorylated proteins, they can be eluted and analyzed by Western blotting. Less commonly used approaches include mass spectrometry antibody arrays, and phosphoprotein dyes or stains. MS is used to identify and quantity phosphopeptides after enzymatic digestion proceeding enzymatic digestion of proteins. For example, LC-MS can enable identification and characterization of phosphorylation sites. Antibody arrays allow a high-throughput approach for detection of phosphorylated proteins. They are composed of a group of antibodies immobilized on a solid support, allowing for simultaneous detection of various phosphorylated proteins. Several stains or dyes (e.g. Pro-Q Diamond, Phos-tag) are able to bind specifically to phosphorylated proteins. These dyes allow for visualization and identification in gels or on blots.

Additional resources

Characterization of Phosphorylated Proteins Using Mass Spectrometry

Antibodies and Proteomics

Western Blotting Assays