Blue-native polyacrylamide gel electrophoresis and immunoblotting

Whole parasites (5 × 10⁶) were mixed with 5 μl solubilisation buffer (750 mM aminocaproic acid, 50 mM Bis-Tris–HCl pH 7.0, 0.5 mM EDTA, 1% (w/v) dodecyl maltoside) and incubated on ice for 10 min. The resulting lysate was centrifuged at 18,000 × g at 4 °C for 30 min. Sample buffer was added to the supernatant (NativePAGE™ 5% (w/v) G-250 Sample Additive and NativePAGE™ Sample Buffer (4X) (Invitrogen™), with a final concentration of Coomassie of 0.0625% (w/v)), resulting in a final concentration of 0.25% (w/v) dodecyl maltoside. NativePAGE™ Running Buffer (20X) and NativePAGE™ Cathode Buffer Additive (20X) (Invitrogen™) were mixed to reconstitute the anode, dark and light cathode buffers according to the manufacturer’s instructions. Samples were loaded on 3-12% (v/v) Bis-Tris Gel (Novex- Life technologies) and 5 μl NativeMark™ (Invitrogen) was used as a molecular weight marker. Gels were run for 1 h at 80 V, 10 mA at 4 °C with dark cathode buffer, then for ~2 h at 200 V, 6 mA with light cathode buffer.

Proteins were transferred from the gel onto a methanol-soaked PVDF membrane (0.45 μm, Hybond™). Wet transfer in Towbin buffer (0.025 M Tris 0.192 M glycine 10% (v/v) methanol) was performed for 60 min at 100 V. The membrane was stained with Coomassie solution (50% methanol, 7% (v/v) acetic acid, and 0.05% (w/v) Coomassie R250 (Serva)) to visualise the molecular weight marker, and destained with 50% methanol, 7% acetic acid. Blots were labelled with primary rabbit anti-ATP-β (1:2000, Agrisera) coupled to secondary horseradish peroxidase (HRP) anti-rabbit (Promega) conjugated antibodies (1:10,000) and visualised using the Pierce ECL Western Blotting Substrate (Thermo Scientific).