Goat anti rabbit igg hrp

Proteins were extracted from cells after three passages with PRO-PREP reagent (Intron Biotechnology, Gyeonggi, Korea), incubated on ice, and centrifuged at 13,000 rpm at 4 °C for 5 min. The supernatant was then transferred to fresh tubes on ice. The protein concentration was analyzed with a Protein Assay Kit (Bio-Rad, Hercules, CA, USA).
The proteins in samples containing 30 mg of protein were separated sequentially on 6% and 2% SDS/PAGE gels and transferred to a polyvinylidene fluoride membrane (Immun-Blot® PVDF Membrane, Bio-Rad, Hercules, CA, USA). Blotted membranes were blocked for 60 min with 3% BSA/5% skim milk and washed. The membrane was incubated overnight at 4 °C with each primary antibodies and 70 min at room temperature with each secondary antibodies.
We employed α-smooth muscle actin as a marker for smooth muscle cells, von Willebrand factor and CD34 as markers for endothelial cells, and β-actin as a housekeeping protein. The following primary antibodies were acquired from Abcam: β-actin (mouse, 1:1,000, #8224), anti-CD34 antibody (rabbit, 1:1,000, #81289), anti-α-smooth muscle actin antibody (rabbit, 1:1,000, #5694), and anti-von Willebrand Factor antibody (rabbit, 1:500, #6994). The secondary antibodies were acquired from GEN Depot and were: goat anti-Mouse IgG (H+L)-HRP (1:10,000, #SA001-500) and goat anti-rabbit IgG-HRP (1:10,000, #SA002-500).