title: Rapid preparation of high-purity nuclear proteins from a small number of cultured cells for use in electrophoretic mobility shift assays.
abstract: BACKGROUND
  Highly purified nuclear protein is required when using an electrophoretic mobility shift assay (EMSA) to study transcription factors, e.g. nuclear factor-κB (NF-κB), a major transcription factor that regulates both innate and adaptive immune responses following infection. Although many protocols have been developed for nuclear protein extraction, they are not necessarily optimized for use in EMSA, often require a large number of cells and long processing times, and do not always result in complete separation of the nuclear and cytoplasmic fractions.
  
  
  RESULTS
  We have developed a simple, rapid and cost-effective method to prepare highly purified nuclear proteins from a small number of both suspended and adherent cultured cells that yields nuclear proteins comparable to those prepared by a standard large-scale method. The efficiency of the method was demonstrated by using EMSA to show the successful detection, in multilple concurrent samples, of NF-κB activation upon tetradecanoyl phorbol acetate (TPA) stimulation.
  
  
  CONCLUSIONS
  This method requires only a small number of cells and no specialized equipment. The steps have been simplified, resulting in a short processing time, which allows researchers to process multiple samples simultaneously and quickly. This method is especially optimized for use in EMSA, and may be useful for other applications that include proteomic analysis.
authors.affiliation: Laboratory of Molecular Diagnostics, Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases, 4-2-1 Aoba-cho, Higashimurayama-shi, 189-0002, Tokyo, Japan. yuqianluo31@gmail.com.
authors.forename: Yuqian
authors.initials: Y
authors.lastname: Luo
authors.affiliation: Laboratory of Molecular Diagnostics, Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases, 4-2-1 Aoba-cho, Higashimurayama-shi, 189-0002, Tokyo, Japan. takehara9@gmail.com.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Hara
authors.affiliation: Laboratory of Molecular Diagnostics, Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases, 4-2-1 Aoba-cho, Higashimurayama-shi, 189-0002, Tokyo, Japan. yishido@nih.go.jp.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Ishido
authors.affiliation: Laboratory of Molecular Diagnostics, Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases, 4-2-1 Aoba-cho, Higashimurayama-shi, 189-0002, Tokyo, Japan. aya.yoshihara@med.toho-u.ac.jp.
authors.forename: Aya
authors.initials: A
authors.lastname: Yoshihara
authors.affiliation: Laboratory of Molecular Diagnostics, Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases, 4-2-1 Aoba-cho, Higashimurayama-shi, 189-0002, Tokyo, Japan. kenzaburou.oda@med.toho-u.ac.jp.
authors.forename: Kenzaburo
authors.initials: K
authors.lastname: Oda
authors.affiliation: Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases, 4-2-1 Aoba-cho, Higashimurayama-shi, 189-0002, Tokyo, Japan. mmaki@niid.go.jp.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Makino
authors.affiliation: Leprosy Research Center, National Institute of Infectious Diseases, 4-2-1 Aoba-cho, Higashimurayama-shi, 189-0002, Tokyo, Japan. norishii@nih.go.jp.
authors.forename: Norihisa
authors.initials: N
authors.lastname: Ishii
authors.affiliation: Department of Education Planning and Development, Faculty of Medicine, Toho University, Tokyo, 143-8540, Japan. n-hiroi@med.toho-u.ac.jp.
authors.forename: Naoki
authors.initials: N
authors.lastname: Hiroi
authors.affiliation: Laboratory of Molecular Diagnostics, Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases, 4-2-1 Aoba-cho, Higashimurayama-shi, 189-0002, Tokyo, Japan. koichis@nih.go.jp.
authors.forename: Koichi
authors.initials: K
authors.lastname: Suzuki
mesh_terms: D045744:Cell Line, Tumor; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D016328:NF-kappa B / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D016328:NF-kappa B