Anti vegf rabbit monoclonal antibody

The proteins of the placentas were extracted, and the protein concentration was determined with the bicinchoninic acid protein quantification method. Then, 30 μg of total protein was separated by SDS-PAGE and electrotransferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA) using a semidry western blot transfer system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were incubated with primary rabbit anti-MMP-2 monoclonal antibody (1 : 1,000 dilution), rabbit anti-VEGF monoclonal antibody (1 : 1,000 dilution) (Abcam, Cambridge, MA, USA), and anti-GAPDH (loading control, 1 : 3,000 dilution) at 4°C overnight. After washing with PBST three times, the membranes were incubated with secondary HRP-conjugated goat anti-rabbit antibody (1 : 10,000 dilution; Cell Signalling Technology, Inc.) for 2 h at room temperature. Finally, the signals were developed with a chemiluminescent ECL reagent (Millipore), and the membrane was exposed to X-ray film. The strips were analysed by scanning using Image Lab software. The ratio of the protein band density to the reference GAPDH band density was determined as the relative expression level of the target protein.