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Application of the Bio-Plex Cell Lysis Kit in Western Blot Analysis of Phosphoproteins, Rev A

Heping Han*, Biren Zhao*, Robert Hsueh#, and Susanne Mumby*, *The Antibody Laboratory and the #Cell Preparation and Analysis Laboratory, The Alliance for Cellular Signaling, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, TX 75390 USA; e-mail correspondence to: Heping.Han@UTSouthwestern.edu


Introduction
The Alliance for Cellular Signaling (AfCS) is a multidisciplinary, multiinstitutional research collaboration to study G-protein-mediated and related signaling systems (www.signaling-gateway.org). The Alliance is charting interactions among cellular signaling molecules to produce theoretical models that will describe how cells respond to stimuli. One approach for producing these models is to quantitate changes in protein phosphorylation that occur in response to ligand binding to cell surface receptors. The assay currently used in the AfCS Antibody Laboratory is multiplex western blotting with phosphorylation site-specific antibodies. While this approach has proven useful (Hsueh et al. 2002, OConnell 2003, Anon. 2003), our objective is to reduce the labor involved and substantially expand our capabilities by transitioning to Bio-Rads Bio-Plex phosphoprotein assays, which employ Luminex xMAP technology and Cell Signaling Technologys phosphorylation site-specific antibodies. The Bio-Plex cell lysis kit is used to prepare cell culture or tissue sample lysates for analysis by Bio-Plex phosphoprotein assays. We recently evaluated the Bio-Plex cell lysis kit to determine whether this protein extraction procedure yields western blotting results that are similar to those that we generate by routine cell lysis and protein extraction with the AfCS
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