Assay of DNA-binding proteins with a dsDNA-coupled plate
Assay of DNA-binding proteins with a dsDNA-coupled plate
复制标题
DOI：
10.1016/j.clinbiochem.2005.10.017
复制
发表时间：
2006-02-01
影响因子：
2.8
通讯作者：
Chen, HM
中科院分区：
文献类型：
--
作者：
Wang, JK;Li, JL;Chen, HM
目的：制备一种低成本的ddna偶联板（dcPlate），用于测定dna结合蛋白（DBP）的丰度和dna结合活性。设计与方法：将氨基修饰的寡核苷酸共价固定在n -氧琥珀酰亚胺酯包被的孔中制备dcdplate。采用dcPlate测定DBP的dna结合活性丰度，步骤相同，包括蛋白孵育、一抗结合、酶联二抗结合和比色开发。结果：通过对NF-kappa B、AP1和SP1三种纯化DBPs和HeLa细胞核提取物的检测，以及NF-kappa B p50对5种不同DNA序列的DNA结合活性的测定，证明了dcPlate可以定量测定DBPs的丰度，并以高通量的形式测定DBPs的DNA结合活性。结论：自制的dcPlate为dbp的研究提供了一种简单、通用的平台。(c) 2005加拿大临床化学家学会。版权所有。
Objectives: This paper fabricated a cost-effective dsDNA-coupled plate (dcPlate) and applied it to measure the abundance and DNA-binding activity of a DNA-binding protein (DBP).Design and methods: The dcPlate was manufactured by covalently immobilizing an amino-modified oligonucleotide in wells of the plate coated with N-oxysuccinimide esters. The dcPlate was applied to measure the abundance of DNA-binding activity of a DBP in the same four steps, including protein incubation, primary antibody binding, enzyme-linked secondary antibody binding, and colorimetric development.Results: The detections of three purified DBPs including NF-kappa B, AP1 and SP1, and HeLa cell nuclear extract and assays of DNA-binding activity of NF-kappa B p50 to five various DNA sequences demonstrated that dcPlate can be used to measure the abundance of DBPs quantitatively and assay DNA-binding activity of DBPs in high throughputs format.Conclusions: The homemade cost-effective dcPlate provides a simple and versatile platform for studying DBPs. (c) 2005 The Canadian Society of Clinical Chemists. All rights reserved.