如何提取细胞膜(动物细胞)上的蛋白质

1个回答

  • NRC Institute for Biological Sciences

    Triton X-114 extraction protocol (Hydrophobic protein preparation)

    Ressuspend cells 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 eppendorfs 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

    [triton X114]

    Adjust volume to 10 ml with wate