Development and characterization of a novel monoclonal antibody that recognizes an epitope in the central protein interaction domain of RapGEF1 (C3G)

Mol Biol Rep. 2018 Dec;45(6):1809-1819. doi: 10.1007/s11033-018-4327-0. Epub 2018 Aug 24.


The ubiquitously expressed protein RapGEF1 (C3G) regulates multiple cellular activities and is essential for early embryonic development in mammals. It has functions dependent on its catalytic activity as well as protein interaction domain and regulates β-catenin signaling. This study describes the generation of a novel monoclonal antibody, 3F6mAb and its characterization for recognition of RapGEF1. Mice were immunized with recombinant protein having only the Crk binding region of RapGEF1 and hybridoma clones created by fusion of immunized spleen cells with Sp2/0 myeloma cells. This antibody recognizes human, primate and murine RapGEF1 protein. Based on the recognition of various deletion constructs, we have mapped its epitope to 580-648 amino acids. Isotyping showed that it belongs to IgG1 class of heavy chain and Kappa light chain. 3F6mAb is suitable for detecting cellular RapGEF1 by western-blotting, immunofluorescence and immunoprecipitation. It has an advantage over most of the commercially available antibodies as it can detect N- and C-terminal truncated forms of RapGEF1. Using this antibody to detect mobility shift, we show that RapGEF1 is phosphorylated on tyrosine as well as S/T residues in its Crk binding domain. This monoclonal antibody is a valuable tool that will aid in understanding functions of cellular RapGEF1.

Keywords: C3G; Crk-binding; Hybridoma; Monoclonal antibody; RapGEF1.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / isolation & purification*
  • Antibodies, Monoclonal / metabolism
  • Blotting, Western
  • Cell Line
  • Epitopes
  • Guanine Nucleotide-Releasing Factor 2 / immunology*
  • Humans
  • Hybridomas
  • Immunoprecipitation
  • Mice
  • Phosphorylation
  • Protein Domains / immunology
  • Protein Engineering / methods
  • Protein Interaction Domains and Motifs / immunology
  • Protein Processing, Post-Translational
  • Recombinant Proteins / immunology
  • Signal Transduction


  • Antibodies, Monoclonal
  • Epitopes
  • Guanine Nucleotide-Releasing Factor 2
  • RAPGEF1 protein, human
  • Recombinant Proteins