Two-dimensional electrophoresis/phage panning (2D-PP): a novel technology for direct antibody selection on 2-D blots.
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.
Furuta
Masaru
M
Department of Cortical Function Disorders, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Ogawahigashi, Kodaira, Tokyo 187-8502, Japan.
Ito
Takashi
T
Eguchi
Chikashi
C
Tanaka
Torahiko
T
Wakabayashi-Takai
Eriko
E
Kaneko
Kiyotoshi
K
D000906:Antibodies / Q000276:immunology* / Q000302:isolation & purification* / Q000378:metabolism; D001435:Bacteriophages / Q000378:metabolism*; D003902:Detergents / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting / Q000379:methods*; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism; D019151:Peptide Library; D011506:Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000906:Antibodies; D003902:Detergents; D019151:Peptide Library; D011506:Proteins