Analysis Of Giant Unilamellar Hydration

Liposomes of specific composition were prepared using a standard protocol. Briefly, Overnight hydration of lipid thin film obtained by high pressure vacuum evaporation of lipid mixture was done using Buffer A (Hepes-10 mM, KCL-150 mM, and 2mM EDTA, pH 7.4). This was followed by short periods of shaking and bath sonication for better retrieval of the lipids. Vesicles were generated from this initial mixture by freeze-thawing (10 cycles) and bath sonication for 20 min. 100 nm liposomes were produced by extrusion through AVANTI® Mini-Extruder loaded with 0.1 µm size filters. For QCM-D experiments, liposomes of 50 nm were prepared at 1 mg/mL concentration in Buffer B (Citrate 10mM, 100mM NaCl, and 0.5 mM EGTA, pH 4.6). All liposomes were used within 24h and concentration of the liposomes was measured using phospholipid assay kit (Sigma-Aldrich).

Giant Unilamellar Vesicles (GUVs):
It was achieved by keeping the protein concentration fixed (1µM) and by varying the concentration of total accessible lipids (0.07 to 2250 µM). After 15 min of incubation at room temp., the 100 µl samples were centrifuged at 220000 x g for 1 h at 4C using a Beckman Coulter’s TLA 100 rotor. At stop, the top 80 µL was considered as supernatant (S) and the remaining 20 µL added with 60 µL (Buffer A) as pellet (P). Thus obtained volumes were analyzed on a 10% SDS-PAGE and stained using Coomassie blue for quantification by Image J (National Institutes of Health, MD). Compensating the dilution of the pellet, the true Intensity of the pellet will be, IPellet = Ip – 0.25xIS, while IP & IS being experimental values obtained for pellet and supernatant, respectively. From this, affinity constants for all the proteins were calculated using the Eq. 2, where protein bound to the LUVs, [Protein]B = IPellet/(IP+IS) and K is the association constant or the reciprocal of apparent dissociation constant,