Lipidated APOA1 Isolation by Ultracentrifugation

Lipidated APOA1 was separated by buoyancy in a KBr gradient by ultracentrifugation. Briefly, the lipidated APOA1 was transferred to the bottom of a ½-inch × 2-inch ultracentrifuge tube and topped to 2 ml using PBS containing 0.3 mM EDTA. The density of this solution was adjusted to [2.45–2.5] g/ml using KBr. An additional 2 ml of 1.063 g/ml PBS containing 0.3 mM EDTA was layered on top. This was then centrifuged at 35,000 rpm for 24 h at 4°Cin a Beckman Optima LE-80 Ultracentrifuge using a SW55Ti rotor with maximum acceleration and without breaking at the end of the spin. Next, 200 μl fractions were collected, and density was calculated by measuring the weight of the 200 μl fraction. Absorbances at 280 nm and 555 nm were measured using the NanoDrop™ 2000/2000c (Thermo Fisher Scientific) to determine the presence of APOA1 and DiI (respectively) in each fraction. Fractions corresponding to the appropriate densities containing sufficient APOA1 were pooled and dialyzed to remove the dialysis.