Goat anti mouse horseradish peroxidase conjugates

Antibodies against cyclinD1, matrixmellatoproteinase-9 (MMP-9), PARP, cellular inhibitor of apoptosis 2 (c-IAP2), Bcl-2, Bcl-xl, c-Myc, β-actin and caspase-3 were obtained from Santa Cruz Biotechnology (USA). Survivin antibody and antibody against cellular FLICE-inhibitory protein (c-FLIP) were purchased from R & D Systems and Imgenex (San Diego, CA, USA), respectively. Secondary antibodies i.e., goat anti-rabbit and goat anti-mouse horseradish peroxidase conjugates were purchased from Bio-Rad (Hercules, CA, USA). Penicillin, streptomycin, DMEM, RPMI-1640, Iscove's DMEM and fetal bovine serum were obtained from Invitrogen (USA).

A supported nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) was first soaked in distilled water, followed by PBS, for 10 s. Samples were applied to dried membrane in a volume of 2 µL. After complete drying of the membrane, it was incubated in a PBST (PBS with 0.1% Tween-20) blocking solution containing 5% Blotting Grade Blocker Non-Fat Dry Milk (Bio-Rad, Hercules, CA, USA) for 1 h, followed by a PBST wash for 5 min. The membrane was then incubated in primary antibody solution (diluted in blocking solution to 1 µg/mL) for 1 h. The membrane was washed in PBST (15 min twice) and then incubated with labelled secondary antibodies (diluted in blocking solution to 1 µg/mL) for 1 h. The secondary antibodies were goat anti-mouse horseradish peroxidase conjugates (Bio-Rad, Hercules, CA, USA). The membrane was washed with PBST (10 min twice). Results were visualized using the Clarity Western ECL Substrate kit (Bio-Rad, Hercules, CA, USA) and the ChemiDoc XRS+ imaging station.