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Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay
Published on: November 11, 2018
Evaluation of the selectivity and sensitivity of isoform- and mutation-specific RAS antibodies
Andrew M Waters1,2, Irem Ozkan-Dagliyan3, Angelina V Vaseva1,4
1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
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.
Insights
Reliable antibodies are crucial for cancer research on RAS proteins. This study validated 22 anti-RAS antibodies, finding significant variability in their performance for detecting RAS isoforms and mutants.
Area of Science:
- Oncology
- Molecular Biology
- Immunology
Background:
- RAS proteins are key oncogenic drivers in numerous cancers.
- Targeting RAS offers a promising therapeutic strategy.
- Understanding RAS isoform-specific functions is vital for effective cancer therapy.
Purpose of the Study:
- To evaluate the specificity and selectivity of commercially available anti-RAS antibodies.
- To identify reliable antibodies for detecting RAS isoforms and mutants.
- To facilitate accurate interpretation of experimental data in RAS-related cancer research.
Main Methods:
- Screening of 22 commercial anti-RAS antibodies using distinct reagents and cell lines.
- Assessment of antibody specificity and selectivity for RAS isoforms and mutants.
- Validation across different techniques including Western blotting, immunofluorescence, and immunohistochemistry.
Main Results:
- Substantial variability in antibody reliability was observed.
- Some antibodies failed to recognize intended targets or showed cross-reactivity.
- No antibodies were validated for immunofluorescence or immunohistochemical analyses, despite some utility in Western blotting for specific mutants.
- Non-specific binding was detected in some cases, including in cells lacking RAS expression.
Conclusions:
- The validation of anti-RAS antibodies is critical for reproducible cancer research.
- Current antibody availability presents challenges for accurate RAS isoform and mutant detection.
- Validated antibodies are essential tools for advancing therapeutic strategies against RAS-driven cancers.
