4.5 Article

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

Journal

SCIENCE SIGNALING
Volume 10, Issue 498, Pages -

Publisher

AMER ASSOC ADVANCEMENT SCIENCE
DOI: 10.1126/scisignal.aao3332

Keywords

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Funding

  1. NIH/NCI [T32 CA009156, F32 CA200313]
  2. Pancreatic Cancer Action Network-American Association for Cancer Research
  3. Department of Defense
  4. Lustgarten Pancreatic Cancer Foundation
  5. NIC/NIH [HHSN261200800001E]
  6. [CA42978]
  7. [CA179193]
  8. [CA175747]
  9. [CA199235]
  10. [CA197709]
  11. [CA165995]

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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 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.

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