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Applied biochemistry and biotechnology

Domain-Specific Monoclonal Antibodies Against Human Rev-erbβ.


PMID 27987190

Abstract

The nuclear receptor Rev-erbβ is a potent transcriptional factor whose functional study has been limited by the lack of suitable antibodies against it. To better understand Rev-erbβ's biological roles, we generated five hybridoma cell lines secreting antibodies against human Rev-erbβ in mice immunized with the purified, prokaryotically expressed recombinant Rev-erbβ-6His fusion protein. Using Western blotting and immunofluorescence analyses, all the five monoclonal antibodies (MAbs) showed strong immunoreactivity to both prokaryotically and eukaryotically expressed recombinant Rev-erbβ. An immunoprecipitation study showed that all five monoclonal antibodies against Rev-erbβ were able to pull down the recombinant Rev-erbβ-Flag protein, but only one of the MAbs against Rev-erbβ, 37H8, could pull down the endogenous Rev-erbβ protein. Furthermore, domain specificity of these MAbs was characterized. Due to the high similarities between Rev-erbα and Rev-erbβ in the C and E domains, those C and E domain-specific anti-Rev-erbβ antibodies can react with human Rev-erbα as well. The MAbs produced in the study will provide a valuable tool for investigating the function of Rev-erbβ.