Standard globular proteins

Reconstituted HDL (rHDL) was prepared using the sodium cholate dialysis method [43 (link)] at an initial molar ratio of 95:5:1:0, 95:5:1:0.5, 95:5:1:1, and 95:5:1:2 for POPC:cholesterol:apoA-I:Aβ, respectively. The size and hydrodynamic diameter of the rHDL particles were determined by 8–25% native polyacrylamide gradient gel electrophoresis (PAGGE, Pharmacia Phast system) by a comparison with the standard globular proteins (GE Healthcare, Uppsala, Sweden). After dialysis, all rHDL showed a similar range of residual endotoxin levels, less than 3.1 to 3.3 EU/mL, based on endotoxin quantification using a commercially available test kit (BioWhittaker, Walkersville, MD, USA).

Reconstituted HDL (rHDL) was prepared using the sodium cholate dialysis method [47 (link)] at an initial molar ratio of 95:5:1:0, 95:5:1:0.5, 95:5:1:1, and 95:5:1:2 for POPC:cholesterol:apoA-I:SAA, respectively. The physiological concentration of SAA, around 0.269 mg/mL [10 (link)], was mimicked using 0.23 mg and 0.46 mg of SAA for the 1:0.5 and 1:1 molar ratio, respectively, of apoA-I:SAA in 1 mL. The size and hydrodynamic diameter of the rHDL particles were determined by 8–25% native polyacrylamide gradient gel electrophoresis (PAGGE, Pharmacia Phast system) and by a comparison with standard globular proteins (GE healthcare, Cat# 17-0445-01). The relative migrations were compared via densitometric scanning analysis using a Gel Doc^® XR (Bio-Rad, Hercules, CA, USA) with Quantity One software, version 4.5.2.