The purpose of the Biochemistry Core laboratory is to centralize tasks such as protein purification, production and characterization of antibodies, preparation of Fab fragments of antibodies, and analysis requiring high performance liquid chromatography (HPLC). In addition, the core will set up routine analytical techniques. We will: I): a) maintain stable supplies of dog kininogens to be used in kininogenase assays and maintain stable supplies of rat glandular kallikrein, SEV and related proteins. b) produce polyclonal antibodies against peptides present in external domains of the kinin receptor and prepare and purify antibodies against rat glandular kallikrein and submandibular enzymatic vasoconstrictor. Antibodies will be used to perform or develop RIAs and for immunocytochemical localization studies. II): maintain hybridomas and produce and purify monoclonal antibodies and Fab fragments to proteins and peptides as required by the different project investigators. We will maintain supplies of monoclonal antibodies against kinins, ANF and rat glandular kallikrein, purify immunoglobulins and produce the corresponding Fab fragments. Antibodies will be produced in large quantities using the Bioreactor technique. III) employ reverse-phase and gel-exclusion HPLC techniques to identify and analyze peptides. IV): perform analytical and quantitative molecular biology techniques as required by participant investigators. Techniques requiring polyacrylamide gel electrophoresis, blotting and autoradiography will also be performed by the Core. V): a) maintain established cell lines in culture. Cell lines, such as M1 and bovine pulmonary artery endothelial cells, will be maintained as required by participant investigators. VI): perform analytical techniques such as measurement of enzymatic activities, protein, electrolytes, inulin and p- amino hippurate. Close communication among the project investigators and Biochemistry Core personnel will permit efficient planning and allow better coordination of the research efforts. Methodological variability will be minimized, and integration of biological data will be enhanced. In this way we will increase efficiency and reduce costs.
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