Quantitative analysis of binding of transcription factor complex to biotinylated DNA probe by a streptavidin-agarose pulldown assay
Quantitative analysis of binding of transcription factor complex to biotinylated DNA probe by a streptavidin-agarose pulldown assay
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DOI：
10.1016/j.ab.2003.08.007
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发表时间：
2003-12-01
影响因子：
2.9
通讯作者：
Wu, KK
中科院分区：
文献类型：
--
作者：
Deng, WG;Zhu, Y;Wu, KK
基因表达受与基因的启动子/增强子区域结合的蛋白质的大复合物调节。我们确定是否链霉亲和素珠结合试验可能是有用的，在复杂的反式激活因子，共激活因子，介质，和一般的转录因子检测个别蛋白质。我们使用生物素标记的环氧合酶-2启动子探针作为模型。将从用或不用激动剂处理的人成纤维细胞获得的核提取物与5 ′-生物素化探针和链霉亲和素-琼脂糖珠在室温下孵育1小时。离心后，洗涤沉淀，并通过免疫印迹评估复合物中的蛋白质。在基础状态下，在同一批沉淀中可同时检测到转录因子阵列。p300及其相关因子PCAF水平增加，而不是Srb 7，Med 7，或TFIIB由佛波酯或肿瘤坏死因子刺激。仅检测到微量CREB结合蛋白。这些结果表明p300和PCAF是考克斯-2启动子激活的主要共激活因子。我们的研究结果表明，链霉亲和素珠下拉试验是有价值的，用于确定大量的转录因子的结合启动子/增强子和评估蛋白质结合与基因表达调控的关系。(C)2003年爱思唯尔公司All rights reserved.
Gene expression is regulated by a large complex of proteins that bind to the promoter/enhancer region of a gene. We determined whether a streptavidin-bead binding assay might be useful in detecting individual proteins in the complex comprising transactivators, coactivators, mediators, and general transcription factors. We used biotinylated cyclooxygenase-2 promoter probes as a model. Nuclear extracts obtained from human fibroblasts treated with or without an agonist were incubated with a 5'-biotinylated probe and streptavidin-agarose beads at room temperature for 1 h. After centrifugation, the pellet was washed and proteins in the complex were assessed by immunoblots. An array of transcription factors was detectable concurrently in the same batch of pellets at basal state. p300 and its associated factor PCAF levels but not Srb7, Med7, or TFIIB were increased by phorbol ester or tumor necrosis factor a stimulation. Only trace of CREB-binding protein was detected. These results suggest that p300 and PCAF are the predominant coactivators for COX-2 promoter activation. Our findings indicate that the streptavidin-bead pulldown assay is valuable for determining the binding of a large number of transcription factors to promoter/enhancer and evaluating the relationship of protein binding with regulation of gene expression. (C) 2003 Elsevier Inc. All rights reserved.