Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription.
Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription.
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DOI：
10.3791/62723
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发表时间：
2021-08-11
期刊：
影响因子：
--
通讯作者：
Sun J
中科院分区：
文献类型：
--
作者：
Wibisono P;Sun J
秀丽隐杆线虫自1963年被发现以来，一直是生物学研究的重要模型系统。然而，秀丽隐杆线虫尚未被充分利用于利用其核提取物进行生物反应的生化研究，如体外转录和DNA复制。在生化研究中使用秀丽隐杆线虫的一个重要障碍是在不牺牲核提取物活性的情况下破坏线虫厚的外层角质层。虽然有几种方法用于破坏角质层，如Dounce均质或超声，但它们通常会导致蛋白质不稳定。目前尚无从秀丽隐杆线虫幼虫或成虫中分离活性核蛋白用于体外反应的既定方案。在这里，该协议详细描述了幼虫期4秀丽隐杆线虫的匀浆使用Balch匀浆机。Balch均质机使用压力缓慢地迫使动物通过一个狭窄的间隙，在此过程中打破角质层。Balch均质机的均匀设计和精密加工允许在实验之间一致地研磨动物。从Balch匀浆器中分离得到的匀浆得到功能活性的核提取物，可用于体外方法测定秀丽隐杆线虫的转录活性。
Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully utilized in the biochemical study of biological reactions using its nuclear extracts such as in vitro transcription and DNA replication. A significant hurdle for using C. elegans in biochemical studies is disrupting the nematode’s thick outer cuticle without sacrificing the activity of the nuclear extract. While several methods are used to break the cuticle, such as Dounce homogenization or sonication, they often lead to protein instability. There are no established protocols for isolating active nuclear proteins from larva or adult C. elegans for in vitro reactions. Here, the protocol describes in detail the homogenization of larval stage 4 C. elegans using a Balch homogenizer. The Balch homogenizer uses pressure to slowly force the animals through a narrow gap breaking the cuticle in the process. The uniform design and precise machining of the Balch homogenizer allow for consistent grinding of animals between experiments. Fractionating the homogenate obtained from the Balch homogenizer yields functionally active nuclear extract that can be used in an in vitro method for assaying transcription activity of C. elegans.
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影响因子：
3.3
作者：
Corsi AK;Wightman B;Chalfie M
通讯作者：
Chalfie M
影响因子：
2.8
作者：
Wibisono P;Liu Y;Sun J
通讯作者：
Sun J
影响因子：
--
作者：
Voss, Cristina;Schmitt, Brita;Anderl, Jan
通讯作者：
Anderl, Jan
影响因子：
11.4
作者：
LICHTSTEINER, S;TJIAN, R
通讯作者：
TJIAN, R
DOI：
10.1073/pnas.90.20.9673
发表时间：
1993-10-15
影响因子：
11.1
作者：
LICHTSTEINER, S;TJIAN, R
通讯作者：
TJIAN, R