Gel electrophoresis for phosphorylated proteins: a brief introduction

SUMMARY Protein phosphorylation is a key mechanism that regulates cellular physiological functions such as proliferation, migration, cell cycle progression, apoptosis, and differentiation. Aberrations in kinase activity and subsequent dysregulation of protein phosphorylation occur in the process of carcinogenesis and cancer progression and are considered to be therapeutic targets and biomarkers in oncology. Gel electrophoresis has versatile utility in the study of protein phosphorylation. Phosphorylated proteins can be enriched prior to gel electrophoresis, and the proteins separated by gel electrophoresis are visualized by colorimetric methods or western blotting with antibodies, which specifically detect phosphorylation. Phosphorylated proteins migrate differently from non-phosphorylated proteins in gels containing substrates with affinity for phosphorylation. All these methods can be combined in multiple ways, generating unique data from different viewpoints. The identification of separated proteins can be achieved by mass spectrometry, making it possible to integrate protein and genetic data. Peptide array allow the evaluation of kinase activity in heterogeneous samples. As protein phosphorylation and kinase activity are under the regulation of multiple mechanisms and their statuses influence the structures and functions of the other proteins, a multidisciplinary approach is required, and gel electrophoresis will play an important role in the study of protein phosphorylation.