Lahiri D K, Ge Y
Laboratory of Molecular Neurogenetics, Institute of Psychiatric Research, Department of Psychiatry, Indiana University School of Medicine, 791 Union Drive, Indianapolis, IN 46202, USA.
Brain Res Brain Res Protoc. 2000 Jul;5(3):257-65. doi: 10.1016/s1385-299x(00)00021-0.
The electrophoretic mobility shift assay (EMSA) is generally used to study the interaction of transcription factors to specific DNA sequences. The preparation of high quality nuclear extracts is an important step before performing the assay. Here we describe a rapid method for the isolation of good-quality DNA-binding proteins from cultured cell lines and autopsy tissue samples from the human brain. The 'rapid method' (RM) utilizes the low salt/detergent lysis steps followed by high salt extraction of nuclei. To test and compare the activity of nuclear extracts prepared by the standard and 'rapid' methods for its ability to form the specific DNA-protein complex, EMSA was carried out with three different oligonucleotide probes: AP1, NF-kappaB and URE. A comparative study indicates that the capacity to form the specific DNA-protein complex with these oligonucleotide probes by standard and RM nuclear extracts was very similar. Each nuclear extract formed the corresponding DNA-protein complex, the specificity of which was checked by the competition experiment. In some cases unspecific bands were observed and which were present in nuclear extracts from both preparations. Thus the simplicity of the 'rapid method' permits the preparation of nuclear extracts from several cell lines and tissue samples at the same time at much shorter time than the 'standard' method without compromising the DNA-binding activity. The RM can be applied to determine the cell type or tissue specificity of transcription factors in an efficient, economical and consistent manner.
电泳迁移率变动分析（EMSA）通常用于研究转录因子与特定DNA序列的相互作用。在进行该分析之前，制备高质量的核提取物是重要的一步。在此，我们描述一种从培养的细胞系和人脑尸检组织样本中快速分离高质量DNA结合蛋白的方法。“快速方法”（RM）采用低盐/去污剂裂解步骤，随后进行高盐提取细胞核。为了测试和比较通过标准方法和“快速”方法制备的核提取物形成特异性DNA-蛋白质复合物的能力，使用三种不同的寡核苷酸探针（AP1、NF-κB和URE）进行了EMSA。一项比较研究表明，标准方法和RM核提取物与这些寡核苷酸探针形成特异性DNA-蛋白质复合物的能力非常相似。每种核提取物都形成了相应的DNA-蛋白质复合物，并通过竞争实验检查了其特异性。在某些情况下，观察到了非特异性条带，两种制备方法的核提取物中都存在这些条带。因此，“快速方法”的简便性使得能够在比“标准”方法短得多的时间内同时从多个细胞系和组织样本中制备核提取物，而不会损害DNA结合活性。RM可用于以高效、经济和一致的方式确定转录因子的细胞类型或组织特异性。
