Methods to separate nuclear soluble fractions reflecting localizations in living cells
Methods to separate nuclear soluble fractions reflecting localizations in living cells
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DOI：
10.1016/j.isci.2021.103503
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发表时间：
2021-12-17
期刊：
影响因子：
5.8
通讯作者：
Imamoto，Naoko
中科院分区：
文献类型：
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作者：
Ogawa，Yutaka;Imamoto，Naoko
为了了解各种核内功能，重要的是要知道蛋白质何时，什么以及如何进入细胞核。虽然已经开发了许多用于核分级分离的方法和商业试剂盒，但是仍然没有获得完整的核蛋白质组的方法。可溶性核蛋白在分离过程中经常丢失。我们开发了显着改进的方法，通过优化质膜的选择性透化的条件，以获得核可溶性组分。结果，1000万个细胞可以在1.5 mL试管中更精确地分离为细胞质和核可溶性部分。此外，添加抑制剂以防止从细胞核泄漏，使小蛋白保留在细胞核中。由于该方法操作简单，易于多样本应用，有望应用于动态核蛋白时空变化的研究，如信号转导等。
To understand various intranuclear functions, it is important to know when, what, and how proteins enter the nucleus. Although many methods and commercial kits for nuclear fractionation have been developed, there are still no methods for obtaining a complete nuclear proteome. Soluble nuclear proteins are often lost during fractionation. We developed remarkably improved methods to obtain nuclear soluble fractions by optimizing the conditions of selective permeabilization of the plasma membrane. As a result, 10 million cells could be separated into the cytoplasmic and nuclear soluble fractions more precisely in a 1.5-mL test tube. Moreover, the addition of an inhibitor to prevent leakage from the nucleus retained small proteins in the nucleus. Because of the simple protocols and easy application for multiple samples, our methods are expected to be applied to various studies on spatiotemporal changes of dynamic nuclear proteins, such as signal transduction.