Western blotting luminol reagent

To lyse the cells, the harvested cells were washed with cold 1 × PBS, and then resuspended for sonication in RIPA buffer (iNtRON Biotechnology, Korea). Equal amounts of protein samples were resolved by 6−15% sodium dodecyl sulfate polyacrylamide gel electrophoresis after determining the protein concentration of the cell lysates using a Bio-Rad DC Protein Assay kit (USA), The resolved proteins were transferred to poly vinylidene fluoride membranes (EMD Millipore). Three percent or 5% skim milk in 1 × PBST (PBS with 0.1% Tween 20) was used to block the membranes by incubation for 2 h at RT. The respective primary antibodies were used to probe the membranes. The diluted primary antibodies (1:1,000 dilution in 1 × PBST) were incubated with the membranes either 2 h at RT or overnight at 4°C. Next, the membranes were washed with PBST, then probed with the secondary antibodies conjugated with horseradish peroxidase (1:4,000 – 6,000 dilution) by incubation for 1 h at RT. Visualization of the Western blots was done using Santa Cruz Western blotting luminol reagent (USA), and ImageQuant LAS 500 from GE Healthcare (Sweden) was used to detect the images.

Total cell lysate was prepared by lysing the cells in RIPA lysis buffer (150 mM NaCl, 0.1% SDS, 0.5% Sodium Deoxycholate, 1% NP-40, and 50 mM Tris-Cl, pH 7.5) supplemented with the Protease Inhibitors Cocktail Set III and 1 mM PMSF at a density of 1 x 10⁷ cells/ml buffer. The cell suspension was agitated for 30 min and then centrifuged at 12,000 rpm for 20 min at 4°C. Then the supernatant was transferred to a new tube and stored at -20°C. The protein concentration was measured using DC protein assay kit (Bio-Rad, CA, USA). Next, 50 μg of protein from the cell lysates were separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto nitrocellulose membranes (Bio-Rad, CA, USA). The membrane was blocked with 5% non-fat milk (Nestle, Switzerland) in PBS with 0.05% tween-20. This was followed by incubation overnight with a diluted solution of primary antibody. This was followed by incubation at room temperature with HRP-conjugated antibody (anti-mouse IgG or anti-rabbit IgG) (Cell Signaling Technology Beverly, MA) for 1 h. At least two independent experiments were performed. The blots were visualized by enhanced chemiluminescence (ECL) using the Western Blotting Luminol Reagent (GE Healthcare, UK). Images developed were captured with an LAS-4000 gel documentation system (Fuji Film, Tokyo, Japan).