The purpose of the Metal Ion Core is to provide the instrumentation, technical support, and to generate reagents necessary to fulfill the goals of this application. The core will provide atomic absorption analysis of metals; equipment and expertise to perform two-dimensional electrophoresis analysis of proteins, a mass spectrometry facility for the analysis of proteins and peptides;and a confocal microscope facility for the immunolocalization of proteins in cells and tissues. The atomic absorption instrument will allow sensitive quantitative analysis of copper and selenium ions necessary to study copper transport and transfer (Projects 1,2, and 4). The mass spectrometer will be used to identify protein interacting with the copper transporters and regulating their intracellular localization and trafficking behavior (Projects 3,4, and 5), as well as to confirm the efficiency of incorporation of selenocysteine into proteins prepared for small angle x-ray diffraction experiments. The confocal microscope will allow direct visualization of the subcellular localization of ion transport proteins so that the mechanisms controlling their trafficking in normal and diseased tissue can be understood (Project 4). The important charge of the core will be to express and purify recombinant proteins for production of antibodies against proteins involved in copper homeostasis and well as to generate proteins for collaborative research between all five projects. These latter category of proteins includes various mutants of ATP7B (Project 5), NBDdelta TMS1,2 (Project 2 and 4), and deltal-63 protein (Projects 4 and 5)
The services provided by the Metal Ion Core and the shared reagents that will be generated in the course of the proposed experiments will facilitate studies by the Program Project. The major goal of the Program Project is to understand the metal ion regulation in human health and disease. The protocols and reagents produced by the Core will also be made available to the biomedical community.
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