Mouse monoclonal anti hif 1α

MCF-7 cells were homogenized in a glass tissue grinder in 20 mM HEPES, pH 7.5, containing 5 mM dithiothreitol, 2 mM EDTA, 0.1% CHAPS detergent, 0.1% Triton X-100, and protease inhibitors; then, they were centrifuged at 15,000 g for 15 min. The supernatant was diluted in loading buffer (2% SDS, 5% glycerol, 0.002% bromophenol blue, 4% β-mercaptoethanol in 0.25 M Tris-HCl, pH 6.8) and denatured by boiling for 3 min. Aliquots corresponding to 80 μg protein were analysed by SDS-PAGE (7.5% gel). Proteins were transferred onto a nitrocellulose membrane for 1 h. This membrane was then saturated with 5% dry milk for 1 h, washed with Tris-buffered saline, and probed overnight at 4°C with the specific primary antibodies (1:500 mouse monoclonal anti-HIF-1α and 1:1,000 mouse monoclonal anti-β-actin; Santa Cruz Biotechnology, Heidelberg, Germany). The membrane was then incubated for 1 h with the secondary antibody (1:2,500 horseradish peroxidase-conjugated species-specific anti-IgG; Santa Cruz Biotechnology).