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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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