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溴化乙锭为鉴定真正的非DNA依赖性蛋白质结合提供了一种简单的工具。

Ethidium bromide provides a simple tool for identifying genuine DNA-independent protein associations.

作者信息

Lai J S, Herr W

机构信息

Cold Spring Harbor Laboratory, NY 11724.

出版信息

Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6958-62. doi: 10.1073/pnas.89.15.6958.

DOI:10.1073/pnas.89.15.6958
PMID:1495986
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC49624/
Abstract

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.

摘要

DNA结合蛋白的DNA依赖性和非DNA依赖性结合在转录调控中很重要。对非DNA依赖性结合的分析通常依赖于在没有靶DNA序列的情况下检测蛋白质相互作用。我们发现蛋白质制剂中的污染DNA可以稳定可能看似非DNA依赖性的DNA依赖性结合。由于细胞提取物中存在污染DNA，三种分子量分别为70 kDa、85 kDa和110 kDa的细胞蛋白与八聚体基序结合蛋白Oct-2共免疫沉淀。此外，在蛋白质亲和层析过程中，污染DNA稳定了Oct-1（或Oct-2）与Pit-1之间的异二聚体形成。在这两种情况下，这些DNA依赖性蛋白质结合在沉淀反应中被溴化乙锭选择性抑制，而对非DNA依赖性蛋白质结合没有任何明显影响。因此，溴化乙锭可作为DNA依赖性和非DNA依赖性蛋白质结合的简单通用指示剂。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2edf/49624/9d5dfc09814c/pnas01089-0308-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2edf/49624/05b0ce00bd33/pnas01089-0306-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2edf/49624/fc8e79520512/pnas01089-0307-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2edf/49624/3ae36a78d1e9/pnas01089-0307-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2edf/49624/e4f213ed6de0/pnas01089-0308-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2edf/49624/9d5dfc09814c/pnas01089-0308-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2edf/49624/05b0ce00bd33/pnas01089-0306-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2edf/49624/fc8e79520512/pnas01089-0307-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2edf/49624/3ae36a78d1e9/pnas01089-0307-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2edf/49624/e4f213ed6de0/pnas01089-0308-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2edf/49624/9d5dfc09814c/pnas01089-0308-b.jpg

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