Cells (5 × 105) were washed with PBS, resuspended in 400 μl of 4 mg/ml digitonin (Sigma) and protease inhibitor cocktail (PIC; Sigma) and incubated on ice for 10 min. Samples were washed twice with 1 ml cold PBS and centrifuged at 10,000g for 10 min at 4 °C. Pellets were resuspended in 1× NativePAGE Sample buffer (Invitrogen) containing 1% DDM (Invitrogen #BN2005) and PIC. After 5 min on ice, the lysates were spun at 22,000g for 30 min at 4 °C. G250 (10 μl; Invitrogen) was added to the supernatant and 25 μl of solubilized complexes were loaded onto a 3–12% native precast gel at 4 °C (Invitrogen). Gels were run in dark blue cathode running buffer (Invitrogen) for 30 min and subsequently in bright blue cathode running buffer (Invitrogen) for 75 min. For OXPHOS complex detection, the total OXPHOS Blue Native WB Antibody Cocktail (abcam, catalogue no. ab110412; dilution 1:2,000) and anti-NDUFS3 antibody (Genetex, catalogue no. GTX105835; dilution 1:1,000) were used. Uncropped and unprocessed scans are provided in Supplementary Fig. 1 .
Native precast gel
Manufactured by Thermo Fisher Scientific
Native precast gel is a laboratory product designed for the separation and analysis of proteins under non-denaturing conditions. It is used to maintain the native structure and function of proteins during electrophoresis. The gel is pre-cast, which means it is prepared and packaged in advance, providing a convenient and ready-to-use solution for researchers.
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5 protocols using native precast gel
1
Native PAGE Analysis of OXPHOS Complexes
2
Assaying Mitochondrial Complex V Activity
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Heart permeabilized mitochondria (20 μg) were run on a 3–12% native precast gel (Invitrogen). CV in gel activity was performed as previously described (Acin‐Perez et al, 2020b (link)). CV in gel Activity was stopped in 50% methanol. Finally, gels were stained with Coomassie to correct for loading. Imaging was performed at different stages: after 3 h and O/N In Gel Activity, after fixing with 50% methanol and after Coomassie staining.
3
Mitochondrial Complex Isolation and Analysis
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Tissue lysates (100–150 µg) were centrifuged at 10,000g for 10 min. The pellet containing mitochondria was resuspended in 30–50 l of MAS, and protein amount was determined by BCA. Digitonin (DIG) incubation (liver, 3 mg DIG/mg protein; heart, 6 mg DIG/mg protein) was performed on ice for 5 min and then centrifuged at 20,000g for 30 min as previously described (Acin-Perez et al, 2008 (link); Acin-Perez et al, 2020a (link)). Cell preparations for BNGE were performed as described (Fernandez-Vizarra & Zeviani, 2021 (link)). Supernatants containing mitochondrial complexes and supercomplexes were mixed with blue native sample buffer (5% Blue G dye in 1 M 6-aminohexanoic acid), loaded, and run on a 3–12% native precast gel (Invitrogen). Gels were run till the blue front ran out of the gel and the gel was transferred to PVDF membranes.
4
Native PAGE Analysis of Mitochondrial Complexes
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Isolated mitochondria were resuspended (0.5 mg/ml) in NativePAGE Sample buffer (Invitrogen) containing 1.1% (w/V) digitonin (Sigma) and protease-inhibitor cocktail (Sigma). After 5 min on ice, the lysate was spun at 20,000xg for 30 min at 4 °C. G250 (5%, 1 µl/100 µg protein, Invitrogen) was added to the supernatant 30–40 µg of protein were loaded onto a 3–12% native precast gel (Invitrogen).
5
Mitochondrial Complex Isolation for BN-PAGE
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Mitochondria derived from tissues were permeabilized with either 8 mg digitonin/mg protein (heart) or 3 mg digitonin/mg protein (gastrocnemius). Digitonin incubation was performed on ice for 5 min and then centrifuged at 20,000 × g for 30 min as previously described (Acin‐Perez et al, 2008 (link), 2020a (link)). Cells preparations for BN‐PAGE were performed as described (Fernandez‐Vizarra & Zeviani, 2021 (link)). Supernatant containing mitochondrial complexes and super complexes were mixed with Blue Native sample buffer (5% Blue G dye in 1 M 6‐amiohexanoic acid), loaded, and run on a 3–12% native precast gel (Invitrogen). Gels were run till the blue front ran out of the gel and gel was transferred to PVDF membranes.
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