Detection of phosphorylated proteins with phosphospecific antibodies and KPL’s immunodetection reagents

Post-translational modifications of proteins, such as phosphorylation, are important events in cell signaling that cannot be predicted from genomic information or from standard proteomic methods. It is estimated that >50% of proteins involved in cancer cell signaling are phosphorylated. Protein kinases, which add phosphate groups to proteins, respectively, are major targets for drug development and there is a significant need for assays that identify and characterize protein phosphorylation events. To this end, we have developed an assay to quantify the amount of phosphorylation on tyrosine, serine, and threonine for a protein. In order to develop this assay, antibodies directed against phosphotyrosine, phosphothreonine, and phosphoserine were screened for specificity in an immunodetection format using BSA specifically phosphorylated on different amino acids. The results demonstrate that not all commercially available antibodies are as specific as claimed. We selected the best antibodies and paired them with KPL’s immunodetection reagents to produce prototype kits for the analysis of protein phosphorylation.