Superose 6 increase 10 300 gl gel filtration column

The NHE3-CHP1 complexes and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid were reconstituted into membrane scaffold protein 1D1 (MSP1D1) nanodiscs at a molar ratio (dimer NHE3-CHP1:MSP1D1:POPC) of 1:5:50. First, the POPC lipid (25 mg/ml) was suspended in the purification buffer supplemented with 2% (w/v) GDN. The NHE3-CHP1 complex protein and MSP1D1 were then added to the lipids in proportion and inverted at 4°C for 1 hour with rotation. Subsequently, the mixture was added into Bio-Beads SM2 (400 mg/ml) to remove detergent and incubated at 4°C for 2 hours with rotation and repeated three times. The mixture was incubated overnight after the final addition of Bio-Beads. Then, the Bio-Beads were removed and the mixture was added into PreScission Protease (PPase) to digest the sfGFP-StrepII tag of NHE3. For further purification, the protein complex was concentrated and subjected to a Superose 6 Increase 10/300 GL gel filtration column (GE Healthcare, USA) pre-equilibrated in the solubilization buffer B without detergent. The peak fractions were pooled and concentrated to about 3.0 mg/ml for cryo-EM sample preparation.