Evaluation of the selectivity and sensitivity of isoform-And mutation-specific RAS antibodies

Andrew M. Waters, Irem Ozkan-Dagliyan, Angelina V. Vaseva, Nicole Fer, Leslie A. Strathern, G. Aaron Hobbs, Basile Tessier-Cloutier, William K. Gillette, Rachel Bagni, Gordon R. Whiteley, James L. Hartley, Frank Mccormick, Adrienne D. Cox, Peter J. Houghton, David G. Huntsman, Mark R. Philips, Channing J. Der

Research output: Contribution to journalArticlepeer-review

22 Scopus citations

Abstract

There is intense interest in developing therapeutic strategies for RAS proteins, themost frequentlymutated oncoprotein family in cancer. 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 BRAFV600E. 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.

Original languageEnglish (US)
Article numbereaao3332
JournalScience signaling
Volume10
Issue number498
DOIs
StatePublished - Sep 26 2017

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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