Nupage 12 bis tris

Cells (10 × 10⁶) were kept on ice for 20 min in 0.15 ml of a buffer containing 150 mM NaCl, 20 mM Tris, 5 mM EDTA-Tris, pH 7.4 with the addition of 1% (v/v) Triton X-100, 10% (v/v) glycerol and protease inhibitor cocktail (Merck). Cell extracts were cleared by centrifugation at 18,000 × g for 20 min, 4 °C. Sample buffer (NuPAGE™ LDS Sample buffer, Invitrogen) supplemented with 12.5% v/v β-mercaptoethanol) was added to supernatants, and samples were separated by polyacrylamide gel (NuPAGE™, 12% Bis-Tris, Invitrogen) electrophoresis and transferred to nitrocellulose membranes. Blocking was performed with a PBS solution containing 5% (w/v) non-fat dry milk (AppliChem, Darmstadt, Germany). Antibodies for OXPHOS (OXPHOS Human WB Antibody Cocktail), prohibitin, Citrate Synthase, CyPD, IF1, and for β, α, b and OSCP subunits were from Abcam (Cambridge, UK), for γ subunit was from Genetex (Alton Pkwy Irvine CA, USA), for TOM20 was from Santa Cruz Biotechnology (Dallas, TX, USA), and the one against GAPDH was from Cell Signaling (Danvers, MA, USA). Detailed information on the specific antibodies used can be found on the key resource table. Band pixels of each replicate are normalized on band pixels of their proper loading control (β, prohibitin or GAPDH). Mean pixel ratios ± SEM are then shown proportionally to the mean of CTR samples, expressed as 100% or 1.

Samples equivalent to OD₆₂₀ = 0.5 were analysed on NuPAGE 12% Bis-Tris (Invitrogen) protein gel set to run at 150 V for 1 h. SeeBlue Plus2 Pre-Stained (Invitrogen) protein standard was used as a ladder. The gels were incubated in 100 mM zinc sulfate solution for 10 min and visualized under UV light for the presence of zinc-induced fluorescence of bilin chromophores (Raps, 1990 (link)). The gels were further stained with 1% (v/v) Coomassie Blue in acetic acid/methanol. For the bands extracted and purified from sucrose gradients (section 2.9) Coomassie staining was not able to clearly visualize the bands. Therefore, the gel was re-stained using silver staining as described in Heukeshoven and Dernick (1985) .

Cells (10 × 10⁶) were kept on ice for 20 min in 0.15 mL of a buffer containing 150 mM NaCl, 20 mM Tris, 5 mM EDTA-Tris, pH 7.4 with the addition of Triton X-100 (1% v/v, Sigma-Aldrich) and glycerol (10% v/v, Sigma-Aldrich). Sample buffer (NuPAGE™ LDS Sample buffer, Invitrogen, Waltham, MA, USA) supplemented with β-mercaptoethanol (12.5% v/v, Sigma-Aldrich) was added to supernatants, and samples were separated by polyacrylamide gel (NuPAGE™, 12% Bis-Tris, Invitrogen) electrophoresis and transferred to nitrocellulose membranes. Blocking was performed with a PBS solution containing 5% (w/v) non-fat dry milk (AppliChem, Darmstadt, Germany). Antibodies for inhibitor factor 1 (IF1), β subunit and OXPHOS (Human WB Antibody Cocktail) were from Abcam (Cambridge, UK). Band pixels of each replicate are normalized on band pixels of their proper loading control. Mean pixel ratios ± SEM are then shown.