METHODOLOGY ARTICLE Rapid preparation of high u h

Among those transcription factors, nuclear factor-κB (NF-κB), which usually exists as a heterodimer formed between The electrophoretic mobility shift assay (EMSA), a common affinity electrophoresis technique used to study Luo et al. BMC Immunology (2014) 15:586 DOI 10.1186/s12865-014-0062-zclear protein, and modified protocols to further separateAoba-cho, Higashimurayama-shi 189-0002, Tokyo, Japan Full list of author information is available at the end of the articlesubunits p50 and RelA/p65, plays a central role in both innate and adaptive immune responses [1-3]. In unstimu-lated cells, NF-κB exists in the cytoplasm as an inactive form sequestered by the inhibitor protein IκB [4]. Upon stimulation by a variety of stimuli, including bacterial lipo-polysaccharide (LPS) [5], tumor necrosis factor α (TNF-α) [6], interleukin-1β (IL-1β) [7] and reactive oxygen species protein-nucleic acid interactions, is often employed to demonstrate the binding of the active form of nuclear NF-κB to its DNA recognition sequence at several time points following a particular stimulation. The first and possibly the most crucial step in ensuring accurate detection of NF-κB binding and stoichiometry by EMSA is purification of nuclear protein extracts that contain DNA-binding proteins with no contamination by the cytoplasmic fraction. Many basic protocols for the extraction of whole nu