S. binding of these antibodies was also perturbed or eliminated when the C terminus was phosphorylated. The results should be eye-opening to casual users of a commercial PP2A C activity assaypublished in dozens of studiesthat deploys one of these monoclonals (1D6) for the first-step immunoprecipitation. More robust clones were verified for immunoblottingnotably, clone 52F8 raised having a peptide slightly upstream of the C-terminus (Fig. 1, ?,CC and ?andD)butD)but none were suitable for PP2A holoenzyme immunoprecipitation. Global PP2A C activity assays of endogenous complexes await better affinity reagents; in the meantime, bulk assays against specific PP2A substrates may be an acceptable substitute for some applications (8). A highly-appreciated quality of these latest papers (2, 3) is the systematic, comparative assessment of commercial and in-house antibodies in the same category. Side-by-side comparisons are the norm for other types of study reagents, such as fluorescent proteins, optogenetic constructs, and tissue-clearing solutions. By contrast, some commercial antibody suppliers are more willing to validate and advertise than to vet their products against the competition, making the evaluation incumbent on investigators. One hopes the Ionomycin publications here will emphasize how important such work is definitely to the broader medical community. The studies will also be refreshingly forthright. In one instance, the authors fresh monoclonal is superior to competing alternatives (2). In the additional, an Ogris-grade monoclonal suffers from the same epitope fragility as those commercially available (3). The results emphasize the annoying combination of best practices and fortune that goes into obtaining a good monoclonal. Together, the two publications Ionomycin of Schchner em et al /em . (2) and Frohner em et al /em . (3) remind that the definition of Ionomycin epitope is usually nebulous. Without detailed structural information about how a monoclonal antibody recognizes its target (9), we cannot know which features of an antigen are critical for the epitope and which are not. A case in point is the 9E10 monoclonal, which binds to the prolonged Myc sequence (Fig. 1A) in an asymmetric 2:1 stoichiometry (10). Hybridoma clones that create research-grade antibodies are stochastic winners in a process of recombination, hypermutation, and selection that we try to control but do not fully understand. Thus, insights can only arise from accidental discoveries (2) and educated guesses (3) about epitope fragility. The Cspg4 information in these papers should be circulated widely to avoid perpetuating unintended errors of the past. Acknowledgments I say thanks to Cheryl Borgman for critiquing this manuscript. Funding: K.A.J. is definitely supported from the NIH (R01-CA214718, U01-CA215794, R01-CA194470) and the David and Ionomycin Lucile Packard Basis (2009-34710). Footnotes Competing interests: The author declares that he has no competing financial interests. References and Notes 1. Bradbury A, Pluckthun A, Reproducibility: Standardize antibodies used in study. Nature 518, 27C29 (2015); published on-line EpubFeb 5 (10.1038/518027a). [PubMed] Ionomycin [Google Scholar] 2. Schchner S, Behm C, Mudrak I, Ogris E, The 9E10 Myc tag monoclonal antibody displays highly variable epitope acknowledgement dependent on neighboring sequence context. Sci Transmission 13, eaax9730 (2019). [PubMed] [Google Scholar] 3. Frohner IE, Mudrak I, Kronlachner S, Schchner S, Ogris E, Antibodies realizing the carboxy-terminus of PP2A catalytic subunit are unsuitable to study PP2A activity and holoenzyme composition. Sci Transmission 13, eaax6490 (2019). [PubMed] [Google Scholar] 4. Evan GI, Lewis GK, Ramsay G,.