S******e
发帖数: 393 | 1 将S1 (cytosolic protein) 和 S3 (nuclear soluble protein?) 去掉后，剩下的
pellet再用0.1%的Triton extract, 收集到的上清暂称为X，想知道X是些什么蛋白？
Chromatin associated protein?
方法如下：
1. Cells were extracted with Buffer A (10mM Hepes pH7.9, 10mM KCl, 1.5mM
MgCl2, 0.34mM Sucrose, 10% Glycerol, 1mM DTT plus protease inhibitors)
containing 0.1% Triton for 5 min on ice, centrifuge 1300g X5 min, discard
supernatant (S1), wash once with BufferA and discard supernatant.
2. Extract cell pellet with Buffer B (3mM EDTA, 0.2mM EGTA, 1mM DTT plus
protease inhibitors) for 30 min on ice, centrifuge 1700g x 5 min, discard
supernatant (S3).
3. Wash once with Buffer B, centrifuge and discard supernatant, the pellet
was extracted again with BufferA containing 0.1% Triton for 30min,
centrifuge 13000rpm x 20 min, collect supernatant (X).
Reference(我把最后的P fraction用0.1%的triton extract):
http://www.lamondlab.com/pdf/chromatinisolation.pdf
谢谢。 | j******n
发帖数: 941 | |
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