Bn page sample buffer

Cells were lysed using 2% digitonin buffer (50 mM BisTris pH 7, 1x protease inhibitor cocktail [Roche], 100 mM NaCl and 10% Glycerol) for 45 min. Samples were than diluted to a final concentration of 1% digitonin and 50 mM NaCl. Samples were pelleted at 20,000g for 20 min using refrigerated centrifuge. Supernatant was collected, mixed with BN-PAGE sample buffer (Invitrogen) and 5% G520 (Sigma). To run purified protein, samples were mixed in 1% digitonin buffer (50 mM BisTris pH 7, 1x protease inhibitor, 50 mM NaCl and 10% Glycerol) with BN-PAGE sample buffer and 5% G520.
Samples were run using 3–12% BN-PAGE Novex Bis-Tris (Invitrogen) gel at 150 V for 1 hr with dark blue buffer (50 mM Tricine pH 7, 50 mM BisTris pH 7% and 0.02%% G250) at room temperature and then exchange with light blue buffer (50 mM Tricine pH 7, 50 mM BisTris pH 7% and 0.002%% G250) for 4 hr in the cold room. To probe the Sec61 translocon, the gels were run for 1 hr with dark blue buffer at room temperature and 2 hr 45 min with light blue buffer in the cold room. After electrophoresis, gel was gently shaken in 1x Tris-Glycine-SDS transfer buffer for 20 min to remove residual blue dye. Transfer was performed using PVDF membrane (EMD Millipore) for 1 hr and 30 min at 85V. After transfer, the membrane was fixed with 4% acetic acid and followed with a standard western blotting procedure.

Methodology for hepatic mitochondrial extraction has been previously described [25 (link)]. Briefly, liver samples were sectioned into small pieces and mixed with sucrose (0.25 M), EDTA (1.0 M), Hepes (10 mM of 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid), pH 7.4 at 0.1g fresh weight tissue / 0.25ml buffer. The tissue mixtures were homogenized with mortar and pestle for 3 min, and then sonicated for 3 cycles at 60 Hz during 5s (VibraCell 50-sonics & Material Inc. Danbury, CT, USA). Mitochondrial fractions were obtained by differential centrifugation. The homogenate was first centrifuged for 10 min, at 1000xg at 4°C to discard cell debris. A second centrifugation for 25 min at 20000xg at 4°C was performed to obtain a mitochondrial pellet. The pellet was re-suspended and subjected to a second centrifugation at 20000xg to wash away cytosolic protein contaminants. Mitochondria pellet was re-suspended in commercial BN-PAGE sample buffer (Thermofisher, BN2003) at 0.1g fresh weight sample tissue per 0.25ml buffer. Protein concentrations within samples to be used on Blue Native PAGE were determined using the Bradford Method [26 (link)]. Extracts were stored at -80°C until further analysis.

The samples were lysed in BN PAGE sample buffer (Thermo Fisher Catalog# BN2008) containing 1% digitonin and protease inhibitors following the manufacturer’s recommendation. The lysates were treated with benzonase at room temperature for 30 minutes to shear the DNA, and cleared by centrifugation for 15 mins. Prior to electrophoresis, the samples were mixed with Coomassie G-250 and resolved in 3–12% NativePAGE Bis-Tris gel (Invitrogen Catalog #BN1003BOX) as per the manufacturer’s recommendations. The gels were then transferred on PVDF membranes (Immunobilon-FL, Millipore) following the manufacturer’s recommendations. After transfer, the membranes were incubated in 8% acetic acid for 15 minutes to fix the proteins, rinsed with deionized water, and air-dried. The lane containing the size standard was cut from the membrane and stained with Ponceau S, and imaged on Amersham Typhoon Phosphorimager (GE Healthcare) using the Cy2 filter set. The membranes were blocked, probed with antibodies, and imaged as described above in the denaturing gel electrophoresis protocol.