Secondary anti rabbit igg labelled with horseradish peroxidase

Whole-cell protein extracts were prepared on ice using a lysis buffer (Cell Signalling) supplemented with Complete EDTA-free Protease Inhibitor Cocktail (Roche). Then, after 10 min on ice and centrifugation at 4 °C (14,000 rpm) for 10 min, the supernatant was collected. Protein concentration was measured using Bradford reagent (Bio-RAD) according to the manufacturer's instructions. Proteins were denatured at 100 °C for 5 min with standard SDS-PAGE Loading Buffer (200 mM Tris-Cl, pH 6.8), 400 mM DTT, 8% SDS, 0.4% bromophenol blue, 40% glycerol). Proteins were separated on a 10% SDS polyacrylamide gel using electrophoresis and transferred to PVDF membranes (GE Healthcare). Membranes were blocked with 0.1% TBST (Tris-Buffered Saline, Tween20) containing 5% non-fat milk for 1 h at room temperature, incubated with primary antibodies against ubiquitin (Cell Signalling Technology, cat. no. 39335) and beta-tubulin (Cell Signalling Technology, cat. no. 21465) and then with a secondary anti-rabbit IgG labelled with horseradish peroxidase (Cell Signalling Technology, cat. no. 70745). Finally, an electrochemiluminescence (ECL ™ Western Blotting Reagents, GE Healthcare) system was used to detect proteins.