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.