Thalamic membranes from 5 male and 5 female A/A or G/G mice were combined. Membrane proteins (2-3 mg) were solubilized in 0.8 ml TTSEC buffer (50 mM Tris-HCl/pH7.4, 2% Triton X-100, 150 mM NaCl, 5 mM EDTA and Roche tablet of protease inhibitors at 1 pill per 10 ml) with 1mM PMSF at 4°C for 3 h. Supernatants were collected after centrifugation at 100,000 g for 30 min and mixed with 50 μl of wheat germ lectin (WGL) Sepharose 6MB at 4°C for 1 h. The beads were washed with cold TTSEC with 0.2% Triton X-100 for three times. The WGL beads-associated proteins were dissociated/eluted in 30 μl 10 × denaturation solution [5% SDS, 0.4M dithiothrietol (DTT)] by incubation for 10 min at 37°C.
