標題: A New Microsphere-Based Immunoassay for Measuring the Activity of Transcription Factors
作者: Lin, Yu-Ling
Lai, Yun-Ju
Tsai, Nu-Man
Peng, Tai-Chu
Liu, Yen-Ku
Lee, Ru-Ping
Tsai, Chueh-Jen
Liao, Kuang-Wen
生物科技學系
分子醫學與生物工程研究所
Department of Biological Science and Technology
Institute of Molecular Medicine and Bioengineering
關鍵字: transcription factor;microsphere-based immunoassay;NF-kappa B;HIF-1
公開日期: 1-Dec-2010
摘要: 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-1 alpha (HIF-1 alpha) and nuclear factor-kappa B (NF-kappa B) 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-kappa B or HIF within 50 or 100 ng of nuclear extract. Furthermore, MIA-TF could detect the changes in NF-kappa B 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.
URI: http://dx.doi.org/10.1007/s12575-010-9030-z
http://hdl.handle.net/11536/26286
ISSN: 1480-9222
DOI: 10.1007/s12575-010-9030-z
期刊: BIOLOGICAL PROCEDURES ONLINE
Volume: 12
Issue: 1
起始頁: 18
結束頁: 26
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