A quantitative comparison of antibodies against epitope tags for immunofluorescence detection

Abstract

Epitope tags recognized by specific antibodies have been widely used over the last few decades, notably to localize tagged proteins within cells by immunofluorescence. The diversity of tags and antibodies usually prevents a side‐by‐side comparison of the efficiency with which each antibody recognizes its cognate tag. We expressed chimeric proteins, each composed of an invariant domain (IL2Ra) associated with a specific epitope tag. Double immunofluorescence allowed us to quantify in parallel the reference signal generated by the anti‐IL2Ra antibody and the signal generated by the anti‐epitope tag antibody. Since all antibodies used in this study were recombinant antibodies fused to the same mouse Fc domain, the generated signals were directly comparable. Three groups of tags/antibodies were revealed: ‘good’ antibodies generated high signals even when used at a low concentration (50 ng·mL−1), ‘fair’ antibodies generated a high signal only at high concentrations (5000 ng·mL−1), and ‘mediocre’ antibodies generated positive but weak signals. Except for an anti‐myc antibody, similar results were obtained when cells were fixed in paraformaldehyde or methanol. These results provide a side‐by‐side quantitative evaluation of different tag/antibody pairs. This information will be useful to optimize the choice of epitope tags and to choose optimal antibodies.

Keywords

• 6xHis
• ABCD database
• Epitope tags
• FLAG
• HA
• Myc