Nativemark unstained protein standard

Mass photometry measurements were performed on a 2MP-0132 mass photometer (Refeyn Ltd). For the measurements, coverslips (No. 1.5 H thickness, 24 × 50 mm, VWR) were cleaned by sequential dipping in Milli-Q-water, isopropanol and Milli-Q-water followed by drying under a stream of gaseous nitrogen. Subsequently, silicone gaskets (CultureWell^™ Reusable Gasket, Grace Bio-Labs) were placed on the cleaned coverslips to create wells for sample loading. For mass measurements, gaskets were filled with 18 μl equilibration buffer (50.mM Tris–HCl, pH 7.5 and 100 mM NaCl) to allow focusing the microscope onto the coverslip surface. Subsequently, 2 μl protein solution was added into the 18 μl droplets and mixed to obtain 50–200 nM final protein concentration. Sample binding to the coverslip surface was monitored by recording a movie for 1 minute using AcquireMP (Refeyn Ltd) software. Data analysis was performed using DiscoverMP (Refeyn Ltd). To convert the measured optical reflection-interference contrast into a molecular mass, a known protein size marker (NativeMark^™ Unstained Protein Standard, Invitrogen) was measured.

Native gel electrophoresis was performed using NativePAGE Bis-Tris Gels according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). Hsp60 and GroEL protein samples were diluted in NativePAGE Sample Buffer (1x) containing 1% digitonin and 0.5% n-dodecyl-β-D-maltoside (DDM) detergent solutions at pH 7.2. Samples and molecular weight marker (NativeMark Unstained Protein Standard-Invitrogen) were loaded on precasted 4–16% Novex NativePAGE Bis-Tris Gels that resolve proteins in the molecular weight range of 15–1,000 kDa. lectrophoresis was performed at 150 V constant voltage for 120 min, using XCell SureLock Mini-Cell (Invitrogen). Gels were stained by Coomassie G-250 staining. Gels were destained in 8% acetic acid until the desired background was obtained and scanned, using Gel Doc XR (Bio-Rad) molecular imager. Protein molecular weights were determined by the Quantity One software (Bio-Rad).

For BN-PAGE, samples extracted as detailed above were diluted as per the manufacturer’s instructions by adding NativePAGE 5% G-250 sample additive, 4× sample buffer, and water. After dilution, samples were loaded and run on NativePAGE 3 to 12% bis-tris gels alongside either NativeMark unstained protein standard (Invitrogen) or SERVA Native Marker (SERVA). The proteins were then transferred to polyvinylidene difluoride membranes using the NuPAGE Transfer Buffer using a Trans-Blot Turbo transfer system (Bio-Rad) as per the manufacturer’s instructions. Proteins were fixed to the membranes by incubating with 8% acetic acid for 15 min, washed with water, and left to dry. Membranes were subsequently reactivated with methanol to correctly visualize the unstained native protein marker. Membranes were immunoblotted as described below.

Native PAGE was performed with DDM-solubilized crude mitochondrial membrane fractions and separated on 3–12% native PAGE gradient gels (Invitrogen Reinach, Switzerland) at 4°C (Wittig et al., 2006 (link)). Proteins were transferred onto nitrocellulose membranes (Thermo Scientific) using a semi-dry protein blotting system (BioRad, Cressier, Switzerland). After blocking in TBS containing 5% (w/v) milk powder, membranes were exposed to primary antibodies rabbit anti-Cox4, rabbit anti-Cyt c1, rabbit anti-ATP synthase subunit β, or mouse anti-Hsp60 antibody (kindly provided by André Schneider, University of Bern, Bern, Switzerland), diluted 1:1,000 in TBS containing 5% (w/v) milk powder. Horseradish peroxidase-conjugated (HRP) anti-mouse, anti-rabbit (Dako, Glostrup, Denmark), anti-HA (HA.11, 16B12, Enzo Life Sciences, Lausen, Switzerland) or anti-cMyc (Santa Cruz Biotechnology) were used at dilutions of 1:5,000, and detected using an enhanced chemiluminescence detection kit (Thermo Scientific). Protein sizes were determined using NativeMark™ Unstained Protein Standard (Invitrogen, Reinach, Switzerland). Bands on blots were quantified using the gel analyzer function of Fiji (Schindelin et al., 2012 (link)). Signal intensities of multiple blots and different exposure times were analyzed with the multiple t-test function in GraphPad Prism software (Version 6.0 g).

Blue native-PAGE was performed using the Native PAGE Novex bis-Tris Gel System (Invitrogen). Five micrograms of protein analytes were mixed with NativePAGE sample buffer (Invitrogen) and NativePAGE 5% G-250 sample additive (Invitrogen). Mixtures were incubated for 30 min on ice. After incubation, analytes were applied to NativePAGE Novex 4–16% bis-Tris gel (Invitrogen), and electrophoresis was performed at 150 V. Preparation of running buffer and other conditions on Blue Native-PAGE were carried out in accordance with the manufacturer’s instructions. NativeMark unstained protein standard (Invitrogen) was used as a molecular weight marker for Blue Native-PAGE. Gels were stained with CBB R-250.

Blue native‐PAGE was performed as indicated by the manufacturer. Protein extracts and immunoprecipitation (IP) eluates were added with 4× NativePAGE™ Sample Buffer (Invitrogen™, BN2003) and NativePAGE™ 5% G‐250 Sample Additive (Invitrogen™, BN2004) to a final concentration of 0.125%. Then, samples were loaded on NativePAGE™ Novex® 3–12% Bis‐Tris Gels (Invitrogen™, BN1001) and run at 150 V in cathode buffer containing Coomassie G‐250 (by adding NativePAGE™ Cathode Buffer Additive to 1/200 dilution, Invitrogen™, BN2002 to NativePAGE™ Running Buffer, Invitrogen™, BN2001). NativeMark™ Unstained Protein Standard (Invitrogen™, LC0725) or SERVA Native Marker (SERVA, 39219.01) were loaded to predict the size of detected protein species.