Basic science shows how a single mutation causes ataxia univgroningen NatureComms
proteins, one aliquot of membrane vesicles was solubilised in 50 mM Tris-HCl pH 8.0, 300 mM KCl, 1% n-dodecyl-β-D-maltoside for 45 min at 4 °C. The insoluble fraction was removed by ultracentrifugation for 30 min at 265,000 ×at 4 °C and the soluble fraction was supplemented with 15 mM imidazole pH 8.0 and incubated with 0.5 mL Ni-sepharose resin on a rocking platform at 4 °C for 1 h.
After removing Bio-beads, the proteoliposomes were centrifuged at 298,906 ×at 4 °C for 20 min, resuspended to 20 mg/mL in internal lumen buffer . Internal buffer was exchanged by three cycles of freeze-thawing. Finally, the aliquots were stored in liquid nitrogen until further use.lipids were destabilised with 30 mM β-DDM, left shaking at 20 °C for 30 s and incubated on a rocking platform for at least 3 h.
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