RT Journal Article
SR Electronic
T1 The Myc tag monoclonal antibody 9E10 displays highly variable epitope recognition dependent on neighboring sequence context
JF Science Signaling
JO Sci. Signal.
FD American Association for the Advancement of Science
SP eaax9730
DO 10.1126/scisignal.aax9730
VO 13
IS 616
A1 Schüchner, Stefan
A1 Behm, Christian
A1 Mudrak, Ingrid
A1 Ogris, Egon
YR 2020
UL http://stke.sciencemag.org/content/13/616/eaax9730.abstract
AB Knowing whether a given antibody is specific for its intended target or is sensitive to posttranslational modifications of that target is critical for interpreting experimental data generated with antibody reagents. Two Research Resources from the Ogris group highlight the importance of knowing the capabilities and limitations of antibody reagents. Frohner et al. found that various antibodies raised against the catalytic (C) subunit of protein phosphatase 2A (PP2A) were sensitive to methylation and phosphorylation of PP2A C, cross-reacted with related phosphatases, and failed to immunoprecipitate major subsets of trimeric holoenzymes. This implies that findings using a common commercial phosphatase assay kit that relies on one of these antibodies should be reevaluated. Schüchner et al. found that the recognition of Myc-tagged proteins by a widely used Myc-specific antibody varied depending on sequences adjacent to the tag. A Focus by Janes highlights the importance of systemic validation of research antibodies.Epitope tags are short, linear antibody recognition sequences that enable detection of tagged fusion proteins by antibodies. Epitope tag position and neighboring sequences potentially affect its recognition by antibodies, and such context-dependent differences in tag binding may have a wide-ranging effect on data interpretation. We tested by Western blotting six antibodies that recognize the c-Myc epitope tag, including monoclonal antibodies 9E10, 4A6, 9B11, and 71D10 and polyclonal antibodies 9106 and A-14. All displayed context-dependent differences in their ability to detect N- or C-terminal Myc-tagged proteins. In particular, clone 9E10, the most cited Myc-tag antibody, displayed high context-dependent detection variability, whereas others, notably 4A6 and 9B11, showed much less context sensitivity in their detection of Myc-tagged proteins. The very high context sensitivity of 9E10 was further substantiated by peptide microarray analyses. We conclude that recently developed, purpose-made monoclonal antibodies specific for Myc have much more uniform reactivity in diverse assays and are much less context sensitive than is the legacy antibody 9E10.