(198d) Generation of Phosphorylated Protein Standards for 2D Gel Western Blotting

Differentiation pathways in higher organisms are regulated by key protein kinases that phosphorylate and thereby modify the functional state of many substrate proteins. While protein arrays exist for measurement of the kinases, identification of the multiple substrate proteins is difficult. One promising approach is 2D gel Western blotting using antibodies against phosphotyrosine, phosphoserine and phosphothreonine. The phosphorylated proteins that light up can subsequently be identified by mass spectrometry. However, while many anti-phosphoprotein antibodies are commercially available they vary widely in specificity. Purified phosphorylated protein standards, not commercially available except for casein, would be useful in screening antibodies and as positive controls for 2D Western blotting, immunoprecipitation and mass spectrometry. In this presentation we described a method for generating purified phosphorylated proteins in vitro using recombinant kinases and substrate proteins in combination with 32P labeling. The labeled proteins are run on 2D gels with Coomassie blue staining and the phosphorylated isoforms unequivocally identified by autoradiography. When the kinase reaction is carried out with unlabeled 32P, 2DE is used to verify that phosphorylation has occurred by matching the cold Coomassie patterns to those from the 32P-labeled sample. Results will be presented for 3 kinase reactions.