Western Blot Detection of Protein Phosphorylation Level

Protein phosphorylation is a key post-transcriptional modification in cells, playing a crucial regulatory role in many cellular processes, such as signal transduction, metabolism, and gene expression. Therefore, accurately detecting the level of protein phosphorylation has become an important research direction in the field of life sciences. Among them, Western Blot (WB) technology, due to its high sensitivity and accuracy, has become the preferred method for assessing the level of protein phosphorylation.

 

WB technology first requires the target protein to be electrophoretically separated by SDS-PAGE. Then, the protein is transferred to a high-affinity membrane, such as polyvinylidene difluoride (PVDF) or nitrocellulose membrane. Then, the protein is detected using a specific antibody. In order to detect the level of protein phosphorylation, researchers use antibodies against specific phosphorylation sites, which can very specifically localize and quantify the phosphorylation state of the protein.

 

Meanwhile, there are also some challenges in the experimental process of phosphorylated protein WB. First, it is crucial to ensure that the antibodies used are highly specific, because background signals or cross-reactions may interfere with the results. Second, phosphorylation is a reversible reaction, so the methods of sample handling and storage need special attention to avoid artificial changes in phosphorylation levels. Adding phosphatase inhibitors and rapid processing of samples can help maintain the phosphorylation state of the sample.