Nonisotopic quantitative analysis of protein-DNA interactions at equilibrium
Nonisotopic quantitative analysis of protein-DNA interactions at equilibrium
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DOI：
10.1006/abio.1997.2231
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发表时间：
1997-08-01
影响因子：
2.9
通讯作者：
Belayew, A
中科院分区：
文献类型：
--
作者：
Benotmane, AM;Hoylaerts, MF;Belayew, A
开发了两种版本的酶联免疫吸附测定型方法来定量平衡时的蛋白质-DNA相互作用。第一种变体包括将DNA结合蛋白固定在微量滴定板上，与生物素化DNA孵育，并用链霉亲和素和生物素取代的辣根过氧化物酶标记结合的DNA。在第二个版本中，将生物素化的DNA固定在链霉亲和素取代的微量滴定板上，与DNA结合蛋白一起孵育，并用特异性抗体定量结合蛋白。为了说明该方法，谷胱甘肽-S-转移酶和解旋酶样转录因子的DNA结合结构域与其顺式元件（纤溶酶原激活物抑制剂-1启动子的B盒）之间的融合蛋白的相互作用用两种版本测定：发现平衡解离常数（Kd）为1 nM的1：1化学计量相互作用，这与电泳迁移率变动测定法测定的值相似，证明了测定的有效性。(C)北京：科学出版社.
Two versions of an enzyme-linked immunosorbent assay-type method to quantify protein-DNA interactions at equilibrium were developed. The first variant comprised immobilization of DNA-binding protein on microtiter plates, incubation with biotinylated DNA, and tagging of bound DNA with streptavidin- and biotin-substituted horseradish peroxidase. In the second version, biotinylated DNA was immobilized on streptavidin-substituted microtiter plates, incubated with DNA-binding protein, and bound protein was quantified with specific antibodies. To illustrate the method, the interaction of a fusion protein between glutathione-S-transferase and the DNA-binding domain of the helicase-like transcription factor with its cis-element (the B box of the plasminogen activator inhibitor-1 promoter) was determined with both versions: a 1:1 stoichiometric interaction with an equilibrium dissociation constant (K-d) Of 1 nM was found, which is similar to the value determined by electrophoretic mobility shift assay, demonstrating the validity of the assays. (C) 1997 Academic Press.