Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
8417479 | Journal of Immunological Methods | 2015 | 10 Pages |
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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Authors
Kazue Terasawa, Anupama R. Rajapakshe, Katarzyna A. Podyma-Inoue, Chiemi Mishima-Tsumagari, Masaki Yanagishita, Miki Hara-Yokoyama,