Signaling to p53: the use of phospho-specific antibodies to probe for in vivo kinase activation.

Phospho-specific antibody technology has been recently adopted to study p53 phosphorylation both in vivo and in vitro. We have developed and carefully characterized p53 phosphospecific reagents directed to major amino- and carboxy-terminal regulatory sites. The specificities of both polyclonal and monoclonal reagents targeting the same phospho-epitope are discussed. We have defined the major chemical binding determinants for specific monoclonal reagents by determining the relative contribution of charge and sequence to epitope recognition. Remarkably, we have found that the utility of these reagents in different assay systems is not universal and depends both on epitope conformation and affinity. This is reflected in the striking differences in their ability to detect endogenous p53 and recombinant protein. Therefore, we conclude that this novel class of reagents is not generally applicable, but that the utility of each reagent must be determined empirically.