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

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

Author information

1
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.
2
Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
3
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.

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

PMID:
25771969
DOI:
10.1016/j.jim.2015.03.006
[Indexed for MEDLINE]

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