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

Science signaling (2017-09-28)
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
RESUMEN

There is intense interest in developing therapeutic strategies for RAS proteins, the most frequently mutated 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.

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