Enhanced chemiluminescence detection method

Samples were mixed with an equal volume of SDS dissociation buffer (125 mM Tris/HCl, pH 6.8, 2% (w/v) SDS, 20% (v/v) glycerol, 100 mM dithiothreitol, 0.002% (w/v) bromophenol blue) and boiled for 5 min. Proteins from mouse brain homogenate (30 μg) were resolved by SDS-PAGE using 7–17% polyacrylamide gradient gels. Resolved proteins were then transferred to Immobilon P polyvinylidene difluoride membrane (Amersham, Little Chalfont, UK). The membrane was blocked by incubation for 1 h with PBS containing 0.1% (v/v) Tween-20 and 5% (w/v) dried milk powder. Antibody incubations were performed in PBS-Tween containing 2% (w/v) BSA. Antibody 6D11 (Eurogentec Ltd., Liege, Belgium) recognises PrP^C, antibody Y188 (Abcam, Cambridge, UK) was used to detect full length APP and antibody AC15 (Sigma, Dorset, UK) was used to detect actin. Horseradish peroxidase (HRP)-conjugated secondary antibodies were used in the same buffer. Bound antibody was detected using the enhanced chemiluminescence detection method (Amersham Biosciences, Amersham, UK).

Rats were anesthetized and the lumbar segments of spinal cord innervated by the L4-5 dorsal roots were rapidly removed. The collected tissue was mechanically homogenized and centrifuged. The supernatant was collected and stored at –80°C. Protein concentrations of the supernatant were determined using the BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Proteins of interest were separated by SDS-PAGE electrophoresis (20 µg of total protein per well), and transferred onto nitrocellulose membranes. The membranes were placed in a blocking solution (Tris-buffered saline with 0.02% Tween and 5% non-fat dry milk powder) for 1 h, and incubated overnight with guinea pig antiserum against MOR (AB1774, 1∶1000; Chemicon). After washing, the membranes were incubated in peroxidase-conjugated secondary antibody (1∶1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h, and then the membranes were detected by the enhanced chemiluminescence detection method (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). The densities of protein blots were analyzed by using Labworks Software (Ultra-Violet Products Ltd., Cambridge, UK) and normalized to β-actin levels.