Antibodies to the rat substance P receptor: production and characterization

M. S. Gilbert, N. W. Bunnett, D. G. Payan

Research output: Contribution to journalArticlepeer-review

Abstract

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 in Escherichia coli driven by the heat-inducible lambda promoter (λPr). 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. 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. 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. This specific SPR antiserum will be a useful tool to investigate further the mechanisms of SP/SPR interactions.

Original languageEnglish (US)
Pages (from-to)529-545
Number of pages17
JournalCellular and Molecular Neurobiology
Volume12
Issue number6
DOIs
StatePublished - Dec 1992

Keywords

  • anti-SPR Fab antiserum
  • protein A-SPR fusion protein
  • substance P
  • substance P receptor (SPR)

ASJC Scopus subject areas

  • Cellular and Molecular Neuroscience
  • Cell Biology

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