title: Preferential recognition of isocitrate dehydrogenase by a rabbit monoclonal antibody (ab124797) against the C-terminal peptide of RANKL.
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.
authors.affiliation: Section of Biochemistry, Department of Hard Tissue Engineering, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan.
authors.forename: Kazue
authors.initials: K
authors.lastname: Terasawa
authors.affiliation: Section of Biochemistry, Department of Hard Tissue Engineering, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan.
authors.forename: Anupama R
authors.initials: AR
authors.lastname: Rajapakshe
authors.affiliation: Section of Biochemistry, Department of Hard Tissue Engineering, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan.
authors.forename: Katarzyna A
authors.initials: KA
authors.lastname: Podyma-Inoue
authors.affiliation: Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
authors.forename: Chiemi
authors.initials: C
authors.lastname: Mishima-Tsumagari
authors.affiliation: Section of Biochemistry, Department of Hard Tissue Engineering, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Yanagishita
authors.affiliation: Section of Biochemistry, Department of Hard Tissue Engineering, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. Electronic address: m.yokoyama.bch@tmd.ac.jp.
authors.forename: Miki
authors.initials: M
authors.lastname: Hara-Yokoyama
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D003429:Cross Reactions; D007521:Isocitrate Dehydrogenase / Q000235:genetics / Q000276:immunology*; D051379:Mice; D017434:Protein Structure, Tertiary; D053245:RANK Ligand / Q000235:genetics / Q000276:immunology*; D011817:Rabbits
chemical_list: D000911:Antibodies, Monoclonal; D053245:RANK Ligand; C505689:Tnfsf11 protein, mouse; D007521:Isocitrate Dehydrogenase
keywords: Isocitrate dehydrogenase; Mitochondria; Peptide; RANKL; Rabbit mAb; ab124797