Native page buffer

DNA–protein interactions were analyzed on 4–16% Bis-Tris native gels (ThermoFisher Scientific, cat. no. BN1002BOX) by Electrophoretic Mobility Shift Assay (EMSA). The DNA fragment was amplified by Expand High Fidelity PCR System (Roche, cat. no. 11732650001) using #1101 and #1146 primers and Msm chromosomal DNA. The resulting 304-bp-long PCR fragment was excised and purified from agarose gel. Binding reactions were performed in 1×STB buffer (50 mM Tris–HCl pH 8.0; 5 mM Mg(C₂H₃O₂)₂; 100 µM DTT; 50 mM KCl; 50 µg/ml BSA) that contained RNAP (25 pmol), HelD (125 pmol) and DNA (0.2 pmol). First, RNAP was pre-incubated in the presence or absence of HelD (at 37 °C, 45 min). Subsequently, DNA was added and samples were incubated at 37 °C for an additional 45 min. Then, NativePage buffer (Invitrogen, cat. no. BN2003) was added, and samples were loaded on a native gel. Electrophoresis was run in a cold room (4 °C). Finally, the gel was stained with DNA stain GelRed (Biotium, cat. no. 41003) in 1×TBS for 25 min and images were taken with an Ingenius UV-light camera (Syngen). Unbound DNA was quantified by the Quantity One software (BIO-RAD). The gel was subsequently stained with Simply Blue (Invitrogen, cat. no. LC6060) for protein visualization.

Blue native gel electrophoresis (BNGE) analysis was performed as described in [33 (link)]. Briefly, 250 μg of isolated muscle mitochondria were resuspended in native page buffer (Invitrogen), protease inhibitors, and 4% digitonin, incubated for 1 h on ice and centrifuged at 20,000 × g at 4 °C. after adding 5% Coomassie G250. 30 μg were separated by 3–12% gradient BNGE and stained with for in-gel activities.

Mitochondria from skeletal muscles were isolated by differential centrifugation (Fernández-Vizarra et al., 2002 (link)) and resuspended in 25 mM sucrose, 75 mM sorbitol, 100 mM KCl, 0.05 mM EDTA, 5 mM MgCl₂, 10 mM Tris–HCl (pH 7.4), and 10 mM H₃PO₄, pH 7.4 (Civiletto et al., 2015 (link)). For BNGE analysis, 100 μg of mitochondria were resuspended in NativePAGE buffer (Invitrogen), protease inhibitors, and 4% digitonin and incubated for 1 h on ice before centrifuging at 20,000 g at 4°C. Coomassie G250 at 5% final concentration was added to the supernatants. Proteins (30 μg aliquots) were separated by pre-cast NativePAGE 3–12% Bis-Tris gels (Thermo Fisher Scientific) according to the manufacturer’s protocol and electroblotted on polyvinylidene fluoride (PVDF) membranes for immunodetection (Civiletto et al., 2015 (link)).

Thylakoids were prepared as described by Jarvi et al. (2011 (link)), using 5 × 5 mm leaf discs of infiltrated leaf areas per sample. Pelleted thylakoids were suspended in 40 μl native PAGE buffer (Invitrogen) with 1 % n-dodecyl-β-d-maltoside (Invitrogen) on ice for 15 min, centrifuged for 15 min at 4 °C, and the supernatants removed and stored at −80 °C. Prior to loading gels, 0.75 μl 5 % G-250 sample additive was added to 15 μl aliquots of thylakoid protein extracts. Electrophoresis was carried out using NativePAGE™ 4–16 % Bis–Tris gels (Invitrogen) at 150 V. For two-dimensional analysis, lanes from native PAGE were excised and proteins denatured in LDS sample buffer containing 0.05 M DTT for 30 min. Proteins in gel slices were analysed by electrophoresis using 12 % SDS Bis–Tris two-dimensional well gels (Novex, Life Technologies). Identification of bands was based on comparisons with earlier NativePAGE analytical data (Liu and Last 2015 (link)).

Fifty micrograms of mitochondria was resuspended in lysis buffer composed of 1% digitonin, 0.1 mM EDTA, 50 mM NaCl, 10% glycerol, 1 mM PMSF, 2 mM Tris-HCl, pH 7.4, and incubated on ice for 10 min. Detritus was pelleted for 10 min at 12,000g and the supernatant was mixed with 10x sample buffer (5% brilliant blue G, 10 mM Bis-Tris, pH 7.0, 500 mM aminocaproic acid). A total volume of 20 μl was loaded on a 3% to 12% NativePAGE gel (Thermo Fisher Scientific), using NativePAGE buffer (Thermo Fisher Scientific). Complexes were first separated at 60 V for 2 h, then the cathode buffer was changed from dark to light blue, followed by 150 V for 1 h. Gels were then incubated in transfer buffer (20% methanol, 0.1% sodium dodecyl sulfate, 190 mM Glycine, 25 mM Tris-HCl, pH 8.3) for 2 h. Gels were transferred to PVDF membranes over night at 25 V. Membranes were then rinsed in pure methanol for 30 s to remove Coomassie dye, blocked for 1 h in TBS-T (140 mM NaCl, 25 mM Tris-HCl, pH 7.4, 0.1% Tween-20) with 5% milk powder. Primary antibodies were anti-DDDK (abcam, ab1257) and total OXPHOS human WB antibody cocktail (ab110411) from Abcam. Secondary antibodies were coupled to HRP, and Clarity Western ECL (Biorad) was used for developing.

Mitochondrial supercomplex assembly was evaluated by two-dimensional PAGE, as described previously (Wakabayashi et al., 2020 (link)). Mitochondrial specimens (50 µg of protein) were suspended in 5% digitonin [digitonin/protein ratio: 8 (g/g); BN 2006, Thermo Fisher Scientific] and NativePAGE™ 4X Sample Buffer (BN20032, Thermo Fisher Scientific). After incubation on ice for 30 min, the suspensions were centrifuged at 20,000 × g for 15 min at 4°C. The supernatants were mixed with NativePAGE™ 5% G-250 Sample Additive (BN 2004, Thermo Fisher Scientific). Samples were loaded on 4–15% gradient gels (4,561,096, Bio-Rad) and were separated by blue native PAGE (BN-PAGE) using native PAGE buffer (BN 2001, Thermo Fisher Scientific). Proteins were then separated by SDS-PAGE and transferred to PVDF membranes. Western blotting was performed using anti-oxidative phosphorylation antibodies (MitoProfile Total OXPHOS Rodent WB Antibody Cocktail; ab110413, Abcam), as described above.