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Simple and Efficient Method for the Preparation of Nuclear Extracts

机译：一种简单高效的核提取物制备方法

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摘要

Gene expression is mediated by the interaction of trans-acting, DNA-bind-ing proteins with specific cis-acting elements in DNA. To study gene expression, nuclear extracts from the tissue or cells of interest are first prepared. The method described byDignam et al. (3) involves cell lysis in a Dounce homogenizer, nuclear pelleting by centrifugation and preparation of nuclear extracts by hypertonic buffer extraction and dialysis. This method is appropriate for cultured cells but is of limited use withtissues that contain organ capsules, vascular structures, connective tissue and proteases. For the preparation of transcriptionally active nuclear extracts from such tissues, a number of elaborate protocols have been described. In these methods, tissuesare homogenized by either motor-driven Teflon~(R) pestle and glass homogenizers or a specially designed Waring~(R) blender that excludes air during grinding. Nuclei are purified by density sedimentation. The nuclei are then lysed and the chromatin is removed by ammonium sulfate precipitation. Finally, the nuclear proteins are precipitated with ammonium sulfate, resuspended and dialyzed. While these methods are quite suitable for nuclei purification from tissues, they are nevertheless quite lengthy because of the repeated ultracentrifugation steps and the time needed for ammonium sulfate precipitation of the chromatin and proteins. Moreover, denaturation of the pelleted proteins usually occurs during resuspension.
机译：基因表达由反式作用的，与DNA结合的蛋白质与DNA中特定的顺式作用元件的相互作用介导。为了研究基因表达，首先要制备目标组织或细胞的核提取物。 Dignam等人描述的方法。 （3）涉及在Dounce均质器中进行细胞裂解，通过离心进行核沉淀以及通过高渗缓冲液提取和透析制备核提取物。此方法适用于培养的细胞，但对于包含器官胶囊，血管结构，结缔组织和蛋白酶的组织使用有限。为了从这些组织制备转录活性核提取物，已经描述了许多精心设计的方案。在这些方法中，通过马达驱动的杵和玻璃匀浆器或专门设计的Waring搅拌器将组织均质化，该搅拌器在研磨过程中排除了空气。核通过密度沉降纯化。然后裂解细胞核，并通过硫酸铵沉淀除去染色质。最后，核蛋白用硫酸铵沉淀，重悬并透析。尽管这些方法非常适合从组织中纯化细胞核，但由于重复的超速离心步骤以及硫酸铵沉淀染色质和蛋白质所需的时间，它们仍然很长。而且，沉淀的蛋白质的变性通常在重悬过程中发生。

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