Novex tris glycine 4 20 gels

Heme transfer was assessed by mixing 10 μg of WT or mouse PGRMC2 heme-binding mutant (3xM) with 10 μg of apo-REV-ERBα protein and incubating for 30 min at 37°C. After incubation, 2X Native Tris-Glycine sample buffer (Life Technologies) was added and samples separated by electrophoresis using Novex Tris-Glycine 4-20% gels and Tris-Glycine Native Running Buffer (Life Technologies) for 6 hr. The gel was washed for 10 min with water and heme staining was performed using the BioFX TMB One Component HRP Microwell Substrate (Surmodics). After imaging the heme stain, the gel was washed overnight with water and counterstained with Coomassie for protein detection.

The proteins were loaded onto Novex Tris-Glycine 4%–20% gels (Life Technologies) and then transferred overnight onto nitrocellulose paper (Bio-Rad Laboratories). After blocking with Li-COR Odyssey Blocking Buffer (Li-COR Biosciences, Lincoln, NE), diluted in Tris buffered saline, primary β-actin rabbit monoclonal antibody from Cell Signaling Technology (Beverly, MA) or mouse β-actin primary polyclonal antibody (Cell Signaling 4970S), and secondary goat anti-rabbit IgG (Li-COR 926-68071) and secondary goat anti-mouse antibodies (Li-COR 926-32210) were added to detect β-actin and the protein of interest. Antibodies from Abcam (Cambridge, UK) were: anti-SDHB (SDH) (ab14714), mitoProfile pyruvate dehydrogenase WB Antibody Cocktail (PDHC), anti-pyruvate dehydrogenase (PDH) cocktail [Abcam (ab110416) for the following proteins: 69 kDa E2, 54 kDa E3, 43.3 kDa E1α, 39.4 kDaE1β], anti-aconitase 2 (ab71440), anti-MDH2 (MDH) and anti-succinate dehydrogenase (ab14714) (28 kDa)]. The membranes were scanned using the LiCOR Odyssey instrument.

2–5 ×10⁶ cells were harvested, washed in cold PBS and lysed for 10min on ice in RIPA lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1x protease and phosphatase inhibitor (Cell Signaling), and 250 units/ml benzonase nuclease (Sigma). Lysates were further clarified by centrifugation for 5min at 16000 × g at 4C. Protein concentration was measured using Pierce 660nm Protein Assay (Thermo Fisher Scientific). 35ug was loaded per well. Electrophoresis was carried out on Novex Tris-Glycine 4–20% gels (Life Technologies) before transfer on a Nitrocellulose Membrane, 0.45 μm (BioRad). Membranes were blocked for 30mins with SEA BLOCK Blocking Buffer (Thermo Fisher Scientific) at RT. Membranes were then incubated with primary antibody, diluted in 3%BSA, for 1h at RT or overnight at 4C. Membranes were then washed at RT 3 times in TBST for 5 mins. The membrane was incubated with goat α-rabbit or α-mouse conjugated to IRDye800 or to IRDye680 (LI-COR Biosciences), diluted in 5% milk, for 1h at RT. Membranes were washed 3 times in TBST for 5mins and were scanned for infrared signal using the Odyssey Imaging System (LI-COR Biosciences). Band intensities were analyzed with Image Studio Lite (LI-COR Biosciences).