细胞膜蛋白质提取方法

 NRC Institute for Biological science s Triton X-114 extraction protocol (Hydrophobic protein preparation) Ressuspend Cell s in Solution A (dil 1/8) and add 15 µl of mammalian cocktail proteases inhibitor (Sigma). Add 1 third of the volume of solution B Incubate on ice for 1 hour with frequent vortexing. Centrifuge at 10 000g for 10 minutes at 4°C to pellet unbroken cells and nuclei. Transfer supernatant in clean eppendorf s then incubate at 30°C for 3 minutes (until sln is cloudy). Centrifuge at 1 300g for 10 minutes at room temperature. Transfer Aqueous phase in new eppendorf (but keep detergent phase at room temperature). Add X volume of triton X-114 to Aqueous phase: Vol of Aq phase = X vol of triton X-114 24.6 Shake well then incubate 3 minutes at 30°C (until cloudy) then transfer on top of detergent phase slowly. Centrifuge at 1 300g for 10 minutes at room temperature. Remove Aqueous phase with pipette down to detergent interphase. Precipitate hydrophobic protein (detergent phase) with 10X volume of acetone. Place overnight at -20°C. Pellet proteins by centrifugation at max speed for 10 minutes at 4°C. Ressuspend proteins in IEF sln (7M urea, 2M thiourea, 4% CHAPS, 1% DTT) then precipitate protein again with 10 volume of acetone. Place 5-10 min at -20°C. Pellet proteins by centrifugation at max speed for 10 minutes at 4°C. Air dry pellet then dissolve protein in IEF solution Solution: Solution A: 80 mM Tris-HCl, pH 7.4 ; 1,2M NaCl 0,63 g Tris-HCl 3,51 g NaCl Dissolve in 30 ml of water Adjust pH to 7.4 Adjust volume to 50 ml with water Solution B: 40 mM Tris-HCl, pH 7.4 ; 600 mM NaCl ; 4% triton X-114 5 ml of sln A Add triton to have a final concentration off 4% 4% x 10 ml = ml