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

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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.… Click to show full abstract

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.

Keywords: phosphorylation specific; positive samples; quality phosphorylation; method; phosphorylation

Journal Title: PLoS ONE
Year Published: 2022

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