Fractionation of nucleoli. Enzymatic and two-dimensional polyacrylamide gel electrophoretic analysis.
Fractionation of nucleoli. Enzymatic and two-dimensional polyacrylamide gel electrophoretic analysis.
复制标题
核仁的分离。
DOI：
10.1021/bi00640a028
复制
发表时间：
1977
期刊：
影响因子：
2.9
通讯作者：
H. Busch
中科院分区：
文献类型：
--
作者：
L. Rothblum;Paula M. Mamrack;H. M. Kunkle;M. Olson;H. Busch
分离核仁的材料与方法。如先前所述，对雄性Holtzman大鼠腹腔内移植的7日龄Novikoff肝癌腹水细胞，通过超声分离核仁（Matsui et al., 1977）。将腹水细胞以1 lOOg离心10分钟，去除红细胞，在Dulbecco盐溶液中洗涤两次。然后将细胞悬浮于2.0 M蔗糖-12 mM MgCl2-10 mM Tris-HCl （pH 7.5）中，在7000g下离心60分钟。将细胞颗粒重新悬于0.88 M蔗糖-12 mMgCl2-10 mM Tris-HCl （pH 7.5）中，以30-s的间隔超声15 s，直到没有细胞核完整，然后以1 lOOg离心20 min。将粗核仁颗粒重悬于0.88 M蔗糖-1 mM MgCl2-10 mM Tris-HCl （pH 7.5）中，并进行共振以分散核仁外染色质。超声在1 lOOg下离心20分钟，核仁颗粒在水中洗涤
Materials and Methods Isolation of Nucleoli. Nucleoli were isolated by sonication of 7-day-old Novikoff hepatoma ascites cells transplanted intraperitoneally in male Holtzman rats as described previously (Matsui et al., 1977). Ascites cells were freed of red blood cells by centrifugation at 1 lOOg for 10 min and washed twice in Dulbecco’s salt solution. These and all subsequent steps were carried outat 4 C. Thecells were then suspended in 2.0 M sucrose-12 mM MgCl2-10 mM Tris-HCl (pH 7.5), and centrifuged at 7000g for 60 min. The cell pellet was resus-pended in 0.88 M sucrose-12 mMgCl2-10 mM Tris-HCl (pH 7.5), sonicated for 15 s at 30-s intervals until no nuclei re-mained intact, and centrifuged at 1 lOOg for 20 min. The crude nucleolar pellet was resuspended in 0.88 M sucrose-1 mM MgCl2-10 mM Tris-HCl (pH 7.5), and resonicated to disperse the extranucleolar chromatin. The sonicate was centrifuged at 1 lOOg for 20 min and the nucleolar pellet was washed in the