This protocol describes the detection of phosphorylated proteins within cells and the identification of their intracellular localization, with a particular focus on mitotic cells. While the detection of phosphorylated proteins can be achieved using radioactive labeling with 32Pi, this method presents experimental challenges due to the requirement for radioisotopes. Alternatively, detection using phosphorylation-specific antibodies is a potent method; however, it necessitates the identification of phosphorylation sites and further requires the generation of antibodies targeting these sites, making it effective only for thoroughly analyzed phosphorylations. Here, we outline a convenient method for detecting intracellular phosphorylation using the Phos-tag technique. Additionally, we discuss the establishment of inducible stable cell lines using lentivirus, synchronization methods for cells in the mitotic phase using thymidine and nocodazole, as well as some tips for immunoprecipitation, Western blotting, and immunostaining from mitotic cells.
Keywords: COPII; Immunoprecipitation; Mitosis; Phos-tag; Phosphorylation; Stable cell lines; Western blotting.
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