Antibody specificity is a thorny issue, which has been discussed in depth (e.g. see Ref. 1) and a source of errors and confusion when nonspecific antibodies are used. MYC is a transcription factor exclusively located in the nucleus, involved in gene and metabolism regulation (2), whose abundance is post-transcriptionally regulated. We characterized MYC expression in normal and malignant B cells (3) by validating 13 monoclonal and polyclonal antibodies, in immunohistochemistry, immunofluorescence, and flow cytometry (4). By using biochemically controlled conditions in control– or phorbol myristate–treated U937 cells, we have shown that the only validated antibody that detects MYC by flow cytometry is the mouse mAb C-33 (Fig. 1, reproduced with permission from Ref. 4). Antibodies 9E10 and other antibodies directed at the EQKLISEEDLN sequence fail to detect MYC by flow cytometry; by immunofluorescence on fixed, frozen, and cultured cells; and by immunohistochemistry.
Figure 1.
Validation of MYC antibodies by flow cytometry. A, exponentially growing (control) or TPA growth-arrested U937 cells are stained by flow cytometry with a negative control (gray shadow) or color-coded anti-MYC mouse monoclonal antibodies. C-33 has a bimodal distribution in growing U937 cells, reflecting the S-phase content (bottom DNA ploidy histograms). The other antibodies do not distinguish growing versus growth-arrested cells in this assay. B, the treated and untreated U937 cells of A were fixed, embedded, and stained with an Rb anti-MYC antibody (sc-764), showing positive staining in growing cells only. Northern blotting of the same cells' RNA extract probed for MYC and a loading control is shown at the right. This research was originally published in the Journal of Pathology. Cattoretti, G. MYC expression and distribution in normal mature lymphoid cells. J. Pathol. 2012; 229:430–440. © Wiley.
In countless experiments with MYC-tagged transfectants in proliferating cell lines, 9E10 failed to identify endogenous MYC, not surprisingly.
Im et al. (5) have used 9E10 to detect MYC in induced stem cells by CYTOF. Unfortunately, 9E10 is the only MYC antibody commercially available as a metal conjugate for mass or image cytometry. But this peculiar format does not change the fact that 9E10 does not detect MYC in any of the single-cell applications. Data produced with this and other unvalid MYC antibodies may represent artifacts.
Footnotes
The author declares that he has no conflicts of interest with the contents of this article.
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