Tethering and sedimentation assays

Liposome-tethering assays were done with 3 μM protein and a total concentration of 2.5 mg/ml liposomes made by mixing mixing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), l-α-phosphatidylinositol (POPI), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS), and 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) dissolved in chloroform. DSPE-PEG2000-biotin and rhodamine-PE liposomes were made with 23.4% POPC, 20.3% POPE, 17.7% PI, 33.6% POPS, 3.9% DOPA, 0.1% rhodamine-PE, and 1% biotin-PE (Avanti Polar Lipids). DID (1,1′ dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine) liposomes were made with 24.5% POPC, 20.3% POPE, 17.7% PI, 33.6% POPS, 3.9% DOPA, and 0.1% DID (Avanti Polar Lipids). For each sample, 50 μl of protein and 25 μl each of Biotin and DID liposomes were incubated with 50 μl preequilibrated MagStrep resin (Novagen) for 30 min and washed with buffer (20 mM Tris, pH 8, 200 mM NaCl). Liposomes and protein bound to resin were analyzed on SDS–PAGE, and the resulting gels were imaged with a Typhoon scanner. All samples were normalized to rhodamine fluorescence to account for the total amount of lipid bound to the resin.

For sedimentation assays, Folch fraction I (Sigma) lipids were dissolved in chloroform, vacuum-dried overnight, resuspended in 20 mM Tris, pH 8, 200 mM NaCl, and sonicated to form small unilamellar vesicles (SUVs). For each sample, 25 µl of 10 µM protein was mixed with 25 µl of 2.5 mg/ml liposomes (or buffer for the negative control). The mixture was incubated at room temperature for 30 min and then pelleted in an ultracentrifuge for 1 h at 50,000 rpm. Pellet and supernatant samples were collected, and the presence of protein was analyzed by SDS–PAGE.