Generation and Application of a Novel InsP₃R₁ Mono-Antibody from Mouse

Inositol 1, 4, 5-Trisphosphate Receptor (InsP₃R) is an intracellular Ca²⁺ release channel, which widely participates in cellular processes. Three isoforms of InsP₃R were identified as InsP₃R₁, InsP₃R_2, and InsP₃R₃. They share 60–0% protein sequence homology and form a channel in a manner of homotetramer or heterotetramer. Several InsP₃R isoform-specific rabbit antibodies have been generated to distinguish their localization and functions. However, there is no report of such a valid antibody raised from other species. In his article, we prepare a mouse monoclonal antibody against a synthetic peptide with rat InsP₃R₁-specific carboxyl terminus sequence. This monoclonal antibody of InsP₃R₁ (R1-mAb) was purified and characterized as IgG2b, which can recognize InsP₃R₁ by Western-blot (WB) analysis and immunoprecipitate (IP) InsP₃R₁ from moue brain lysate tested. Applied in immunofluorescent (IF) and immunohistochemical (IHC) assays, this antibody and rabbit polyclonal antibody could give the consistent results in SH-SY5Y cells, human sperm, and mouse brain paraffin section. In summary, we generate a mouse InsP₃R₁-specific IgG 2b antibody identifying InsP₃R₁ in WB, IF, IHC, and IP analysis, which provides a possible choice for detection of InsP₃R₁, especially in application of co-localization analysis with other InsP₃R isoforms or other proteins.