Characterization of cis-regulatory elements and transcription factor binding: gel mobility shift assay.
Characterization of cis-regulatory elements and transcription factor binding: gel mobility shift assay.
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顺式调控元件和转录因子结合的表征：凝胶迁移率变化测定。
DOI：
10.1007/978-1-59745-030-0_10
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发表时间：
2007
期刊：
影响因子：
--
通讯作者：
Wang,Qin
中科院分区：
文献类型：
--
作者：
Lin,JimJung-Ching;Grosskurth,ShaunE;Harlan,ShannonM;Gustafson-Wagner,ElisabethA;Wang,Qin
为了了解心脏基因表达的调控机制，利用凝胶迁移率变动分析（GMSA）或凝胶阻滞分析（gel retardation assay）来鉴定和鉴定顺式调控元件及其反式作用因子是必不可少的和常用的步骤。除了提供一个通用的协议GMSA，本章描述了一些应用程序，这种检测来表征心脏特异性和泛素化的反式作用因子结合到调节元件[新的TCTG（G/C）直接重复和A/T丰富的区域]的大鼠心肌肌钙蛋白T启动子。在GMSA中，反式作用因子与标记DNA探针结合的特异性应通过在反应混合物中加入未标记的探针来验证。通过比较不同组织蛋白质提取物形成的DNA-蛋白质复合物的迁移特性，可以确定反式作用因子的组织特异性。GMSA与反式作用因子特异性抗体（抗体超移试验）结合，用于鉴定DNA-蛋白质复合物中存在的蛋白质。使用含有略微不同序列的未标记探针的凝胶移位竞争测定是用于评估DNA-蛋白质相互作用的序列特异性和相对结合亲和力的强大技术。GMSA与SDS-PAGE分级分离的蛋白质允许确定结合的反式作用因子的表观分子量。
To understand how cardiac gene expression is regulated, the identification and characterization ofcis-regulatory elements and theirtrans-acting factors by gel mobility shift assay (GMSA) or gel retardation assay are essential and common steps. In addition to providing a general protocol for GMSA, this chapter describes some applications of this assay to characterize cardiac-specific and ubiquitoustrans-acting factors bound to regulatory elements [novel TCTG(G/C) direct repeat and A/T-rich region] of the ratcardiac troponin Tpromoter. In GMSA, the specificity of the binding of trans-acting factor to labeled DNA probe should be verified by the addition of unlabeled probe in the reaction mixture. The migratory property of DNA-protein complexes formed by protein extracts prepared from different tissues can be compared to determine the tissue specificity oftrans-acting factors. GMSA, coupled with specific antibody totrans-acting factor (antibody supershift assay), is used to identify proteins present in the DNA-protein complex. The gel-shift competition assay with an unlabeled probe containing a slightly different sequence is a powerful technique used to assess the sequence specificity and relative binding affinity of a DNA-protein interaction. GMSA with SDS-PAGE fractionated proteins allows for the determination of the apparent molecular mass of boundtrans-acting factor.