Peroxidase conjugated anti mouse igg or anti rabbit igg and enhanced chemiluminescence

Samples were solubilized in lysis buffer [20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 1 mM phenylmethylsulfonyl fluoride, 1μg/ml leupeptin, and 2 μg/ml aprotinin] for 1 h at 4°C. The lysates were then clarified by centrifugation at 15,000 g for 30 min at 4°C, were diluted in Laemmli sample buffer containing 2% SDS and 5% (v/v) 2-mercaptoethanol, and were heated for 5 min at 90°C. The proteins were separated via SDS polyacrylamide gel electrophoresis (PAGE) using 10% or 15% resolving gels followed by transfer to nitrocellulose membranes (Bio-Rad, Hercules, CA) and then probed with antibodies against HIF-1a (Novus), CD31 (Abcam), HGF (Santa cruz), VEGF (Abcam), and FGF2 (Abcam) for 1h at room temperature (Table 2). Peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG and enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) were used as described by the manufacturer for detection. The membranes were scanned to create chemiluminescent images that were then quantified with an image analyzer (Kodak).