Research ResourceTechniques

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

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Sci. Signal.  26 Sep 2017:
Vol. 10, Issue 498, eaao3332
DOI: 10.1126/scisignal.aao3332

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A resource on RAS antibodies

Researchers rely largely on antibodies to measure the abundance, activity, and localization of a protein, information that provides critical insight into both normal and pathological cellular functions. However, antibodies are not always reliable or universally valid for the methods in which they are used; in particular, the reliability of commercial antibodies against RAS is highly variable. Waters et al. rigorously assessed 22 commercially available RAS antibodies for their utility to detect the distinct RAS isoforms in various cell types and for their use in specific analytical methods. Their findings show how reliably one can interpret the data acquired from each reagent.

Abstract

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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