搜搜做题作业网如何提取细胞膜（动物细胞）上的蛋白质
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如何提取细胞膜（动物细胞）上的蛋白质
求提取并纯化动物细胞膜蛋白而避免膜上的脂类等物质的污染的最佳方法.可以以肝细胞为例.
求提取并纯化动物细胞膜蛋白而避免膜上的脂类等物质的污染的最佳方法.可以以肝细胞为例.
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
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