title: Analyzing transcription factor activity using near infrared fluorescent bridge polymerase chain reaction.
abstract: This study has developed a new method, near infrared fluorescent bridge polymerase chain reaction (NIRF-bPCR), for analyzing transcription factor (TF) activity. This method was first used to detect the activity of purified nuclear factor kappa B (NF-κB) p50. The results demonstrated that this method could quantitatively detect the activity of p50 protein at less than 115ng (∼ 2320fmol), and the detection limit reached as little as 6.94ng (∼ 140fmol) of p50 protein. This method was then used to detect TF activity in cell extracts. The results revealed that this method could specifically detect NF-κB activity in HeLa cell nuclear extracts. Finally, this method was used to detect the activities of multiple TFs in a protein sample. The results showed that this method could detect the activities of six TFs-NF-κB, AP-1, TFIID, CREB, NF-E2, and p53-in the TNFα-induced and -uninduced HeLa cell nuclear extracts. Calculation of the fold induction of six TFs revealed that NF-κB, CREB, and AP1 were activated by TNFα induction in HeLa cells, in agreement with the detection results of other methods. Therefore, this study provides a new tool for analyzing TF activity. This study also revealed that NIRF-bPCR may be used as a new method for detecting DNA molecules.
authors: State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China.
authors: Fei
authors: F
authors: Zhou
authors: State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China.
authors: Xiaoqian
authors: X
authors: Ling
authors: State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China.
authors: Junhuan
authors: J
authors: Yin
authors: State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China. Electronic address: wangjinke@seu.edu.cn.
authors: Jinke
authors: J
authors: Wang
mesh_terms: D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D052002:NF-kappa B p50 Subunit / Q000378:metabolism; D016133:Polymerase Chain Reaction; D019265:Spectroscopy, Near-Infrared; D014157:Transcription Factors / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D017931:DNA Primers; D005456:Fluorescent Dyes; D052002:NF-kappa B p50 Subunit; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D004247:DNA
keywords: Bridge PCR; NF-κB; Near infrared fluorescence; Transcription factor