Unmasking of phosphorylation-sensitive epitopes on p53 and Mdm2 by a simple Western-phosphatase procedure

Monoclonal antibodies are widely used for the assessment of protein express ion levels, protein-protein interactions and protein localization. Phosphor ylation of one or more residues within an epitope recognized by a particula r antibody may compromise the ability of that antibody to bind the target p rotein. Inhibition of immunoreactivity by phosphorylation has been reported for many antibody/protein pairs. Here me describe a simple convenient prot ocol for assessing the effect of phosphorylation on immunoreactivity? emplo ying phosphatase treatment of Western blotted membranes. The efficacy of th is protocol is demonstrated for p53 and for Mdm2. This method is useful for obtaining more uniform protein quantification, as well as for rapid assess ment of changes in the extent of phosphorylation within a given epitope in response to defined signals.