Magnetocapture of abalone transcription factor NF-κB: A new strategy for isolation and detection of NF-κB both in vitro and in vivo

Abstract: Rel\NF-κB plays an important role in the immune response. Recently, a homologue of Rel\NF-κB transcription factor was identified from a gastropod abalone, Haliotis diversicolor supertexta, named Ab-Rel. We present here a new strategy, so called magnetocapture, based on magnetic separation that was developed to isolate and detect this abalone transcriptional factor. With this method, Ab-Rel was successfully isolated from abalone haemocytes and the recombinant Ab-Rel protein from an insect cell line. Western blot analysis was performed to verify the effectiveness of this magnetocapture assay. Since only the activated NF-κB can be captured and separated from the whole cell extracts, this method was simultaneously used to detect the NF-κB activity in vivo, as well as the biological activity of the recombinant protein in vitro. In addition, several proteins which might interact with the abalone NF-κB were together isolated, suggesting that the magnetocapture method would be useful for identifying interaction molecules. These results illustrated that the magnetocapture strategy is rapid, reliable and versatile, providing a promising tool for studying Rel\NF-κB protein. [Copyright &y& Elsevier]