N octyl β d glucoside

Soy polar lipid extract (composition (w/w), 45.7% phosphatidylcholine, 22.1% phosphatidylethanolamine, 18.4% phosphatidylinositol, 6.9% phosphatidic acid and 6.9% other soy lipids) in chloroform (Avanti) was dried and resuspended in reconstitution buffer (30 mM HEPES pH 7.4, 140 mM NaCl and 5 mM MgCl₂). Liposomes were homogenized by extruding through 0.4 µm pore size polycarbonate membranes (Millipore) and n-octyl-β-d-glucoside (Anatrace) was added to a final concentration of 1% (v/v). Liposome suspension was sonicated in water bath three times for 1 min, with incubation on ice for 1 min in between each sonication. Purified protein was added to liposomes to a calculated lipid:protein ratio of five. The detergent n-octyl-β-d-glucoside was removed by incubating with 400 mg ml⁻¹ Bio Beads (Bio-Rad) overnight at 4 °C using a rotary shaker. Proteoliposomes (5 mg ml⁻¹) were flash frozen in liquid nitrogen and stored at −80 °C.

The detergent n-dodecyl-β-D-maltoside (DDM) was purchased from Inalco Pharmaceuticals (San Luis Obispo, CA), whereas all other detergents used were from Anatrace (Maumee, OH): n-undecyl-β-D-maltopyranoside (UDM), n-decyl-β-D-maltopyranoside (DM), n-octyl-β-D-glucoside (OG), n-nonyl-β-D-glucoside (NG), 3[(3-cholamidopropyl) dimethylammonio] propanesulfonic acid (CHAPS), n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (AZ), 2,2-dioctylpropane-1,3-bis-β-D-maltopyranoside (DMNG), and n-dodecylphosphocholine (FS-12). For solubilization analysis, 10 μL of detergent mixture (2X of desired concentration (w/v)) dissolved in 500 mM NaCl, 50 mM Tris/HCl, pH 8, with 10% glycerol, was combined with 10 μl of membrane vesicles (4 mg/ml) diluted in the same buffer. The mixture was incubated at 4°C for 2 h with gentle rotation, followed by centrifugation at 125,000 g for 45 min to separate solubilized from unsolubilized material. Samples were subjected to SDS-PAGE and Western blot analysis as described earlier. ImageJ software was used for quantitative analysis.