Development of a sensitive multi-well colorimetric assay for active NFkappaB.
Development of a sensitive multi-well colorimetric assay for active NFkappaB.
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DOI：
10.1093/nar/29.4.e21
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发表时间：
2001-02-15
影响因子：
14.9
通讯作者：
Remacle, J
中科院分区：
文献类型：
--
作者：
Renard, P;Ernest, I;Remacle, J
转录因子核因子kappaB(NFkappaB)是由炎性细胞因子或某些细菌和病毒产物等多种分子触发的免疫反应中的关键因子。这种转录因子代表了抗炎分子开发的新靶点，但由于缺乏一种方便快速的NFkappaB激活筛选方法，这类研究目前受到阻碍。事实上，NFkappaB的DNA结合能力传统上是通过放射性凝胶位移分析来估计的。在这里，我们提出了一种新的DNA结合分析方法，该方法基于多孔板，其中包被含有NFkappaB共同结合位点的冷寡核苷酸。然后用抗NFkappaB抗体检测DNA结合转录因子的存在，并用比色法显示。该方法简便、无放射性、重复性好、对NFkappaB具有特异性，比常规的放射性胶移更灵敏，便于高通量筛选。
The transcription factor nuclear factor kappaB (NFkappaB) is a key factor in the immune response triggered by a wide variety of molecules such as inflammatory cytokines, or some bacterial and viral products. This transcription factor represents a new target for the development of anti-inflammatory molecules, but this type of research is currently hampered by the lack of a convenient and rapid screening assay for NFkappaB activation. Indeed, NFkappaB DNA-binding capacity is traditionally estimated by radioactive gel shift assay. Here we propose a new DNA-binding assay based on the use of multi-well plates coated with a cold oligonucleotide containing the consensus binding site for NFkappaB. The presence of the DNA-bound transcription factor is then detected by anti-NFkappaB antibodies and revealed by colorimetry. This assay is easy to use, non-radioactive, highly reproducible, specific for NFkappaB, more sensitive than regular radioactive gel shift and very convenient for high throughput screening.