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发帖数: 263 | 1 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 |
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