Production and characterisation of monoclonal and polyclonal antibodies against the transcription factor NF-kB and its inhibitor I-kB

Abstract

The activation of the transcription factor NF-kB and its inhibition by I-kB has been the subject of much research. However, there are many questions which remain unanswered regarding the functional properties of, and relationship between, the factor and the inhibitor. In this study, antibodies directed against the protein components of NF-kB and I-kB have been produced and used as tools to determine the subcellular localisation of both proteins in different cell lines both before and after activation with various agents. In particular the production and characterisation of monoclonal antibody 3B6 will be described. This antibody was raised against the p50 subunit of NF-kB and recognises an epitope contained within the nuclear localisation signal of the protein. Polyclonal antisera raised against p50 and I-kB were used in Western blots and immunofluorescence experiments and show changes in the localisation of NF-kB subunits and I-kB following activation of cells for various times.