Abstract
Abstract Protein-protein interactions (PPIs) are fundamental to biological processes, from signaling pathways that modulate gene expression, cell proliferation, tumor invasion to cytoskeleton networks that maintain morphological integrity of cells and tissues. Activation of oncogenes and inactivation of tumor suppressor genes in the cell that lead to carcinogenesis are inevitably accompanied by disruption of normal or activation of aberrant PPIs, thus detection and understanding of PPIs may advance the diagnosis and treatment of cancer. Although many methods have been developed to discover and detect PPIs in vivo and ex vivo, few are applicable to in situ detection of PPIs, particularly in formalin-fixed, paraffin-embedded (FFPE) tissue samples. Here we report a sensitive in situ PPI detection method for FFPE tissues that utilizes biotin ligase (BirA), an enzyme that covalently labels its specific peptide target substrate (BTS) with biotin. We developed BirA and BTS conjugated antibodies, each of which binds specifically to one of the interacting protein pair and whose close proximity permits BirA to biotinylate the BTS for subsequent visualization. Using both artificial system such as hapten-labeled proteins and known biological PPI models such as E-cadherin and beta-catenin and HER2/HER3 dimers in the context of FFPE tissues, we have demonstrated that the assay is both sensitive and robust. Moreover, the assay has been fully automated and is easily adaptable for detecting protein dimers, fusion proteins, or other novel binary interactions in tissue specimens. As understanding details of signaling pathways becomes increasingly important in cancer biology, in situ proximity assay may offer novel mechanistic and therapeutic insights and provide a powerful tool for cancer diagnostics. Citation Format: Zeyu (David) Jiang, Rui Hong, Mike Farrell. An in-situ proximity assay using biotin ligase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3493. doi:10.1158/1538-7445.AM2014-3493
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