A simplified probe preparation for ELISA-based NF-κB activity assay
A simplified probe preparation for ELISA-based NF-κB activity assay
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基于 ELISA 的 NF-κB 活性测定的简化探针制备
DOI：
10.1016/j.jbbm.2005.08.006
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发表时间：
2005-10-31
影响因子：
--
通讯作者：
Hu, QH
中科院分区：
文献类型：
--
作者：
Jin, S;Lu, DQ;Hu, QH
核因子 kappa B (NF-kappa B) 关键参与许多基因以及多种生物和病理过程的转录调控。为了有效监测和快速筛选 NF-kappa B 转录活性，自首次演示和最近开发以来，基于 ELISA 的测定已作为一种新方法越来越多地成功应用于各种细胞系和实验模型。在基于 ELISA 的测定中，NF-kappa B 被预先连接在多孔板上的双链 DNA 探针捕获。通常，DNA探针包含活性NF-κB的双链共有结合序列和连接共有结合序列与板的另一个双链序列（接头序列）。由于核因子与单链DNA没有结合活性，我们将探针结构修改为包含双链共有结合序列和单链接头序列。我们的结果表明，这种探针对于NF-κB活性测定具有高度的敏感性和特异性，而且这种探针的制备也更加方便。单链接头序列可以很大程度上减少非特异性蛋白质结合，从而提高该测定的灵敏度。 (c) 2005 Elsevier B.V. 保留所有权利。
Nuclear factor-kappa B (NF-kappa B) is critically involved in the transcriptional regulation of many genes and multiple biological and pathobiological processes. To efficiently monitor and to rapidly screen NF-kappa B transcriptional activity, an ELISA-based assay has been increasingly and successfully employed as a new method in a variety of cell lines and experimental models since its first demonstration and recent development. In the ELISA-based assay, NF-kappa B is captured by a double-stranded DNA probe pre-linked on multi-well plates. Typically, the DNA probe contains the double-stranded consensus binding sequence for active NF-kappa B and another double-stranded sequence linking the consensus binding sequence with the plate (linker sequence). Since nuclear factor has no binding activity with single-stranded DNA, we modified the probe construction as containing the double-stranded consensus binding sequence and a single-stranded-linker sequence. Our results show that this kind of probe is highly sensitive and specific for NF-kappa B activity assay, whereas the preparation of this kind of probe is much more convenient. A single-stranded-linker sequence may largely decrease nonspecific protein binding and thus increase the sensitivity of this assay. (c) 2005 Elsevier B.V. All rights reserved.