This section details a core facility for procedures in protein chemistry that can be utilized by all members of the program. The major functions of this core component will be: 1) Protein purification of S100beta from bovine brain, rat brain, or other sources as necessary; 2) Synthesis of peptides to be used as antigens, enzyme substrates, and neuroactive agents; 3) Preparation of polyclonal and monoclonal antibodies. Purified S100beta is required by all members of the program. It is used as a standard for radioimmunoassays, receptor binding assays, neurotrophic assays, other cellular assays (e.g. mitogenesis) and in gel electrophoresis. In addition to purification of S100beta, the particular disulfide dimer forms will be separated from monomer forms using preparative high performance electrophoresis. Peptides will be in high demand by all member of the program as antigens for polyclonal antisera, neuropeptides for physiological experiments, and enzyme substrates including protein kinase substrates. Preparation of antisera will be of great importance to all sections of the program. Site-directed antisera to against domains of S100beta, receptor molecules including the putative S100beta receptor and the 5-HT1A receptor, structural domains of S100beta including chemically synthesized S100beta, and neuronal proteins involved in the S100beta response, such as protein kinases and transcription factors. Advanced methods of protein chemistry are essential to all projects of the program to develop and provide standardized protein and peptide reagents. Many of the protein chemical techniques require training in areas outside the expertise of some of the project leaders, such as synthetic organic chemistry for peptides. Facilities for procedures in protein chemistry are most efficiently organized as a central core because the capital expenditure and maintenance costs are prohibit duplication at five sites. In addition, the quality and reproducibility of the reagents produced by the Core will be carefully controlled to assure consistency among the projects. For example, protein concentrations of S100beta standard solutions will be done by amino acid analysis, eliminating variability in colorimetric assays. Peptides are screened by mass spectrometry for evidence of contamination and accuracy of synthetic procedures. Antibodies are prepared with consistent protocols, and sera are titered, stored, and distributed under uniform conditions.
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