Characterization of a high-intensity band that cross-reacts with FLAG-M2 antibodies in immunoblots in a subset of laboratory strains of Saccharomyces cerevisiae

Epitope tags are commonly used for various purposes in research labs. The DYKDDDDK-peptide epitope, trademarked as the FLAG epitope, is a commonly used epitope tag. It is often used for monitoring protein levels and for affinity chromatography. Multiple DYKDDDDK-binding antibodies are available; however, the mouse monoclonal anti-FLAG M2 is widely used due to its commercial availability in several formats. Many laboratory Saccharomyces cerevisiae strains, including the BY4741 strain that was used in multiple systematic deletion and tagging libraries, have a high-intensity band that cross-reacts with the FLAG-M2 antibody. The presence of this high-intensity cross-reactive band can be problematic in some applications. Here, we show that despite high-intensity in immunoblots, the cross-reacting band is not enriched by FLAG-M2 affinity beads under native conditions. We also report the fortuitous identification of a strain closely related to BY4741 that lacks the high-intensity cross-reactive band. Finally, contrary to anecdotal reports, we determined that the high-intensity cross-reacting band is not Rtf1. These findings and resources should assist other researchers using the DYKDDDDK-epitope for immunoblots and affinity chromatography.