Horseradish peroxidase hrp labeled anti rabbit immunoglobulin g igg anti body

The total microsomal protein was extracted from rat livers prepared by diluting liver microsomal suspension with 1 volume of immunoprecipitation assay lysis buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxy-cholic acid, 0.1% SDS, and 50 mM Tris-Cl, pH 7.5) for 15 min and then centrifuged at 13,000 rpm 4 °C for 10 min. Aliquots containing 20 µg of protein were separated by SDS-PAGE on a 10% gradient gel (Invitrogen, USA) and transferred to a PVDF membrane that was blocked with 5% non-fat dry milk in Tris-buffered saline with Tween 20 (200 mM Tris–HCl, 1.37 M NaCl, 0.1% Tween 20, pH 7.6) for 1 h. The membrane was then incubated with rabbit anti-rat CYP1A2 polyclonal antibody (1:2000 dilution), rabbit anti-rat CYP3A4 polyclonal antibody (1:1000 dilution) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:2000 dilution), overnight at 4 °C. After rinsing five times with buffer TBST at room temperature, the membrane was incubated with horseradish peroxidase (HRP)-labeled anti-rabbit immunoglobulin G (IgG) anti-body (Santa Cruz Biotechnology, USA). The immunoreactive bands were visualized using an enhanced chemiluminescence detection system (ChemiDoc™XRS + , Bio-Rad, Hercules, CA, USA) and quantified by using densitometry Image J 1.41 software.

The total protein extract was prepared by diluting 50 μL liver microsome suspension with 1 volume of radioimmunoprecipitation assay (RIPA) lysis buffer (150 mM NaCl, 1% Nonidet™ P-40 (NP-40), 0.5% sodium deoxycholic acid, 0.1% SDS, and 50 mM Tris-Cl, pH 7.5) for 15 min and then centrifuging at 13,000 rpm 4 °C for 10 min. Aliquots containing 20 μg of protein were separated by SDS-PAGE on a 10% gradient gel (Invitrogen, Carlsbad, CA, USA) and transferred to a PVDF membrane which was blocked with 5% non-fat dry milk in Tris-buffered saline with Tween 20 (TBST) (200 mM Tris–HCl, 1.37 M NaCl, 0.1% Tween 20, pH 7.6) for 1 h. The membrane was then incubated with rabbit anti-rat CYP2C19 polyclonal antibody (1:2000 dilution), rabbit anti-rat CYP2E1 polyclonal antibody (1:1000 dilution) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:2000 dilution), overnight at 4 °C. After rinsing five times with TBST, for 5 min each time, at 20 °C, the membrane was incubated with horseradish peroxidase (HRP)-labeled anti-rabbit immunoglobulin G (IgG) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The immunoreactive bands were visualized using an enhanced chemiluminescence detection system (ChemiDoc™ XRS+, Bio-Rad, Hercules, CA, USA) and quantified by densitometry using ImageJ 1.41 (National Institutes of Health, Bethesda, Maryland, USA) software.