Protein phosphorylation is a modification in which a serine, a threonine or a tyrosine residue is phosphorylated by a protein kinase by the addition of a covalently bound phosphate group. Protein phosphorylation is one of the most common modes of regulating protein function. In most cases, the protein will switch between a phosphorylated (active) and an unphosphorylated (inactive) form. Abnormal protein phosphorylation in some cases can result in cancer, diabetes, and inflammatory disease. There are various techniques used to study protein phosphorylation including western blotting, ELISA, and purification kits.
Western blot is a widely used technique for assessing the phosphorylation state of a protein. It uses gel electrophoresis to separate native or denatured proteins by length of the polypeptide. Proteins are then transferred to a membrane (usually PVDF or nitrocellulose), and then a phospho-specific antibody can be used to identify the protein of interest.
Enzyme-Linked Immunosorbent Assay (ELISA) is another tool used to measure protein phosphorylation. This format uses a capture antibody specific for the desired protein, independent of the phosphorylation state. The target protein is added allowing it to bind to the antibody-coated plate. A detection antibody is then added that will specifically bind to the phosphorylation site of the target protein. The results are analyzed by colorimetric or fluorometric detection.
Another useful tool is phosphoprotein purification kits. Very often the levels of endogenous phosphoprotein in various cell lysates are extremely low, usually 10% or less of all cellular proteins. These low levels often cannot be detected even with high concentrations of antibody and long exposure times. In this case a purification/enrichment of these phosphoproteins can be quite helpful. Phosphoprotein purification kits can offer this quick purification/enrichment of phosphoproteins from various samples.