Abstract
A single spin density gradient ultracentrifugation method in a swinging bucket rotor has been applied for the detection and isolation of low density lipoprotein (LDL) subfractions. The visualization of the LDL heterogeneity was facilitated by prestaining the serum with Coomassie Brilliant Blue R prior to density gradient ultracentrifugation for 19.5 hr. A total of 13 human serum pools was analyzed. In each pool, two LDL subfractions, a lighter LDL1 subfraction, occasionally showing a subdivision into two bands, LDL1A and LDL1B, and a heavier LDL2 could be clearly distinguished by the banding pattern in the density gradient. Physicochemical characteristics of the isolated LDL subfractions were determined. The simple method for detection and isolation of these subfractions presented here may facilitate future studies on LDL heterogeneity.
Highlights
In this report we describe a density gradient method for the isolation of low density lipoprotein (LDL) subfractions from 3.4ml of serum, which is suitable for hypertriglyceridemic serum, in
The apoprotein composition of the LDL subfractions including apoB-100 and other high molecular weight proteins was studied with SDS gel electrophoresis using 3%/4% discontinuous polyacrylamide disc gels [16]
LDL subfractions were analyzed without further dilution (Lowry protein range 800 to 1000 mg/l) against a serum pool of known apoprotein concentrations in suitable dilutions
Summary
Agarose gel electrophoresis was performed in 0.8% agarose in barbital buffer, pH 8.6, as previously describvd [15]. The apoprotein composition of the LDL subfractions including apoB-100 and other high molecular weight proteins was studied with SDS gel electrophoresis using 3%/4% discontinuous polyacrylamide disc gels [16]. Lipoproteins mixed with SDS-phosphate buffer and dithiothreitol as a reducing reagent were boiled and immediately loaded onto the gels. This resulted in complete delipidation of the apoproteins. ApoA-I, and apoE were determined by rocket immunoelectrophoresis. ApoA-I and apoE were purified by Sephacryl S-200 column chromatography (Pharmacia, Uppsala, Sweden). LDL subfractions were analyzed without further dilution (Lowry protein range 800 to 1000 mg/l) against a serum pool of known apoprotein concentrations in suitable dilutions
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