生物制药

以下是我们实验室用到的方法，屡试不爽，相关成果发表在Molecular Cell上。
Chromatin Fractionation
1．about 3 × 106 cells were washed with PBS and resuspended in 200 ul of buffer A (10 mM HEPES (pH 7.9), 10mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 1 mM dithiothreitol,0.1% Triton X-100 and protease inhibitor mixture (Roche Molecular Biochemicals).
2. the cells were incubated for 5 min on ice.
3.Nuclei were collected in the pellet (P1)by low speed centrifugation (1500 × g, 4 min, 4 °C).
4.The supernatant(S1) was further clarified by high speed centrifugation (13,000 ×g, 10min, 4 °C) to remove cell debris and insoluble aggregates. The supernatant was designated S2.
5.Nuclei were washed once with buffer A without 0.1% Triton X-100 and then lysed in 200 ul of buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM dithiothreitol, and protease inhibitor mixture).
6.After a 10min incubation on ice, soluble nuclear proteins (S3) were separated from chromatin by centrifugation (2000 ×g, 4 min).
7.Isolated chromatin (P3) was washed once with buffer B and spun down at high speed (13,000 ×g,1 min).
8.remove soup and restore the pellet(P3) in 200ul sample buffer.
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相关疾病：
46XX性发育睾丸疾病

没什么特别的，很多文章用。PMID: 18184650，12499369