Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu, Taiwan, Republic of China.
School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, Taiwan, Republic of China.
Biol Proced Online. 2010 Apr 14;12(1):18-26. doi: 10.1007/s12575-010-9030-z.
There are several traditional and well-developed methods for analyzing the activity of transcription factors, such as EMSA, enzyme-linked immunosorbent assay, and reporter gene activity assays. All of these methods have their own distinct disadvantages, but none can analyze the changes in transcription factors in the few cells that are cultured in the wells of 96-well titer plates. Thus, a new microsphere-based immunoassay to measure the activity of transcription factors (MIA-TF) was developed. In MIA-TF, NeutrAvidin-labeled microspheres were used as the solid phase to capture biotin-labeled double-strand DNA fragments which contain certain transcription factor binding elements. The activity of transcription factors was detected by immunoassay using a transcription factor-specific antibody to monitor the binding with the DNA probe. Next, analysis was performed by flow cytometry. The targets hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-kappa B (NF-κB) were applied and detected in this MIA-TF method; the results that we obtained demonstrated that this method could be used to monitor the changes of NF-κB or HIF within 50 or 100 ng of nuclear extract. Furthermore, MIA-TF could detect the changes in NF-κB or HIF in cells that were cultured in wells of a 96-well plate without purification of the nuclear protein, an important consideration for applying this method to high-throughput assays in the future. The development of MIA-TF would support further progress in clinical analysis and drug screening systems. Overall, MIA-TF is a method with high potential to detect the activity of transcription factors.
有几种传统且成熟的方法可用于分析转录因子的活性，如 EMSA、酶联免疫吸附测定和报告基因活性测定。所有这些方法都有其独特的缺点，但都无法分析培养在 96 孔微量滴定板孔中的少数细胞中转录因子的变化。因此，开发了一种基于微球的测量转录因子活性的新方法（MIA-TF）。在 MIA-TF 中，链霉亲和素标记的微球被用作固相，以捕获含有某些转录因子结合元件的生物素标记的双链 DNA 片段。通过使用转录因子特异性抗体进行免疫测定来检测转录因子的活性，以监测与 DNA 探针的结合。接下来，通过流式细胞术进行分析。该方法应用并检测了缺氧诱导因子-1α（HIF-1α）和核因子-κB（NF-κB）这两个靶标；我们获得的结果表明，该方法可用于监测 50 或 100ng 核提取物中 NF-κB 或 HIF 的变化。此外，MIA-TF 无需纯化核蛋白即可检测培养在 96 孔板孔中的细胞中 NF-κB 或 HIF 的变化，这是未来将该方法应用于高通量测定的重要考虑因素。MIA-TF 的发展将支持临床分析和药物筛选系统的进一步发展。总体而言，MIA-TF 是一种具有高潜力的检测转录因子活性的方法。
