This Core provides basic support for routine laboratory procedures as required by the individual Projects. Support includes routine protein determinations such as are performed by wet chemical techniques or UV spectroscopy; gel electrophoresis, which includes the separation of proteins using native PAGE, urea, IEF, 2D and agarose gels in a variety of formats such as large gels, mini-gels, tube gels, and the small PhastSystem (Pharmacia) gels; immunological techniques include western blotting, dot blotting, and ELISA. Lipoprotein separations, notably the procurement of LDL and HDL are performed by sequential ultracentrifugation. From HDL, apoA-I, apoA-II and the apoCs are all purified using an FPLC System (Pharmacia). ApoA-I single isoform is given to the Project for structural studies, including crystallization, and apoA-I and the apoCs are given to the Project for binding studies. Thrombin cleaved fragments of apoB from LDL are separated and purified using a Mwt sieving column and intact LDL is used by all the Projects. HPLC purification of the consensus sequence peptide of apoA-I (from a peptide synthesizer) was performed for the Atkinson Project and collaborative NMR studies with the Project by Hamilton. Currently, for the Hamilton Project, this Core is performing the limited pepsin digest of BSA in the presence of octanoic acid. The two major fragments produced by this digestion are purified by FPLC. Additionally, purification of FABP has been performed for the Hamilton project. Lipid and protein analysis of lipoproteins, complexes and membranes are also carried out as needed for all Projects. The Core also oversees the general management of the Program Project laboratories, ensures an adequate inventory of 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 this Core staff to ensure uniformity and consistency within the Program Project.
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