推荐产品
公司新闻/正文
古朵生物讲述:细胞膜蛋白质提取方法
人阅读 发布时间：2018-08-20 16:16
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 water
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 water
询价列表
暂时没有已询价产品
快捷询价 发送名片
当你希望让更多商家联系你时，可以勾选后发送询价，平台会将你的询价消息推荐给更多商家。