A simplified probe preparation for ELISA-based NF-κB activity assay

Nuclear factor-κB (NF-κB) is critically involved in the transcriptional regulation of many genes and multiple biological and pathobiological processes. To efficiently monitor and to rapidly screen NF-κB transcriptional activity, an ELISA-based assay has been increasingly and successfully employed as a new method in a variety of cell lines and experimental models since its first demonstration and recent development. In the ELISA-based assay, NF-κB is captured by a double-stranded DNA probe pre-linked on multi-well plates. Typically, the DNA probe contains the double-stranded consensus binding sequence for active NF-κB and another double-stranded sequence linking the consensus binding sequence with the plate (linker sequence). Since nuclear factor has no binding activity with single-stranded DNA, we modified the probe construction as containing the double-stranded consensus binding sequence and a single-stranded-linker sequence. Our results show that this kind of probe is highly sensitive and specific for NF-κB activity assay, whereas the preparation of this kind of probe is much more convenient. A single-stranded-linker sequence may largely decrease nonspecific protein binding and thus increase the sensitivity of this assay.