Sodium cholate

The stock solution of 50 mM POPC (1-palmitoyl-2-oleoyl-sn-3-glycero-phosphatidylcholine) was prepared by firstly drying an appropriate amount of chloroform dissolved POPC (Avanti polar lipids, USA) under nitrogen followed by overnight removal of residual chloroform in a vacuum desiccator. This lipid cake was then resuspended in MSP standard buffer (25 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.5 mM EDTA) supplemented with sodium cholate (Anatrace, USA) to a final concentration of 100 mM, so that the final POPC: sodium cholate molar ratio was 1:2. C-terminal His6-tagged FLAP was added to the resuspended lipids in a molar ratio of 1:70 (FLAP:POPC) and MSP1E3D1 was added in a molar ratio of 2:1 (MSP:FLAP). This reconstitution mixture was incubated on ice for one hour. The final concentration of sodium cholate was adjusted to 20 mM. Reconstitution of FLAP into nanodiscs was initiated by adding the Biobeads (Biorad) 0.5 mg/ml and incubating in a rotary incubator for 16 hours at 4°C [18 (link)]. This reconstitution mixture was then purified by Ni-sepharose beads to fish our FND using the His-tag in FLAP. This was followed by clarification of eluates by centrifugation at 13000 x g for 10 min at 4°C and sample purification by gel-filtration using a GE Superdex 10/300 column equilibrated with MSP standard buffer. The peak fractions were collected and concentrated using centrifugal filters.