Hybond c extra nitrocellulose membrane

Cell pellets were lysed in 50 mM Tris pH 7.9/8 M Urea/1%Chaps and incubated at 4 °C with agitation for at least 30 min. The lysate was cleared by centrifugation and the supernatant was collected. The protein concentration of the extracts was measured by Bradford assay. About 25 μg of supernatants were resolved by SDS-polyacrylamide gel electrophoresis and transferred onto a Hybond-C Extra Nitrocellulose membrane (Fisher Scientific UK, Loughborough, UK). The membrane was blocked in 5% milk, 0.1% Tween-20 in TBS buffer for 1 h and probed overnight with antibodies against BRG1 (Santa Cruz, sc-17796, dilution 1:2000), a-tubulin (Abcam Ab7291, dilution 1:5000), p21 (Cell Signalling, 2947 S, dilution 1:2000), INO80 (Bethyl, A303-371A, dilution 1:2000) in 5% milk, 0.1% Tween-20 in TBS buffer. The membrane was washed three times with 0.1% Tween-20 in TBS and incubated for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibodies in 5% milk, 0.1% Tween-20 in TBS. The secondary antibodies used were Rabbit anti-Mouse HRP (Dako, P0260, dilution 1:5000) and Goat anti-Rabbit HRP (Dako, P0448, dilution 1:5000). Proteins were visualised by using in-house ECL reagent or SuperSignal West Pico Chemiluminescent Substrate (Life Technologies).