Protein phosphorylation is a major mechanism that regulates protein activity in the cell. It is involved both directly and indirectly in regulating nearly all cellular processes where it serves as a molecular "switch" to turn protein activity on and off through cycles of phosphorylation/dephosphorylation events. Establishing that protein phosphorylation is a mechanism of cellular regulation in a particular system includes showing that the protein is phosphorylated, that the phosphorylation event changes the activity of the protein, and defining which amino acid(s) in the protein that the phosphate moiety is attached.

Phosphospecific antibodies have emerged as key tools for studying phosphorylation events by a variety of techniques including western blot, ELISA, flow cytometry, and immunohistochemical analysis. These antibodies are directed specifically against phosphorylated antigens and a key attribute is that they do not recognize the unphosphorylated form of the protein. In some experiments it may advantageous to use both the phosphospecific and pan (nonphosphospecific) antibodies. The phosphospecific antibody will detect the fraction of protein that is phosphorylated, whereas the pan antibody will detect the total amount of the substrate. By using both antibodies researchers can verify the presence of a particular protein with the pan antibody irrespective of whether phosphorylation is present. Researchers can also determine associations between the quantity of total protein and phosphorylation using phosphodetection ELISAs such as the IkBa ELISA (IMGENEX, Cat. No. IMK-501).