Core A will provide standardized measurements of lipids, lipoproteins, lipoprotein subclasses and apoproteins in plasma and lipoprotein subfractions on mice used in research project studies. Immunochemical support of these studies will include development of apoprotein and enzyme specific immunoassays and immunoaffinity separation procedures. Specialize ultracentrifugation and gel filtration procedures will be performed for isolation of lipoproteins subspecies. Function: 1. Lipid analyses - Lipid concentrations of individual lipds will be determined by standardized, micro-scale enzyme-endpoint assay methods and gas-lipid chromatography (GLC). 2. Lipoprotein analyses - Whole plasma and lipoprotein fractions will be analyzed by nondenaturing gradient gel electrophoresis and computer-based densitometry. Plasma lipoprotein fractions and subfractions will be obtained within standard ultracentrifugal density intervals and by gel filtration techniques. Plasma lipoproteins will be separated by electrophoretic mobility on agarose gels followed by lipid staining and/or determination of apoprotein associations using Western blotting procedures. Gel filtration will be performed on mouse plasma to obtain fractions for analysis of lipid, apoprotein and enzyme concentrations. 3. Apoprotein analyses - Quantification of apoproteins B, CIII, AI, AII, E, Lp(a), and albumin will be performed using standardized enzyme-linked immunoassay (ELISA). Apoprotein associations on the same lipoprotein particle will be quantified in plasma and isolated lipoprotein fractions using magnetized immunoaffinity beads for lipoprotein capture and routine ELISA measurements to identify associated apoproteins by difference from the total sample apoprotein measurement. This technique will be used to identify apoprotein-associated enzyme activity and mass levels, as well. 4. Immunoassay and research support - In support of the immunochemical procedures, purified apoprotein and apoprotein- specific antisera stocks will be prepared and characterized for use in immunoassays and immunoaffinity separation procedures, and will be available to research projects for Western and dot blotting procedures. ELISA quantitation of human and mouse apoproteins, enzymes, and proteins of interest to research projects will be standardized in accordance with research project requirements. Standardized and quality controlled non-denaturing gradients gels in both 2-14% and 3- 31% acrylamide concentrations will be provided to research projects as needed. The Core A centrifugation facility will provide maintenance and repair of ultracentrifuges, centrifuge rotors, and training in standard and specialized density separations. Databases for storage and retrieval of analytic data will be maintained.
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