Sucrose Density Gradient

Sucrose stocks of 3, 2, 1, 0.7, and 0.3 M were prepared in hydration buffer [50 mM Hepes and 150 mM NaCl (pH 7.3)]. The protein:liposome ratio in the sample was 1:10; 10 mM liposomes and 1 mM protein were mixed with 3 M sucrose to yield a final sucrose concentration of 1.6 M. The sucrose density layers were pipetted into a 4 mL Ultra-Clear centrifuge tube (Beckman Coulter) using a Hamilton syringe. The layers were pipetted from the top down, adding the denser layers underneath the previous layer. The final gradient consisted of 250 μL of 2 M sucrose, 500 μL of 1.6 M sucrose (the sample), 250 μL of 1 M sucrose, 2500 μL of 0.7 M sucrose, 250 μL of 0.3 M sucrose, and 250 μL of hydration buffer. The sucrose gradient was centrifuged at 50000 rpm for 17 h at 4 °C (SW60 Ti, Beckman Coulter). After centrifugation, the samples were immediately fractionated into ∼290 μL fractions by a Hamilton syringe, from top to bottom.

A 20 μL sample of each fraction was diluted 10 times in hydration buffer, and the absorbance (280 nm) and emission (533 nm) spectra were recorded (Magellan).

An 11 μL sample was mixed with loading dye and loaded onto a 10% or 4–12% Bis-Tris sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel (NuPAGE, Invitrogen) run at 180 V in MOPS buffer together with a PageRuler Plus prestained protein ladder (ThermoFisher Scientific). The gels were stained by silver staining. The gels were soaked for 2 h in a fix solution (40% EtOH and 10% acetic acid) and washed for 3 × 20 min with 30% EtOH. Thereafter, the gels were washed with a thiosulfate solution (0.02%) for 1 min and then subsequently washed for 3 × 20 s in dH₂O before a 1 h incubation in AgNO₃ (0.2%). The gels were washed again, 3 × 20 s with dH₂O, and developed for 1–10 min in a developing solution (0.0004% N₂S₂O₃, 3% Na₂CO₃, and 0.05% H₂CO). The development was quenched by exchanging the development solution for a stop solution (5% glycine), and the mixture incubated for 5 min prior to a final wash with dH₂O. The gels were imaged using a Konica Minolta Bizhub c458 instrument, and the gel band density was analyzed by ImageJ.⁴²