Phospho-protein Analysis in Adherent Cells Using Flow Cytometry

Protein phosphorylation is one of the most important post-translational modifications, which acts as a reversible on or off switch for the activity of a large number of proteins. Analyzing the phosphorylation status of different proteins can reveal the alterations in the state of the cells in response to cellular damage, cancer and pharmaceutical drugs. Techniques such as mass spectrometry, radiolabeling, 2D-gel electrophoresis and western blotting are used to quantify protein phosphorylation. These assays can quantify phosphorylation in the bulk population of cells, however, flow cytometry can couple cell surface marker expression data with phosphorylation data to understand differential signaling in a sub-population within a heterogeneous population of cells. Our protocol describes the use of flow-cytometry for rapid and single cell-based quantification of intracellular phospho-protein with the help of anti-phospho protein specific antibody.