Posttranslational modifications (PTMs) happen after or during protein translation. Small Ubiquitin-like Modifier (SUMO) proteins are covalently attached to certain lysine residues of the target proteins to modify their activity, stability, or localization. This process is called SUMOylation, which is a reversible PTM: SUMO protease enzymes can cleave SUMOs off the target protein backbone. Although many ubiquitinated proteins are targeted for degradation, SUMOylation does not necessary lead to the degradation of the modified protein but lead to the regulation of various physiological responses. SUMOylation of the examined protein cannot simply be monitored by immunoblotting techniques performed on total protein extracts, due to the SUMO-specific signals derived from other modified molecules. Furthermore, the fact that only a limited fraction of the target protein pool is SUMOylated makes the detection of SUMOylated proteins challenging. This protocol shows how SUMOylated phytochrome B (phyB) molecules can be detected using homologous and heterologous experimental systems in planta.
Keywords: Immunoblot; Immunoprecipitation; Posttranslational modification; SUMO; SUMOylation.