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Molecular & Cellular Proteomics 5:553-559, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Technology

Differential Phosphoprotein Labeling (DIPPL), a Method for Comparing Live Cell Phosphoproteomes Using Simultaneous Analysis of ³³P- and ³²P-Labeled Proteins^*

Andreas Wyttenbach and Aviva M. Tolkovsky

From the Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, United Kingdom

We developed a differential method to reveal kinase-specific phosphorylation events in live cells. In this method, cells in which the specified kinase is inactive are labeled with ³²Pi, whereas cells in which the kinase is active are labeled with ³³Pi. The two cell extracts are then mixed, and proteins are separated on a single two-dimensional gel. The dried gel is exposed twice. The first exposure reveals both ³²P- and ³³P-labeled proteins; the kinase-specific spots are revealed because of ³³P labeling. The second exposure is conducted with two acetate sheets intervening between the gel and the detection plate. This maneuver screens out the less energetic ³³P-labeled proteins while allowing the more energetic ³²P-labeled proteins to be detected, thus leaving only those spots that were phosphorylated independently of the specified kinase. We demonstrate the utility of this method for detecting kinase substrates in rare tissue by focusing on extracellular signal-regulated kinase-specific phosphorylation of stathmin/OP18 in primary rat sympathetic neurons.

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