Generation and application of a novel InsP(3)R(1) mono-antibody from mouse.

Abstract

Inositol 1, 4, 5-Trisphosphate Receptor (InsP3R) is an intracellular Ca(2+) release channel, which widely participates in cellular processes. Three isoforms of InsP3R were identified as InsP3R1, InsP3R2, 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.