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

Journal of immunological methods (2015-03-17)
Kazue Terasawa, Anupama R Rajapakshe, Katarzyna A Podyma-Inoue, Chiemi Mishima-Tsumagari, Masaki Yanagishita, Miki Hara-Yokoyama
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.

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