A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA splicing

Gene analysis techniques Pub Date : 1988-03-01 DOI:10.1016/0735-0651(88)90023-4

Kevin A.W. Lee, Albrecht Bindereif, Michael R. Green

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引用次数: 432

Abstract

A convenient and rapid method for preparing soluble extracts from the nuclei of as few as 3 × 10⁷ mammalian cells (miniextract procedure) is described. By several criteria, miniextracts are comparable to nuclear extracts prepared from large numbers of cells by the conventional procedure. Miniextracts are able to support efficient transcription of a variety of class II promoters. In addition, DNase I footprinting and gel retardation assays can be performed directly in miniextracts, enabling the detection of sequence-specific DNA-binding proteins. Besides transcription, miniextracts efficiently carry out pre-mRNA splicing and allow formation and fractionation of previously characterized splicing complexes. The small-scale procedure enables simultaneous preparation of multiple extracts from a variety of cell types under different experimental conditions. Moreover, the use of small amounts of cells allows minimal expenditure of valuable or expensive materials such as radioactive compounds. Consequently, the procedure is highly advantageous for biochemical analysis of transcription and RNA processing in mammalian cells.

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支持高效转录和前mrna剪接的核提取物制备的小规模程序

本文描述了一种方便、快速地从3 × 107个哺乳动物细胞的细胞核中制备可溶性提取物的方法(微量提取程序)。通过几个标准，微萃取物可与传统方法从大量细胞中制备的核萃取物相媲美。微型提取物能够支持多种II类启动子的高效转录。此外，dna酶I足迹和凝胶阻滞试验可以直接在微萃取物中进行，从而能够检测序列特异性dna结合蛋白。除了转录外，微提取物还能有效地进行pre-mRNA剪接，并允许先前表征的剪接复合物的形成和分离。这种小规模的程序能够在不同的实验条件下同时制备来自多种细胞类型的多种提取物。此外，使用少量的电池可以将宝贵或昂贵的材料(如放射性化合物)的消耗降到最低。因此，该方法对哺乳动物细胞中转录和RNA加工的生化分析非常有利。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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Gene analysis techniques

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