Quantifying Hepatic and Intestinal Proteins

Hepatic and intestinal microsomes (20–40 μg of protein per lane) were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane for 1 h. For immunodetection, the blots were incubated on a rotary shaker overnight at 4 °C with primary antibodies against CYP1A1/2, CYP2B1/2, CYP2C11, CYP2D1, CYP3A1/2, and P-gp diluted in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) and 5% bovine serum albumin (1:2000). The blots were washed and incubated for 1 h at room temperature with a horseradish peroxide-conjugated secondary antibody diluted 1:10,000 in TBS-T containing 5% skim milk. Protein expression was visualized by enhanced chemiluminescence (Bio-Rad) using an Image Quant LAS 4000 Mini (GE Healthcare Life Sciences, Piscataway, NJ, USA) and the band density was measured using ImageJ 1.45 s software (NIH, Bethesda, MA, USA). β-Actin was used as a loading control [26 (link)].