Biomax l film

The Oxyblot Oxidized Protein Detection Kit was purchased from Chemicon (S7150). The procedure was according to our recent study [11 (link), 42 (link), 45 (link)]. DNPH derivatization was carried out on 6 μg of protein for 15 minutes according to manufacturer's instructions. One-dimensional electrophoresis was carried out on 12% SDS/polyacrylamide gel after DNPH derivatization. Proteins were transferred to nitrocellulose membranes, which were then incubated in the primary antibody solution (anti-DNP 1:150) for 2 hours, followed by incubation with secondary antibody solution (1:300) for one hour at room temperature. The washing procedure was repeated eight times within 40 minutes. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL), which was then exposed to Biomax L film (Fuji). For quantification, ECL signals were digitized using Labwork software (UVP). On each gel, a standard control sample was loaded.

The procedure and protocol of western blot were according to our recent reports [7 (link), 8 (link)]. Briefly, equal amounts (10–30 μg) of protein extracts from ischemic quadriceps of the animals (n = 6 for each group) were loaded and separated by SDS-PAGE using 7% or 12% acrylamide gradients. The membranes were incubated with monoclonal antibodies against CD31 (1 : 1000, Abcam), CXCR4 (1 : 1000, Abcam), vascular endothelial growth factor (VEGF) (1 : 1000, Abcam), and stromal cell-derived growth factor- (SDF-) 1α (1 : 1000, Cell Signaling). Signals were detected with HRP conjugated goat anti-rabbit IgG. Proteins were transferred to nitrocellulose membranes which were then incubated in the primary antibody solution (anti-DNP 1 : 150) for two hours, followed by incubation with secondary antibody solution (1 : 300) for one hour at room temperature. The washing procedure was repeated eight times within 40 minutes. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences) which was then exposed to Biomax L film (Fuji). For quantification, ECL signals were digitized using Labwork software (UVP).