A simple method for the preparation of positive samples to preliminarily determine the quality of phosphorylation-specific antibody

by Yang Han, Lin Zhong, Fuli Ren

Protein phosphorylation is one of the most common and important post-translational modifications and is involved in many biological processes, including DNA damage repair, transcriptional regulation, signal transduction, and apoptosis regulation. The use of antibodies targeting phosphorylated protein is a convenient method to detect protein phosphorylation. Therefore, high-quality antibodies are essential, and uniform and effective standards are urgently needed to evaluate the quality of these phosphorylation-specific antibodies. In this study, we established a simple, broad-spectrum system for the preparation of phosphorylation-positive samples. The positive samples for evaluation of phosphorylation-specific antibodies were then validated in cells from different species and tissues, and also been proven effectively in western blot, enzyme-linked immunosorbent assays, LC-MS/MS and immunofluorescence analysis. Overall, our findings established a novel approach for evaluation of the quality of phosphorylation-specific antibodies and may have applications in various biomedical fields.