Mammalian in vitro splicing assays.
Mammalian in vitro splicing assays.
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哺乳动物体外剪接测定。
DOI：
10.1385/1-59259-676-2:315
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发表时间：
1999
影响因子：
--
通讯作者：
A. Krainer
中科院分区：
文献类型：
--
作者：
A. Mayeda;A. Krainer
剪接反应通常使用核提取物、补充有SR蛋白的S100提取物或源自粗提取物的部分纯化的级分进行。浸提液制备程序见第24章。最常用的是HeLa细胞提取物（见注1）。前体mRNA底物通常通过噬菌体聚合酶的体外径流转录制备（见第1章）。剪接的中间体和产物最方便地通过尿素/聚丙烯酰胺凝胶电泳（urea-PAGE）和放射自显影来可视化，这需要使用标记的前mRNA底物。此处提供的方案基于参考文献1，并在参考文献中进行了修改。2，3，并已在我们的实验室常规使用多年。
Splicing reactions are typically carried out using nuclear extracts, S100 extracts complemented with SR proteins, or partially purified fractions derived from the crude extracts. The extract preparation procedures are described in Chapter 24. Extracts derived from HeLa cells are used most commonly (see Note 1). The pre-mRNA substrates are usually prepared by in vitro runoff transcription with a bacteriophage polymerase (see Chapter 1). The intermediates and products of splicing are most conveniently visualized by urea/polyacrylamide gel electrophoresis (urea-PAGE) and autoradiography, which requires the use of labeled pre-mRNA substrate. The protocols provided here are based on ref. 1, with modifications introduced in refs. 2,3, and have been routinely used in our laboratory for many years.