Overexpressed membrane fractions preparation:
1. Remove tissue of interest from animal and flash freeze in liquid nitrogen. Store tissue at -80°C until further use.
2. Suspend frozen tissue in 5 volumes of ice-cold lysis buffer [4 mM HEPES pH 7, 320 mM Sucrose, 5 mM EDTA pH8, protease inhibitor cocktail (Complete, EDTA-free, Roche)].
3. Homogenize the tissue with a polytron homogenizer. Centrifuge homogenates10 min. at 2000 x g, 4°C. Discard large debris.
4. Transfer the supernatant to a clean tube and resuspend the pellet in 2 volumes lysis buffer and re-homogenize.
5. Centrifuge the homogenate 10 min. at 2000 x g, 4°C. Combine supernatant with that of step 4.
6. Centrifuge the supernatant (from steps 4 and 5) 1 hr at 100,000 x g, 4°C.
7. Discard the supernatant. Resuspend the pellet (which contains tissue membranes) in 2 volumes lysis buffer and briefly homogenize with a polytron homogenizer.
8. Measure protein concentration using the Bradford method. Adjust protein concentration to 3 mg/ml with lysis buffer. Store protein samples at -80°C until further use.