Two-Dimensional Electrophoresis/Phage Panning (2D-PP): A Novel Technology for Direct Antibody Selection on 2-D Blots

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Author(s)

    • Furuta Masaru FURUTA Masaru
    • Department of Cortical Function Disorders, National Institute of Neuroscience, National Center of Neurology and Psychiatry
    • Ito Takashi ITO Takashi
    • Department of Cortical Function Disorders, National Institute of Neuroscience, National Center of Neurology and Psychiatry
    • TANAKA Torahiko
    • Department of Cortical Function Disorders, National Institute of Neuroscience, National Center of Neurology and Psychiatry
    • WAKABAYASHI-TAKAI Eriko
    • Department of Cortical Function Disorders, National Institute of Neuroscience, National Center of Neurology and Psychiatry
    • KANEKO Kiyotoshi
    • Department of Cortical Function Disorders, National Institute of Neuroscience, National Center of Neurology and Psychiatry

Abstract

We describe a novel method, two-dimensional electrophoresis/phage panning (2D-PP), for the generation of antibodies against proteins in crude biochemical samples, such as cellular membrane fractions. These sources have traditionally presented problems as to the development of antibodies by conventional techniques. 2D-PP involves two-dimensional resolution of proteins, blotting of the proteins onto a nitrocellulose membrane, and screening of a phage antibody library and isolation of corresponding antibodies. By 2D-PP with detergent-insoluble “lipid rafts” as a target protein complex, we obtained specific phage pools against eight antigen spots (from a total of 39 spots). These antibodies were functional in Western blotting, enzyme-linked immunosorbent assaying (ELISA), and immunoscreening of a cDNA expression library. Propagation of anti-nitrocellulose phages was the major problem in 2D-PP, but was overcome by the use of the soluble anti-nitrocellulose antibody fragment. 2D-PP constitutes a key tool for functional analysis of proteins in complex fractions.

Journal

  • The Journal of Biochemistry

    The Journal of Biochemistry 132(2), 245-251, 2002-08-01

    The Japanese Biochemical Society

References:  34

Cited by:  2

Codes

  • NII Article ID (NAID)
    10010650479
  • NII NACSIS-CAT ID (NCID)
    AA00694073
  • Text Lang
    ENG
  • Article Type
    Journal Article
  • ISSN
    0021924X
  • NDL Article ID
    6255020
  • NDL Source Classification
    ZR2(科学技術--生物学--生化学)
  • NDL Call No.
    Z53-B472
  • Data Source
    CJP  CJPref  NDL  J-STAGE 
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