Nativepage bis tris gel system 4 16

Serum samples were separated on 4%–15% Tris-Glycine precasted gels (BioRad), for the denaturant PAGE and on NativePAGE Bis-Tris Gel System 4%–16% (Invitrogen, Thermo Fisher Scientific), for the native PAGE, according to the manufacturer's instructions.
In both cases, serum proteins were transferred from the gel to PVDF membranes and probed with anti-human ApoA-I antibodies (64308, Abcam, for denaturant blot and Q0496, Dako, Agilent Technologies, for native blot). Detection was performed by using HRP-conjugated secondary antibodies (GE Healthcare) and a chemiluminescence detection substrate (Super-Signal West Femto, Thermo Fisher Scientific). Blots were imaged using the Odyssey Fc system (LI-COR Biosciences).

Lyophilized POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, Avanti Polar Lipids) and cholesterol (unesterified free cholesterol [FC]) (Avanti Polar Lipids) were dissolved in 3:1 chloroform-methanol, and the solvent was evaporated by overnight incubation under a stream of nitrogen gas. POPC and FC were dissolved in phosphate buffer saline (PBS), and lipoparticles were generated by using the cholate dialysis method (22 (link)). For this, POPC and POPC-FC lipoparticles were produced by incubating POPC and cholesterol, diluted in sodium deoxycholate, with ApoA-I variants at 80:4:1 or 40:2:1 M ratio (to produce 9.6- and 8.4-nm particles, respectively) and at a 1 mg/ml protein concentration. Mixtures were incubated at 37°C for 1 h and then dialyzed against PBS for 72 h. At the end of the incubation, homogeneous 9.6-nm and 8.4-nm POPC and POPC-FC-ApoA-I particles were isolated by size-exclusion chromatography by using a preparative Superose 6 increase 10/300 GL column (GE Healthcare). Samples were eluted at a flow rate of 0.5 ml/min, in PBS, and analyzed by Blue Native PAGE using the NativePAGE Bis-Tris Gel System 4%–16% (Invitrogen, Thermo Fisher Scientific) according to the manufacturer's instructions, flushed with nitrogen and stored at –80°C prior to experiments. The stability and integrity of the particles after freezing is shown in supplemental Figure S1.