Plasma samples were mixed with an equal volume of native sample buffer and 5 μl were separated by 4 to 16% polyacrylamide gradient gel electrophoresis in native running buffer (Invitrogen, Carlsbad, CA). Electrophoresis was carried out at 60 V constant voltage at 10°C for 18 h. Separated lipoproteins were electrotransferred to polyvinylidene difluoride membranes. Plasma samples were prestained with a lipophilic dialkylaminostyryl fluorophore (N-(3-Sulfopropyl)-4-(4-(didecylamino)styryl)pyridinium, inner salt [Di10-ASP-PS] (Molecular Probes)). Fluorescent-stained lipoproteins were detected with a Typhoon scanner (GE Healthcare) and analyzed with ImageQuant software (GE Healthcare).
ApoA-I and ApoE-containing lipoproteins were detected by Western blot analysis with polyclonal antibodies against mouse ApoA-I (PAB 10089, Abnova) and ApoE (ab947, Chemicon). Molecular weights (particle sizes) were determined using High Molecular Weight Native Marker Kit (GE Healthcare: thyroglobulin (669 kDa, 17 nm), ferritin (440 kDa, 12.2 nm), catalase (232 kDa, 9.2 nm), lactate dehydrogenase (140 kDa, 8.1 nm), and bovine serum albumin (67 kDa, 7.1 nm)).
ApoA-I and ApoE-containing lipoproteins were detected by Western blot analysis with polyclonal antibodies against mouse ApoA-I (PAB 10089, Abnova) and ApoE (ab947, Chemicon). Molecular weights (particle sizes) were determined using High Molecular Weight Native Marker Kit (GE Healthcare: thyroglobulin (669 kDa, 17 nm), ferritin (440 kDa, 12.2 nm), catalase (232 kDa, 9.2 nm), lactate dehydrogenase (140 kDa, 8.1 nm), and bovine serum albumin (67 kDa, 7.1 nm)).