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Antibodies to the rat substance P receptor: production and characterization

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Summary

  1. 1.

    A protein A-rat substance P receptor (SPR) fusion protein was genetically engineered and used as an immunogen to raise a polyclonal antiserum to the SPR. The fusion protein was expressed inEscherichia coli driven by the heat-inducible lambda promoter (λPr).

  2. 2.

    The fusion protein was purified using an IgG-Sepharose column, which specifically binds proteins containing the protein A moiety. The IgG fraction obtained after the immunization was cleaved to produce Fab fragments, which were subsequently purified using a fusion protein affinity column. The serum (anti-SPR Fab serum) was analyzed by fluorescence-activated cell sorting (FACS) and immunohistochemistry on both a constitutive cell line for the SPR (AR42J) and a cell line transfected with the SPR (KNRKSPR).

  3. 3.

    Specificity of the antiserum for SPR was confirmed by immunohistochemistry on cells using antiserum that had been preincubated with the protein A fusion protein (blocked).

  4. 4.

    The Ca2+ signal normally observed on stimulation of SPR with SP in AR42J cells and SP binding to KNRKSPR cells was shown to be diminished in the presence of anti-SPR Fab serum. SPR from both cell lines was immunoprecipitated using the anti-SPR Fab serum. The antiserum itself did not induce intracellular Ca2+ mobilization normally observed when cells were incubated with SP.

  5. 5.

    This specific SPR antiserum will be a useful tool to investigate further the mechanisms of SP/SPR interactions.

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Authors and Affiliations

  1. Department of Microbiology-Immunology, University of California, San Francisco, 94143, San Francisco, California, USA

    M. S. Gilbert & D. G. Payan

  2. Department of Physiology, University of California, San Francisco, 94143, San Francisco, California, USA

    N. W. Bunnett

  3. Department of Surgery, University of California, San Francisco, 94143, San Francisco, California, USA

    N. W. Bunnett

  4. Howard Hughes Medical Institute, University of California, San Francisco, 94143, San Francisco, California, USA

    D. G. Payan

  5. Department of Medicine, University of California, San Francisco, 94143, San Francisco, California, USA

    D. G. Payan

  6. University of California, San Francisco, Third and Paranassus Avenue, 94143-0724, San Francisco, California, USA

    D. G. Payan

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  1. M. S. Gilbert
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  2. N. W. Bunnett
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  3. D. G. Payan
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Gilbert, M.S., Bunnett, N.W. & Payan, D.G. Antibodies to the rat substance P receptor: production and characterization. Cell Mol Neurobiol 12, 529–545 (1992). https://doi.org/10.1007/BF00711233

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  • DOI: https://doi.org/10.1007/BF00711233

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