Use of vertical slab isoelectric focusing and immunoblotting to evaluate steady-state phosphorylation of eIF2 alpha in cultured cells.
Use of vertical slab isoelectric focusing and immunoblotting to evaluate steady-state phosphorylation of eIF2 alpha in cultured cells.
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使用垂直平板等电聚焦和免疫印迹评估培养细胞中 eIF2 α 的稳态磷酸化。
DOI：
10.1006/meth.1996.0438
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Jagus,R
中科院分区：
文献类型：
--
作者：
Savinova,O;Jagus,R
垂直、一维等电聚焦和免疫印迹的组合对于使用网织红细胞裂解物或纯化的eIF2评价eIF2 α亚基的磷酸化状态非常有效。然而，该方法更难以应用于培养细胞中eIF2α磷酸化的分析。这在一定程度上反映了培养细胞提取物的蛋白质含量很少与网织红细胞提取物中发现的蛋白质含量一样高的事实，这在一定程度上反映了细胞提取物的某些组分干扰eIF2α进入等电聚焦凝胶的事实。为了克服这些困难，我们已经修改了早期的方法，包括免疫沉淀的eIF2从细胞提取物之前，等电聚焦，以及低十二烷基硫酸钠浓度的等电聚焦样品缓冲液。由于PKR激活状态和eIF2α磷酸化状态随细胞密度和营养状态而变化，因此我们常规设置一致的补料时间表，并建议在一系列细胞密度范围内收集数据。
The combination of vertical, one-dimensional isoelectric focusing and immunoblotting works very well for the evaluation of the phosphorylation state of the α-subunit of eIF2 using reticulocyte lysate or purified eIF2. However, the method is more difficult to apply to the analysis of eIF2α phosphorylation in cultured cells. In part this reflects the fact that the protein content of cultured cell extracts is rarely as high as that found in extracts produced from reticulocytes, and in part this reflects the fact that some component(s) of cell extracts interferes with the entry of eIF2α into the isoelectric focusing gel. To overcome these difficulties, we have modified the earlier method to include immunoprecipitation of eIF2 from cell extracts prior to isoelectric focusing, as well as a low sodium dodecyl sulfate concentration in the isoelectric focusing sample buffer. Since the PKR activation state and therefore the eIF2α phosphorylation state change with cell density and nutritional status, we routinely set up consistent feeding schedules and recommend the collection of data over a range of cell densities.