Anti goat igg antibodies

Western blot analysis was performed in cytosolic and nuclear extracts prepared from liver homogenates. The supernatant fraction was collected and stored at -80°C in aliquots until use. Protein concentration was measured by the Bradford assay [20 (link)]. Lysate proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TTBS) for 1 h at room temperature and probed overnight at 4°C with polyclonal anti-NQO1, anti-Keap1, anti-Nrf2, anti-ATF6, and anti BIP/GRP78 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1 : 200-1 : 1.000 dilution with TTBS in 5% nonfat dry milk. Bound primary antibody was detected (HRP—with anti-mouse IgG, anti-rabbit IgG, or anti-goat IgG antibodies) (Sigma-Aldrich, St. Louis, MO, USA). Protein detection was performed via chemiluminescence using a commercial ECL kit (Amersham Pharmacia Biotech, Little Chalfont, Great Britain) [23 (link)]. The density of the specific bands was quantified with imaging densitometer software (Scion Image, Maryland, MA).

MC3T3-E1 cells were seeded at a density of 8 × 10⁴ cells/well on SPL cell-culture slides and cultured in α-MEM-containing 10% FBS. After incubation for 2 d, the growth medium was changed to 2% FBS; each concentration of IF-I (100 μg/mL) or LF-II (10 μg/mL, 30 μg/mL, and 100 μg/mL), 50 μg/mL ascorbic acid, and 10 mM β-GP-containing medium was differentiated. Cells were cultured for 2 weeks, then stained with anti-OPN primary antibody (R&D Systems, Inc., Minneapolis, MN, USA) and anti-goat IgG antibodies (Sigma-Aldrich Co., LLC., St. Louis, MO, USA). Fluorescence microscopy was used to observe OPN expression.