ETHIDIUM-BROMIDE PROVIDES A SIMPLE TOOL FOR IDENTIFYING GENUINE DNA-INDEPENDENT PROTEIN ASSOCIATIONS
ETHIDIUM-BROMIDE PROVIDES A SIMPLE TOOL FOR IDENTIFYING GENUINE DNA-INDEPENDENT PROTEIN ASSOCIATIONS
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DOI：
10.1073/pnas.89.15.6958
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发表时间：
1992-08-01
影响因子：
11.1
通讯作者：
HERR, W
中科院分区：
文献类型：
--
作者：
LAI, JS;HERR, W
DNA结合蛋白的DNA依赖性和DNA非依赖性缔合在转录调控中是重要的。DNA非依赖性关联的分析通常依赖于在不存在靶DNA序列的情况下测定蛋白质相互作用。我们已经发现，蛋白质制剂中的污染DNA可以稳定DNA依赖的协会，可能出现DNA独立。三个细胞蛋白的70，85，和110 kDa的共免疫沉淀的八聚体基序结合蛋白Oct-2，因为在细胞提取物中存在污染的DNA。此外，Oct-1（或Oct-2）和Pit-1之间的异二聚体形成蛋白质亲和层析过程中稳定的污染DNA。在这两种情况下，这些DNA依赖性蛋白质协会选择性抑制溴化乙锭在沉淀反应中没有任何明显的影响，DNA独立的蛋白质协会。因此，溴化乙锭可作为DNA依赖性和DNA非依赖性蛋白质缔合的简单和一般的指示剂。
DNA-dependent and DNA-independent associations of DNA-binding proteins are important in transcriptional regulation. The analysis of DNA-independent associations frequently relies on assaying protein interaction in the absence of target DNA sequences. We have found that contaminating DNA in protein preparations can stabilize DNA-dependent associations that may appear DNA-independent. Three cellular proteins of 70, 85, and 110 kDa coimmunoprecipitated with the octamer motif-binding protein Oct-2 because of the presence of contaminating DNA in the cell extracts. In addition, heterodimer formation between Oct-1 (or Oct-2) and Pit-1 during protein-affinity chromatography was stabilized by the contaminating DNA. In both instances, these DNA-dependent protein associations were selectively inhibited by ethidium bromide in the precipitation reaction without any evident effect on DNA-independent protein associations. Thus, ethidium bromide may serve as a simple and general indicator of DNA-dependent and DNA-independent protein associations.