Lipoprotein fractions were purified by isopycnic salt gradient ultracentrifugation (Figure 5) [3 (link),4 (link)]. Briefly, 0.9 mL of plasma sample, brought to d = 1.3 g/mL with solid NaBr (472.2 mg NaBr/mL plasma), were gently overlaid with 2.1 mL of a d = 1.006 g/mL solution (0.6% NaCl) (Figure 5a), and centrifuged at 541,000× g for 3 h at 4 °C in a TL-100 series ultracentrifuge equipped with a TLA-100 fixed-angle rotor (Beckman Coulter, Indianapolis, IN, USA) (Figure 5b). Afterwards, VLDL (d = 1.006–1.063 g/mL), LDL (d = 1.063–1.19 g/mL) and HDL (d = 1.19–1.21 g/mL) fractions were collected and further purified by a second centrifugation step, performed at 541,000× g for 2 h in saline solutions at density 1.006, 1.063, and 1.21 g/mL (Figure 5c), respectively, followed by desalting and concentration using Amicon Ultra-0.5 mL centrifugal filter units (10 KDa MWCO, Merck-Millipore, Darmstadt, Germany). The degree of purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), as previously described [3 (link)].
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