Rapid electrophoretic analysis for histone phosphorylation. A reinvestigation of phosphorylation of lysine-rich histone during rat liver regeneration.
Rapid electrophoretic analysis for histone phosphorylation. A reinvestigation of phosphorylation of lysine-rich histone during rat liver regeneration.
复制标题
组蛋白磷酸化的快速电泳分析。
DOI：
10.1021/bi00797a024
复制
发表时间：
1971
期刊：
影响因子：
2.9
通讯作者：
R. Chalkley
中科院分区：
文献类型：
--
作者：
R. Balhorn;W. O. Rieke;R. Chalkley
材料与方法肝部分切除术。用Higgins和安德森（1931）的方法切除了体重100-200 g的Holtzman大鼠的部分肝脏，切除了大约三分之二的肝脏。进行所有肝切除术，使得再生肝的移除发生在上午11：00和下午1：00之间，防止实验之间有丝分裂活性的可能的昼夜变化。大鼠自由进食，并在肝再生的适当间隔后取出剩余的肝脏。组蛋白的分离。用Panyim和Chalkley（1969）的方法从纯化的细胞核中分离出完整的组蛋白。为了获得足够干净的核，有必要将Triton X-100的浓度增加到1%。使用Johns（1964）的高氯酸方法从染色质或整个组蛋白中选择性地提取富含赖氨酸（Fi）的组蛋白。富含赖氨酸的组蛋白在用95%乙醇透析沉淀之前，先用0.4N硫酸在4 ℃下充分透析。电泳将组蛋白溶于15%蔗糖-0.9n乙酸中，在25 ℃下电泳24小时，在4 ℃下电泳40小时，电泳在25-cm的预处理凝胶（15%丙烯酰胺）上进行，凝胶中含有2.5m尿素和0.9n乙酸（pH2.8）。在200 V下电泳后，通过破坏管除去凝胶。它们被阿米多染色了-
Materials and MethodsPartial Hepatectomy. Holtzman rats weighing 100-200 g were partially hepatectomizedby the method of Higgins and Anderson (1931), removing approximately two-thirds of the liver. All hepatectomies were performed such that the removal of the regenerated liver occurred between 11: 00 am and 1: 00 pm, preventing possible diurnal variations in mitotic activity between experiments. The rats were fed ad libitum and the remaining liver removed following the appropriate interval of liver regeneration. Isolation of Histone. Whole histone was isolated from puri-fied nuclei by the method of Panyim and Chalkley (1969). It was necessary to increase the concentration of Triton X-100 to 1% in order to obtain sufficiently clean nuclei. Lysine-rich (Fi) histone was selectively extracted either from chromatin or from whole histone using the perchloric acid method of Johns (1964). The lysine-rich histone was then dialyzed extensively against 0.4 n sulfuric acidat 4 prior to precipitation by dialysisagainst 95% ethanol. Electrophoresis. Electrophoresisof histone dissolved in 15% sucrose-0.9 n acetic acid was performed for 24 hr at 25 and for 40 hr at 4 on 25-cm preelectrophoresed gels (15% acrylamide) containing 2.5 m urea in 0.9 n acetic acid (pH 2.8). After electrophoresis at 200 V, the gels were removed by breaking the tubes. They were stained in Amido-