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.