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2D Gel Western Blotting Using Antibodies against Acetyl, Sumo and Ubiquinin Groups on Proteins Is a Useful Tool for Studying Disease Processes

Nancy Kendrick, Matthew Hoelter, and Jon Johansen. Kendrick Labs Inc, 1202 Ann St, Madison, WI 53713

Understanding protein interactions within a cell is key to unraveling disease processes. Genomic studies alone will not suffice. Many methods exist for resolving proteins mixtures including high throughput HPLC and capillary electrophoresis, and low throughput SDS PAGE, and 2D electrophoresis (2DE). Different proteins have dramatically different characteristics imparted by rigid tertiary structure dependent on amino acid order, so no one method suffices to resolve all the proteins within cells. Of the known methods, 2DE has the highest resolution by far for resolving protein mixtures, with the capability of separating several thousand proteins on a single gel. But although the proteins are resolved, they cannot be easily visualized and identified. General staining methods including Coomassie blue, fluorescence and silver have notoriously poor sensitivity.

Western blotting may be the solution. This method of immunostaining is very sensitive, on the order of 10-100 fold higher than any staining method depending on the antibody, and can delve very deep into the proteome. While most antibodies are generated against specific proteins, new classes of antibodies are now being developed against post-translational modifications (PTM) of proteins such as phosphorylation and glycosylation. Subsets of proteins with specific PTMs affected by disease processes can be found by 2DE Western blotting and identified by mass spectrometry. In this presentation we will report results of Western blotting experiments using anti-acetyl, anti-ubiquitin, and anti-SUMO antibodies. Validation results will be presented and sensitivity of the method described along with problems and applications. Western blotting with new antibodies against acetyl, ubiquitin, and SUMO groups should be an important method for studying protein turnover within cells.

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