Core C provides basic support for laboratory procedures as required by the individual Projects 1-4. Support includes routine protein determinations using wet chemical techniques or UV spectroscopy; gel electrophoresis including the separation of proteins using native PAGE, SDS, urea, IEF, 2D and agarose gels in a variety of forms such as large gels, mini-gels, tube gels, and PhastSystem (Pharmacia) gels; immunological techniques include Western blotting, dot blotting, and ELISA. Lipoprotein separations from human plasma (LDL and HDL) are performed by sequential ultracentrifugation. LDL is provided to Projects 3 and 1. ApoA-I, A-II, and CI-I from HDL are provided to Project 3 for structural studies. Recombinant apo-B, notably apoB-5.9, is purified by immobilized metal affinity and affinity chromatography for high resolution NMR studies ongoing in Project 2. Other apoB constructs (apoB-17, 32.5 and 37) are purified from conditioned media either as the free protein or in association with lipids by a combination of chromatographic protocols for Project 2. ApoA-I mutants and truncated forms of apoB produced in Core D are purified for Project 3 for structural studies. Additionally, purification of recombinant FABPs are performed for Project 4. Lipid and protein analysis of lipoprotein, complexes and membranes are carried out as needed for Projects 1-4. Core C will assist Project 1 in the purification and isolation of the holo-LDL and insulin receptors and their sub-domains from expression systems developed in Core D. Core C also oversees the general management of the Program laboratories, ensures an adequate inventory of basic supplies, supervises the proper use of common equipment and training of new users, and guarantees compliance to all fire, radioisotope and safety regulations. Graduate students, post-doctoral fellows, and technicians are trained in analytic procedures by Core C staff to ensure uniformity and consistency within the Program Project.
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