Preparation of nuclear and cytoplasmic extracts from mammalian cells.
Preparation of nuclear and cytoplasmic extracts from mammalian cells.
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DOI：
10.1002/0471142727.mb1201s75
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发表时间：
2006-12-01
影响因子：
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通讯作者：
Workman, Jerry L
中科院分区：
文献类型：
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作者：
Abmayr, Susan M;Yao, Tingting;Workman, Jerry L
从培养细胞的分离细胞核制备的提取物有助于剖析转录和mRNA加工发生的机制。这些提取物能够在体外重现准确的转录起始和剪接，这在直接功能研究中是有用的。它们还可以作为蛋白质纯化的起始材料，然后可以在功能研究中重新组装或进行更详细的生物化学检查。本单元描述了从培养细胞中制备核提取物和从HeLa细胞中优化生产转录活性提取物。附加的协议描述了优化的方法，以增加特定蛋白质的产量，适应下游应用的方法，如亲和纯化，和制备的细胞质（S-100）fraction.Extracts制备从培养细胞的分离的细胞核已有助于解剖转录和mRNA加工发生的机制。这些提取物能够在体外重现准确的转录起始和剪接，这在直接功能研究中是有用的。它们还可以作为蛋白质纯化的起始材料，然后可以在功能研究中重新组装或进行更详细的生物化学检查。本单元描述了从培养细胞中制备核提取物和从HeLa细胞中优化生产转录活性提取物。其他方案描述了优化方法以增加特定蛋白质的产率，调整方法用于下游应用，如亲和纯化和制备细胞质（S-100）级分。
Extracts prepared from the isolated nuclei of cultured cells have been instrumental in dissecting the mechanisms by which transcription and mRNA processing occur. These extracts are able to recapitulate accurate transcription initiation and splicing in vitro, which has been useful in direct functional studies. They also serve as the starting material for purification of proteins that can then be reassembled in functional studies or examined in more detail biochemically. This unit describes the preparation of nuclear extracts from cultured cells and optimized production of transcriptionally active extracts from HeLa cells. Additional protocols describe optimization of the method to increase the yield of specific proteins, adaptation of the method for downstream applications such as affinity purification, and preparation of the cytoplasmic (S-100) fraction.Extracts prepared from the isolated nuclei of cultured cells have been instrumental in dissecting the mechanisms by which transcription and mRNA processing occur. These extracts are able to recapitulate accurate transcription initiation and splicing in vitro, which has been useful in direct functional studies. They also serve as the starting material for purification of proteins that can then be reassembled in functional studies or examined in more detail biochemically. This unit describes the preparation of nuclear extracts from cultured cells and optimized production of transcriptionally active extracts from HeLa cells. Additional protocols describe optimization of the method to increase the yield of specific proteins, adaptation of the method for downstream applications such as affinity purification, and preparation of the cytoplasmic (S-100) fraction.