Nativepage novex 3 12 bis tris protein gels

Purified gH/gL monomer or dimer protein (previously described in [28 ]) were incubated with 3G16, MSL-109 or 13H11 Fabs in various combinations at a ratio of 10 μg gH/gL to 5 μg each Fab for 1 h at room temperature (RT) before being resolved on NativePAGE Novex 3–12% Bis-Tris Protein Gels (Invitrogen Inc.) along with NativeMark Unstained Protein Standard (Invitrogen Inc.). The gels were subsequently stained with Coomassie Blue to visualize the bands.

The BN-PAGE procedure for detecting the exosome complex has been adapted from Fasken et al.³⁰ (link) Fibroblast pellets were re-suspended in cold BN-PAGE lysis buffer (10 mM Tris-HCl [pH 8]; 150 mM NaCl; 0.1% NP40, supplemented with protease inhibitors) and lysed on ice for 30 min. Lysates were centrifuged at 17,000 × g for 10 min, and 50–100 μg total protein of the supernatant was loaded on pre-cast BN-PAGE gels. NativePAGE Novex 3–12% Bis-Tris Protein Gels, NativePAGE Running Buffer, Cathode Buffer Additive, 4× Sample Buffer, and 5% G-250 Sample Additive (all ThermoFisher Scientific) were utilized for electrophoresis. Transfer and antibody detection were performed as described above.

Total protein extracts from cultured cells or fractions were resolved by SDS–PAGE using Novex NuPAGE 4–12% Bis‐Tris Precast Gels (Thermo Fisher Scientific).
Samples for blue‐native gel electrophoresis (BNGE) were prepared either from digitonized cellular extracts (Nijtmans et al, 2002) or from isolated mitochondria (Fernández‐Vizarra et al, 2010). For the solubilizations, either 1.6 mg DDM/mg protein or 4 mg digitonin/mg protein was used (Wittig et al, 2006; Acin‐Perez et al, 2008). Approximately 50 μg of protein was loaded into Native PAGE Novex 3–12% Bis‐Tris Protein Gels (Thermo Fisher Scientific) and electrophoresed in the conditions indicated by the manufacturer.
Proteins were electroblotted to PVDF membranes and immunodetected using commercial antibodies (Table 1). Immunoreactive bands were visualized using ECL Western Blotting Detection Reagents (GE Healthcare) and X‐Ray films (Fuji) or using a digital Amersham Imager 680 (GE Healthcare). Signal intensities were quantified by densitometry using ImageJ.

MCF7 and MDA-MB231 cells were seeded at density 3 × 10⁶/100 mm dish. After overnight incubation cells were treated as required. Mitochondria from MCF-7 and MDA-MB231 cells were isolated in Tris-Sucrose buffer as described above and 50 μg pellets were solubilized as per manufacturer’s protocol (Thermo Fisher Scientific) and BN-PAGE was performed on Native PAGE Novex3%–12% Bis-Tris Protein Gels (ThermoFisher Scientific). In-gel enzyme activity of different OXPHOS complexes was analyzed on gradient Bis-Tris gel.
Substrate: for complex I, 1 mg NADH and 25 mg NTB was  prepared in 2 mM Tris-HCl (pH 7.4), and for complex IV, 5 mg DAB and 10 mg cytochrome C in 50 mM potassium phosphate buffer (pH 7.4) was used for in-gel activity. For complex III and complex IV combined, 10 mg 3,3′ diaminobenzidine tetrachloride (DAB) and 25 mg cytochrome C in 25 ml of 50 mM sodium phosphate buffer (pH 7.2) was used.

Samples were prepared in the native lysis buffer (50 mM HEPES (pH 7.4), 50 mM NaCl, 2 mM MgCl₂, 0.5% NP40, Benzonase 1 U/μl, proteinase inhibitor cocktail). NativePAGE 5% G-250 Sample Additive (ThermoFisher, BN2004) was added to the samples at a final concentration of 0.125%. Samples were separated on NativePAGE Novex 3–12% Bis–Tris Protein Gels (ThermoFisher, BN1001BOX) for 2 h at 150 V. Dark blue cathode buffer was replaced with light blue cathode buffer during the run. Proteins transferred to PVDF membrane were fixed with 8% acetic acid for 15 min and processed for western blotting.

The extracted and solubilized mitochondria were separated by electrophoresis in native gels (NativePAGE Novex 3–12% Bis-Tris Protein Gels, Thermo Scientific) in order to evaluate the arrangement of the complexes in the electron transport chain. The mitochondrial samples were loaded in 10% (v/v) loading buffer (5% (v/v) Serva-Blue G; 1 M 6-aminohexanoic acid). The electrophoresis was carried out at 100 V for 1 h in the presence of blue cathode buffer (50 mM tricine; 15 mM Bis-Tris/HCl; 0.02% (w/v) Coomassie Brilliant Blue G, pH 7) in the cathode chamber, and anode buffer (50 mM Bis-Tris, pH 7) in the anode chamber, followed by buffer replacement (removal of Coomassie blue dye) in the cathode chamber and electrophoresis at 40 V overnight at 4 °C. The gel containing the separated proteins was then incubated at 4 °C in carbonated transfer buffer (10 mM NaHCO₃; 3 mM Na₂CO₃·10 H₂O, pH 9.5–10) for 20 min with agitation to ensure that the gel was fully saturated with said transfer buffer. The transfer was then carried out onto polyvinylidene difluoride membranes (Thermo Scientific) (previously activated with methanol) at 60 V for 90 min at 4 °C in the Mini-Transblot system (Bio-Rad, USA). The membranes were then subjected to normal western blot protocol by using anti-NDUFS1 primary antibody (1:500) (Sc-50132, Santa Cruz) and anti-goat (1:10000) (ab6741, Abcam) as a secondary antibody.