Molecular Probes Incorporated (Eugene OR) proposes to invent new technology that permits quantitative, multi-color fluorescence detection of proteins in 2-D gels. This methodology will allow the parallel determination of both protein expression level changes and altered post-translational modification patterns ( e.g., glycosylation and phosphorylation) within a single 2-D gel experiment. The linear responses of the fluorescence dyes utilized, allow rigorous quantitation of even very small changes in protein expression ( ~2O% or less) and have an incredibly broad linear dynamic range (linear over lOOOX). By combining 2-D gel technology with our unique non-overlapping fluorescent dyes (designed as both total-protein and post- I translational indicators), a complete """"""""snapshot"""""""" of changes in cellular content can be imaged in a very efficient I and high-throughput manner. Additional dye and pre-fractionation technology will also be developed for ultrasensitive detection of hydrophobic integral membrane proteins. Current 2-D protein visualization technology (e.g., silver staining and other colorimetric methods) are extremely limited in dynamic range (~lOX), linearity, and are intrinsically """"""""single-color"""""""", greatly limiting the quantitative and throughput capabilities of these approaches. The new quantitative fluorescence approaches described herein, will be applied to whole tissue extracts of normal and cancerous tissues, so that altered protein expression levels and post-translational modification patterns can be determined. Special emphasis will be placed on the plasma membrane fraction of cancerous versus normal tissues, although studies on entire tissue proteomes will also be explored. This application describes advances in detection methodologies that will radically increase the information content of2-D gel experiments. This new information will greatly enhance the applicability of this technique to address key, fundamental questions associated with proteome-wide changes related to cancer. The technologies described in this application can be immediately dispersed to any laboratory capable of performing 2-D gel electrophoresis, greatly expanding the number of researchers involved in proteomic/cancer studies.
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