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 expression levels, protein-protein interactions and protein localization. Phosphorylation of one or more residues within an epitope recognized by a particular antibody may compromise the ability of that antibody to bind the target protein. Inhibition of immunoreactivity by phosphorylation has been reported for many antibody/protein pairs. Here we describe a simple convenient protocol for assessing the effect of phosphorylation on immunoreactivity, employing phosphatase treatment of Western blotted membranes. The efficacy of this protocol is demonstrated for p53 and for Mdm2. This method is useful for obtaining more uniform protein quantification, as well as for rapid assessment of changes in the extent of phosphorylation within a given epitope in response to defined signals.
Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, Israel.
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D000469:Alkaline Phosphatase / Q000378:metabolism*; D015153:Blotting, Western / Q000379:methods; D018985:Epitopes, B-Lymphocyte / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D018985:Epitopes, B-Lymphocyte; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D000469:Alkaline Phosphatase