Unveiling Phosphorylation Modification Using Phos-tag SDS-PAGE Gel Electrophoresis and <em>In Vitro</em> Kinase Assay

Phosphorylation is a classic post-translational modification that regulates protein function. Proteins typically contain multiple potential phosphorylation sites, which can be modified by various kinases at different locations. Studying phosphorylation changes of target proteins in disease often requires phospho-specific antibodies. However, commercial options may be limited to a single site or entirely unavailable. Here, a method is described to detect changes in phosphorylation modifications of target proteins in clinical samples using the Phos-tag gel electrophoresis and to identify detailed phosphorylation sites through in vitro kinase assays. Phosphorylated proteins bound to Phos-tag exhibit slower migration rates in SDS-PAGE gel electrophoresis, enabling semi-quantitative analysis of phosphorylation changes in disease tissues based on the mean pixel intensity of the slowly migrating bands. By combining Phos-tag SDS-PAGE with immunoblotting using pan-specific antibodies against multiple candidate proteins, researchers can efficiently identify target proteins with phosphorylation changes. Following the screening of candidate kinases, in vitro kinase assays are performed with the target protein, and the resulting phosphorylated products are subjected to mass spectrometry for precise site identification. This method does not require specific phosphorylated protein antibodies, allowing large-scale screening of tissue samples to identify altered phosphorylation states in disease. Furthermore, the identified phosphorylation sites can be used to develop specific antibodies for quantitative and localization analysis in disease tissues, providing deeper insights into their functional roles.