Preferential recognition of isocitrate dehydrogenase by a rabbit monoclonal antibody (ab124797) against the C-terminal peptide of RANKL

J Immunol Methods. 2015 May;420:1-10. doi: 10.1016/j.jim.2015.03.006. Epub 2015 Mar 13.

Abstract

A rabbit monoclonal antibody (Abcam ab124797), with high affinity for a synthetic peptide corresponding to the C-terminal region of the receptor activator of nuclear factor (NF)-κB ligand (RANKL), specifically recognizes a 37 kDa protein by immunoblotting, in good agreement with the molecular mass of RANKL. However, our mass spectroscopy analysis revealed that the protein recognized by the antibody is the α-subunit of NAD(+)-dependent isocitrate dehydrogenase (ICDH), a key Krebs cycle enzyme in mitochondria. Consistently, immunocytochemical staining with the antibody revealed a network organization characteristic of mitochondria, which overlapped with staining by MitoTracker and was lost after the siRNA-mediated downregulation of ICDH. The C-terminal peptide of ICDH contains similar chemical characteristics to that of the RANKL peptide and interacts with the antibody, although the affinity is a hundred times weaker. The present study provides an example of the preferential recognition of a surrogate protein by a rabbit monoclonal antibody.

Keywords: Isocitrate dehydrogenase; Mitochondria; Peptide; RANKL; Rabbit mAb; ab124797.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology*
  • Antibody Specificity*
  • Cross Reactions
  • Isocitrate Dehydrogenase / genetics
  • Isocitrate Dehydrogenase / immunology*
  • Mice
  • Protein Structure, Tertiary
  • RANK Ligand / genetics
  • RANK Ligand / immunology*
  • Rabbits

Substances

  • Antibodies, Monoclonal
  • RANK Ligand
  • Tnfsf11 protein, mouse
  • Isocitrate Dehydrogenase