Nuclear remodeling assay in Xenopus egg extract.
Nuclear remodeling assay in Xenopus egg extract.
复制标题
非洲爪蟾卵提取物中的核重塑测定。
DOI：
10.1007/978-1-59745-154-3_17
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Kikyo,Nobuaki
中科院分区：
文献类型：
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作者：
Gonda,Koichi;Kikyo,Nobuaki
Xenopusegg提取物是鉴定核重塑活性的理想材料，对核克隆具有重要意义。自从20多年前建立了鸡蛋提取物的制备方案以来，鸡蛋提取物已被广泛用作纯化与许多生理活性相关的因子的来源。鸡蛋很大，容易大量获得，并且富含各种生物活性蛋白质。在细胞核克隆中观察到的体细胞核重塑的许多方面在卵提取物中孵育的体细胞核中重现。我们的体外核重塑试验已被证明可有效纯化两种新的核重塑活性，ISWI，TATA结合蛋白从染色质中解离的关键参与者，以及FRGY2a和FRGY2b，两种能够进行核仁解体的蛋白质。在这里，我们概述了我们的方案，鸡蛋提取物的制备和体外核重塑试验，以及FRGY 2a和FRGY 2b的纯化方法。我们的体外细胞核重塑试验结合鸡蛋提取物将作为一个强大的工具，生物化学揭示参与核克隆的分子事件。
Xenopusegg extract is an ideal material to identify nuclear remodeling activities important for nuclear cloning. Since the protocol for egg extract preparation was established more than 20 yr ago, egg extract has been widely used as a source for the purification of factors associated with a number of physiological activities. The eggs are large, easily obtained in large quantities, and are abundant in a variety of bioactive proteins. Many aspects of somatic nuclear remodeling observed in nuclear cloning are recapitulated in somatic nuclei incubated in egg extract. Our in vitro nuclear remodeling assay has proven effective for the purification of two novel nuclear remodeling activities, ISWI, a key player in the dissociation of TATA binding protein from chromatin, and FRGY2a and FRGY2b, two proteins capable of nucleolar disassembly. Here we outline our protocol of egg extract preparation and in vitro nuclear remodeling assay, as well as the purification method for FRGY2a and FRGY2b. Our in vitro nuclear remodeling assay in combination with egg extract will serve as a powerful tool with which to biochemically uncover molecular events involved in nuclear cloning.