Differential phosphoprotein labeling (DIPPL), a method for comparing live cell phosphoproteomes using simultaneous analysis of (33)P- and (32)P-labeled proteins.

Andreas Wyttenbach; Aviva M Tolkovsky

doi:10.1074/mcp.t500028-mcp200

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Differential phosphoprotein labeling (DIPPL), a method for comparing live cell phosphoproteomes using simultaneous analysis of (33)P- and (32)P-labeled proteins.

Andreas Wyttenbach·Aviva M Tolkovsky

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Abstract

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 (32)Pi, whereas cells in which the kinase is active are labeled with (33)Pi. 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 (32)P- and (33)P-labeled proteins; the kinase-specific spots are revealed because of (33)P 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 (33)P-labeled proteins while allowing the more energetic (32)P-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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Acetates Animals Cell Survival Electrophoresis, Gel, Two-Dimensional Isotope Labeling Phosphoproteins Phosphorus Radioisotopes Phosphorylation Proteome Rats Rats, Wistar Stathmin Comparative Study Journal Article Research Support, Non-U.S. Gov't

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Andreas Wyttenbach, Aviva M Tolkovsky. 2005-11-21. Differential phosphoprotein labeling (DIPPL), a method for comparing live cell phosphoproteomes using simultaneous analysis of (33)P- and (32)P-labeled proteins.. https://doi.org/10.1074/mcp.t500028-mcp200

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