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Science Signaling: Evaluation of the selectivity and sensitivity of isoform- and mutation-specific RAS antibodies

Published On: September 26, 2017
Shared by Brian Phillips

Andrew M. Waters¹,², Irem Ozkan-Dagliyan³, Angelina V. Vaseva¹,⁴, View ORCID ProfileNicole Fer², Leslie A. Strathern², View ORCID ProfileG. Aaron Hobbs¹, View ORCID ProfileBasile Tessier-Cloutier⁵, View ORCID ProfileWilliam K. Gillette², View ORCID ProfileRachel Bagni², View ORCID ProfileGordon R. Whiteley², James L. Hartley², Frank McCormick²,⁶, Adrienne D. Cox¹,³,⁷, Peter J. Houghton⁴, David G. Huntsman⁵, View ORCID ProfileMark R. Philips⁸, and View ORCID ProfileChanning J. Der¹,³,*

Abstract

There is intense interest in developing therapeutic strategies for RAS proteins, the most frequently mutated oncoprotein family in cancer. The development of effective anti-RAS therapies will be aided by the greater appreciation of RAS isoform-specific differences in signaling events that support neoplastic cell growth. However, critical issues that require resolution to facilitate the success of these efforts remain. In particular, the use of well-validated anti-RAS antibodies is essential for accurate interpretation of experimental data. We evaluated 22 commercially available anti-RAS antibodies with a set of distinct reagents and cell lines for their specificity and selectivity in recognizing the intended RAS isoforms and mutants. Reliability varied substantially. For example, we found that some pan- or isoform-selective anti-RAS antibodies did not adequately recognize their intended target or showed greater selectivity for another; some were valid for detecting G12D and G12V mutant RAS proteins in Western blotting, but none were valid for immunofluorescence or immunohistochemical analyses, and some antibodies recognized nonspecific bands in lysates from “Rasless” cells expressing the oncoprotein BRAF^V600E. Using our validated antibodies, we identified RAS isoform-specific siRNAs and shRNAs. Our results may help to ensure the accurate interpretation of future RAS studies.

Article Categories: All News, Research Paper

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