Differential phosphoprotein labeling (DIPPL), a method for comparing live cell phosphoproteomes using simultaneous analysis of P-33- and P-32- labeled proteins
Differential phosphoprotein labeling (DIPPL), a method for comparing live cell phosphoproteomes using simultaneous analysis of P-33- and P-32- labeled proteins
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.
553-559
Wyttenbach, A.
69846a0f-fb60-4a28-84eb-ed865a5e31fa
Tolkovsky, A.M.
8390d11a-729b-47d8-a2be-76c44edfc8e6
21 November 2006
Wyttenbach, A.
69846a0f-fb60-4a28-84eb-ed865a5e31fa
Tolkovsky, A.M.
8390d11a-729b-47d8-a2be-76c44edfc8e6
Wyttenbach, A. and Tolkovsky, A.M.
(2006)
Differential phosphoprotein labeling (DIPPL), a method for comparing live cell phosphoproteomes using simultaneous analysis of P-33- and P-32- labeled proteins.
Molecular & Cellular Proteomics, 5 (3), .
(doi:10.1074/mcp.T500028-MCP200).
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 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.
This record has no associated files available for download.
More information
Published date: 21 November 2006
Identifiers
Local EPrints ID: 55932
URI: http://eprints.soton.ac.uk/id/eprint/55932
ISSN: 1535-9476
PURE UUID: b792b409-58b1-4750-9f97-5d55fc4735b6
Catalogue record
Date deposited: 06 Aug 2008
Last modified: 15 Mar 2024 10:58
Export record
Altmetrics
Contributors
Author:
A. Wyttenbach
Author:
A.M. Tolkovsky
Download statistics
Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.
View more statistics