Nativepage running buffer

Assays were performed as in ref. ^{71 (link)}. Mitochondria were purified from ten thoraces of 7–10-day-old male flies and BN-PAGE was performed using NativePAGE gels from Life Technologies, following the manufacturer’s instructions. Mitochondria were suspended in NativePAGE sample buffer (Life Technologies) supplemented with 1% digitonin and protease inhibitors, incubated on ice for 20 min and centrifuged at 20,000 × g for 30 min at 4 °C, the supernatant was recovered, G-250 (Life Technologies) sample additive and NativePAGE sample buffer was added before loading onto 3–12% precast Bis–Tris NativePAGE gels (Life Technologies). Electrophoreses was performed using the NativePAGE Running buffer (as anode buffer, from Life technologies) and the NativePAGE Running buffer containing 0.4% Coomassie G-250 (cathode buffer). Protein complexes were revealed by staining with Novex Colloidal Blue staining kit (Life Technologies). Complex I activity in native gels was performed by incubating gels in 0.1 mg/mL NADH, 2.5 mg/mL nitrotetrazolium blue chloride, 5 mM Tris-HCl (pH 7.4) overnight at room temperature. Gels were imaged using a BioRad station and densitometry was performed in ImageJ.

Proteins were prepared (9 μl) at the required concentration (10 to 20 μM) in 10 mM tris-HCl (pH 8.0), 5 mM EDTA, and 1% β-OG buffer, to which 0.45 μl of 5% G250 (Thermo Fisher Scientific, BN2004) and 3 μl of 4× NativePAGE sample loading buffer (Thermo Fisher Scientific BN2003) were added. Running buffer (1×) was made from 20× stock of NativePAGE running buffer (Thermo Fisher Scientific, BN2001), and 1× light blue cathode buffer was made from 20× stocks of NativePAGE running buffer and Native PAGE cathode additive (Thermo Fisher Scientific, BN2002). NativePAGE Novex 4 to 16% bis-tris protein gel (Thermo Fisher Scientific, BN1002BOX) and buffer solutions were cooled to 4°C before loading samples (8 μl) and native mark unstained protein ladder (Thermo Fisher Scientific, LC0725). Gels were run at 4°C at 150 V for 1 hour, followed by 1 hour at 250 V. Gels were stained as described above for SDS-PAGE gels, with staining duration increased to 1 hour.