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        细胞膜蛋白质提取方法

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        5235
        相关专题
         
        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
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