Trans blot turbo 0.2 m nitrocellulose membranes

Lysate protein content was determined using the Bio-Rad DC Protein Assay kit (Bio-Rad Laboratories, #500-0113 (Working reagent A); #500-0114 (Working reagent B); #500-0115 (Working reagent S)). Sample protein contents were equalized with ddH₂O, and NuPAGE LDS 4x Sample Buffer (Invitrogen, #NP0007 + 0.5 M Dithiothreitol (DTT) was added. Proteins were separated by SDS-PAGE using Criterion TGX Precast Gels, Tris/Glycine/SDS running buffer (Bio-Rad, #161 0732) and Benchmark ladder (Invitrogen, #10747-012) and transferred to Trans-blot Turbo 0.2 µm nitrocellulose membranes (Bio-Rad, #170-4159) using the Trans-blot Turbo Transfer System (Bio-Rad, #10022518). After staining with Ponceau S (Sigma-Aldrich, #P7170-1L), membranes were blocked (blocking buffer: 5% nonfat dry milk in TBST (0.01 M Tris/HCl, 0.15 M NaCl, 0.1% Tween 20)) for 1 h at 37 °C. Subsequently, they were incubated overnight at 4 °C with primary antibodies, washed in TBST, and incubated with HRP-conjugated secondary antibodies for 1 h, RT. Membranes were washed in TBST and developed using Pierce ECL Western blotting substrate (Thermo Scientific, #32106), on a Fusion FX developer (Vilber Lourmat). Band intensity was quantified using ImageJ software.