Researchers develop model for how the brain acquires essential omega-3 fattyacids NatureComms
). While the cryo-EM maps presented here do display a density at this site, it is unclear at this resolution whether there is indeed a sodium bound. As such, further studies are required to clarify the involvement of the sodium, or lack of, in the lipid flipping mechanism.
Mfsd2a was overexpressed in Sf-9 insect cells, harvested 60 h after infection. The cell pellets were resuspended in lysis buffer containing 20 mM Tris and 150 mM NaCl and supplemented with EDTA-free protease inhibitor cocktail . The Sf-9 cells were lysed using 50 homogenizing cycles on ice. The lysate was clarified by centrifugation at 130,000 ×for 1 h. The pelleted membrane was resuspended in high-salt buffer containing 1.
To purify Mfsd2a, the membrane pellet was solubilized and membrane protein extracted in 2% n-dodecyl-β-d-maltopyranoside , 20 mM Tris , 150 mM NaCl, 5% glycerol and 0.2% cholesteryl hemisuccinate Tris salt for 4 h at 4 °C. After extraction, the insoluble debris were removed by centrifugation for 1 h at 130,000 ×. 20 mM imidazole was added to the supernatant and incubated with TALON beads for 16 h at 4 °C.
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