Optimized Immunoblot Protein Quantification

Extracted proteins for immune-based Western blotting were first separated, according to molecular weight, using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels, followed by electrotransfer to nitrocellulose membranes (Amersham Hybond-ECL, GE Healthcare, Chicago, IL, USA), as described before [26 (link)]. Equal amounts of protein and volume were loaded onto a 12.5% polyacrylamide gel for CYGB, heme oxygenase 1 (HO-1) and NF E2-related factor 2 (NRF2). Membranes were blocked in TBS-T (Tris-buffered saline; 0.1% Tween-20), containing 5% non-fat dry milk, for 1 h at room temperature. After blocking, membranes were incubated overnight at 4 °C with primary antibodies (anti-CYGB, Proteintech, Rosemont, IL, USA; 13317-1-AP; anti-β-2-Microglobulin (B2M), Proteintech, 13511-1-AP; anti-β-Actin (ACTB), Santa Cruz, sc-47778; anti-HO-1, Proteintech, 10701-1-AP; anti-NRF2, Proteintech, 16396-1-AP). The following day, membranes were washed with TBST-T and incubated for 1 h with horseradish-conjugated secondary antibodies (anti-rabbit IgG HRP, Sigma, GENA934-1ML; anti-mouse IgG HRP, Invitrogen, 31430). The signal was revealed using ECL Prime (Amersham, GERPN2232) on an Amersham Imager 680 (GE Life Sciences; Piscataway, NJ, USA) and exported and quantified using the Image Studio™ program (LI-COR Biosciences, Lincoln, NE, USA).