2.10 Western blot analysis
We used Western blot analysis to quantify MOP protein expression in lumbar DRG and spinal cord, small intestine, and periaqueductal gray. Samples of each tissue were separated and homogenized for immunoblotting. The tissues were lysed in ice-cold radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, 0.5 mg∙ml-1BSA). After samples were centrifuged at 13,000g (or 12,000 rpm) for 15 min, the protein concentration was determined by using a detergent-compatible protein assay (Pierce™ BCA Protein Assay Kit) with a BSA standard (Thermo Fisher Scientific, Waltham, MA). Samples were separated on a 7.5% (w/v) sodium dodecyl sulfate-polyacrylamide gel by electrophoresis and transferred onto a nitrocellulose membrane (Amersham, Pittsburgh, PA) with a Trans-Blot Transfer Cell system (Bio-Rad, Hercules, CA). Membranes were incubated with the indicated primary antibody overnight at 4℃, and immunoreactivity was detected by enhanced chemiluminescence (ECL, Amersham). Antibodies against MOP (1:2000, AB5511, EMP Millipore) and GAPDH (1:50,000, ABS16, EMD Millipore) were used. Western blots were imaged with the ImageQuant LAS 4000 (GE Healthcare Life Sciences) and analyzed with ImageJ 1.46a software. GAPDH staining was used as an internal control for protein loading.