Abmayr Susan M, Yao Tingting, Parmely Tari, Workman Jerry L
The Stowers Institute for Medical Research, Kansas City, Missouri, USA.
Curr Protoc Pharmacol. 2006 Dec;Chapter 12:Unit12.3. doi: 10.1002/0471141755.ph1203s35.
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.
从培养细胞的分离细胞核中制备的提取物，在剖析转录和mRNA加工发生的机制方面发挥了重要作用。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很有用。它们还作为蛋白质纯化的起始材料，这些蛋白质随后可在功能研究中重新组装或进行更详细的生化检查。本单元描述了从培养细胞中制备核提取物以及从HeLa细胞中优化生产具有转录活性的提取物的方法。其他方案描述了优化该方法以提高特定蛋白质产量、将该方法应用于下游应用（如亲和纯化）以及制备细胞质（S-100）组分的方法。
