Electrophoretic mobility shift assay on poly(ethylene glycol-modified glass microchips for the study of estrogen responsive element binding

Yen Jun Chuang, Jia Wei Huang, Honest Makamba, Mei-Ling Tsai, Chun Wei Li, Shu-Hui Chen

研究成果: Article

17 引文 斯高帕斯(Scopus)

摘要

The binding of estrogen receptor (ER) to estrogen response element (ERE) is essential for genomic pathways of estrogens and gel-based electrophoretic mobility shift assay (EMSA) is commonly used for analyzing ERE binding. Gel-based EMSA, however, requires the use of hazard radio isotopes and they are slow, labor-intensive and difficult to quantify. Here, we present quantitative affinity assays based on microchip electrophoresis using PEG-modified glass microchannels, which bear neutral surfaces against the adsorption of acidic DNA molecules and basic ER proteins. We first demonstrated the feasibility of the method by measuring binding constants of recombinant ERα and ERβ with a consensus ERE sequence (cERE, 5′-GGTCAGAGTGACC-3′) as well as with an ERE-like sequence (ERE 1576, 5′-GACCGGTCAGCGGACTCAC-3′). Changes in mobility as a function of protein-DNA molar ratios were plotted and the dissociation constants were determined based on non-linear curve fitting. The minimum amount of ER proteins required for one assay was around 0.2 ng and the run time for one chip analysis was less than 2 min. We further measured the estrogenic compound-mediated dissociation constants with recombinant ER proteins as well as with the extracted ERβ from treated and untreated A549 bronchioloalveolar carcinoma cells. Dissociation constants determined by this method agree with the fact that agonist compounds such as 17β-estradiol (1.70nM), diethylstilbestrol (0.14nM), and genistein (0.80 nM) assist ERE binding by decreasing the constants; while antagonist compounds such as testosterone (140.4 nM) and 4-hydroxytamoxifen (10.5 nM) suppress the binding by increasing the dissociation constant.

原文English
頁(從 - 到)4158-4165
頁數8
期刊Electrophoresis
27
發行號21
DOIs
出版狀態Published - 2006 十一月 1

    指紋

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry

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