发信人: laomeng (LaoMeng), 信区: Biology
标 题: Re: sonication vs. freeze-and-thaw
发信站: Unknown Space - 未名空间 (Wed Aug 27 16:04:37 2003), 站内信件

use needle pass-through bah
Use low ionic strength buffer (Tris 20mM pH 7.7, protease inhibitor mix, MeSH)
(i.e. Buffer) and 27.5G needle (i.e. Needle)
1. wash cell with PBS, spin down cell, remove all PBS.
2. resuspend cell in Buffer, pipete 10 times, sit on ice for 15 min.
3. use shringe and Needle, pass the suspended cell through Needle 7 times
4. site on ice for another 10 min.
5. pass through Needle another 7 times
6. spin at 500 g to remove debris, 100k g for membrane fraction

【 在 sunnyday (飞鸟) 的大作中提到: 】
: 哪位贴一个freezing-thawing cycle的protocol?
: 我们的sonicator坏了...
: 555555
: 【 在 windysea (神仙眷侣~像一个小孩只懂在你怀里坏) 的大作中提到: 】
: : to make whole cell extract,
: : which one is better in breaking plasma membrane?
: : which one doesn't destroy protein complex(or minimally)?


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