Horseradish peroxidase hrp linked goat anti rabbit igg

Total protein was isolated from the osteogenic induction group and non-treated group with RIPA buffer and quantified with a BCA protein assay kit (Beyotime, Beijing, China). Equal quantities of samples were separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a hydrophi-lic polyvinylidene fluoride (PVDF) membrane (0.45 μm, Merck Millipore, Boston, MA, USA) with the Bio-Rad protein assay system (Bio-Rad, Hercules, CA, USA). Primary Antibodies against Runx2 (1：1,000, Abcam, Cambridge, UK), Alp (1：1,000, Abcam, Cambridge, UK), and β-ac-tin (1：2,000, Santa Cruz Biotechnology, CA, USA) were incubated with the membrane at 4℃ overnight. The secondary antibodies, horseradish peroxidase (HRP)-linked goat anti-rabbit IgG or goat anti-mouse IgG (Santa Cruz Biotechnology, CA, USA), were incubated with the membrane at room temperature for 1 h. The membrane was immersed with electrochemiluminescence (ECL) response solution (Pierce, Waltham, MA, USA) at room temperature for 1 min. All bands were analyzed with Image Lab Software (version 5.1; Bio-Rad Laboratories, CA, USA), and relative levels against the internal control β-actin were calculated.