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Biotech / Medical : PROTEOMICS -- Ignore unavailable to you. Want to Upgrade?

To: Mike McFarland who wrote (477)	7/22/2003 3:07:02 PM
From: tuck	Read Replies (1) \| Respond to of 539

>>Published online before print July 22, 2003 Proc. Natl. Acad. Sci. USA, 10.1073/pnas.1633513100 Cell Biology Profiling receptor tyrosine kinase activation by using Ab microarrays Ulrik B. Nielsen , Mike H. Cardone , Anthony J. Sinskey , Gavin MacBeath , and Peter K. Sorger *Department of Biology 68-371, Massachusetts Institute of Technology, Cambridge, MA 02139; Bauer Center for Genomics Research, Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138; and Merrimack Pharmaceuticals, Inc., Cambridge, MA 02142 Communicated by Robert T. Sauer, Massachusetts Institute of Technology, Cambridge, MA, June 9, 2003 (received for review December 17, 2002) Signal transduction in mammalian cells is mediated by complex networks of interacting proteins. Understanding these networks at a circuit level requires devices to measure the amounts and activities of multiple proteins in a rapid and accurate manner. Ab microarrays have previously been applied to the quantification of labeled recombinant proteins and proteins in serum. The development of methods to analyze intracellular signaling molecules on microarrays would make Ab arrays widely useful in systems biology. Here we describe the fabrication of multiplex Ab arrays sensitive to the amounts and modification states of signal transduction proteins in crude cell lysates and the integration of these arrays with 96-well microtiter plate technology to create microarrays in microplates. We apply the Ab arrays to monitoring the activation, uptake, and signaling of ErbB receptor tyrosine kinases in human tumor cell lines. Data obtained from multicolor ratiometric microarrays correlate well with data obtained by using traditional approaches, but the arrays are faster and simpler to use. The integration of microplate and microarray methods for crude cell lysates should make it possible to identify and analyze small molecule inhibitors of signal transduction processes with unprecedented speed and precision. We demonstrate the future potential of this approach by characterizing the action of the epidermal growth factor receptor inhibitor PD153035 on cells by using Ab arrays; direct scale-up to array-based screening in 96- and 384-well plates should allow small molecules to be identified with specific inhibitory profiles against a signaling network.<< Cheers, Tuck