Protein phosphorylation is a key post-transcriptional modification in cells that plays a crucial regulatory role in many cellular processes, such as signaling, metabolism, and gene expression. Therefore, accurate detection of protein phosphorylation levels has become an important research direction in the field of life sciences. Among them, Western blot (WB) technology has become the preferred method for assessing protein phosphorylation levels due to its high sensitivity and accuracy.
Figure 1Far-Western blot analysis.
The WB technique first requires electrophoresis separation of the protein of interest by SDS-PAGE. The protein is then transferred to a high-affinity membrane, such as polyvinylidene fluoride (PVDF) or nitrocellulose membrane. Next, the protein is detected using specific antibodies. To detect the phosphorylation level of a protein, researchers use antibodies that target specific phosphorylation sites, which allows for very specific localization and quantification of the phosphorylation status of the protein.
However, there are some challenges in the experimental process of phosphorylated protein WB. First, it is critical to ensure that the antibody used is highly specific, as background signals or cross-reactivity may interfere with the results. Second, phosphorylation is a reversible reaction, so special care is required in the handling and storage of samples to avoid artificial alterations in phosphorylation levels. The addition of phosphatase inhibitors and rapid sample processing can help maintain the phosphorylation status of the sample.