Measuring surface protein expression via biotinylation and western blotting

Twenty-four hours after conclusion of the last drinking session (EtOH n = 9) or control condition (Ctrl n = 7), rats were rapidly decapitated without anesthesia. The NAc was dissected on ice, sliced with a McIllewan tissue chopper and incubated in Sulfo-NHS-SS-Biotin (Pierce) and the reaction quenched by glycine. A portion of the sample lysate was incubated with streptavidin agarose beads. The remainder of the sample was stored as the total protein fraction. Biotinylated proteins attached to streptavidin-coated beads were separated by centrifugation and eluted with Laemli buffer. The amount of target proteins in the total and biotinylated fractions was be analyzed by Western blotting. Briefly, proteins were separated using 10% SDS-PAGE and transferred to PVDF membrane. Membranes were blocked in 5% milk and probed with primary antibody against xCT (Novus 1 : 5000), GLT-1 (EMD Millipore 1 : 1000), GluA1 (EMD Millipore 1 : 2000) and mGlu2/3 (EMD Millipore 1 : 5000). For total protein expression, blots were re-probed with either calnexin (Millipore 1 : 20 000) or beta-tubulin (Abcam, 1 : 50 000) to serve as loading controls. Membranes were incubated with HRP-conjugated secondary antiserum (Jackson ImmunoResearch Lab) at room temperature. Secondary antibody concentrations and species were as follows, xct: anti-rabbit 1 : 50 000; GLT-1: anti-guinea pig 1 : 50 000; GluA1: anti-mouse 1 : 50 000; mGlu2/3: anti-rabbit 1 : 50 000, calnexin: anti-rabbit 1 : 50 000; beta-tubulin: anti-rabbit 1 : 50 000. Bands were visualized using ECL Plus (GE Healthcare). Band density was quantified with NIH IMAGE J software.