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Lipoprotein fractions are isolated using columns arranged in tandem to achieve complete resolution of the major lipoprotein classes from 1-2 ml of plasma. The columns are equilibrated in 50 mM phosphate-buffered saline and calibrated using lipoprotein fractions isolated by ultracentrifugation. Fractions (0.5 ml) are collected and the appropriate tubes containing the desired lipoprotein fraction(s) combined. The position of the major lipoprotein classes are determined by cholesterol (or triglyceride) assay on the column fractions using a microtiter plate enzyme-based assay. As an alternative method lipoproteins can be isolated by fast protein liquid chromatography.
This includes analysis of the composition of the fraction (protein and lipid) as well as morphologic analysis (sizing) by negative stain electron microscopy. For compositional analysis the lipoprotein fractions protein is analyzed using the BCA method with a modification to eliminate lipid interference. The samples are then lyophilized and delipidated using ethanol and ether. Lipid components are separated by TLC and analyzed by GLC and/or colorimetric assays.
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Released July 13, 2020