Differences between epitope, affinity and fluorescent tags

Demystifying tags: epitope, affinity, and fluorescent tags

There are three main categories of fusion tags, each used for a different application: epitope tags, affinity tags, and fluorescent tags.

• Epitope tags tend to be short peptide sequences that can be used for immunological applications, such as western blot and co-immunoprecipitation.
• Affinity tags are generally longer and are used for protein purification or increasing protein solubility.
• Fluorescent tags can be used in both live and dead cells and are largely used for imaging studies, such as cellular localization and co-expression experiments.

C vs N: which terminal?

Whether you choose to fuse your tag to the C or N terminal of your protein of interest largely depends on the protein itself: how it folds and whether the terminus you choose has a functional requirement or not. For example, if the C-terminal is folded inside the protein, you’re unlikely to receive any fusion protein signal; or if your protein is post-translationally cleaved at the terminal your tag is fused to, then your tag will be removed from your protein of interest.

If you have the resources or your experiment is novel, it might be best to clone both C- and N-terminally tagged constructs to determine the best option. One research group found that more C-terminal fusion proteins localize to the intended subcellular compartment than N-terminally tagged fusion proteins. However, it is important to stress that while C-terminally tagged proteins tend to localize and behave as expected, this is not always possible to predict. Immunofluorescence can be used to check that the fusion protein localizes correctly; an immunoblot will help to confirm the fusion protein is the correct size and expressed at the expected levels, and co-immunoprecipitation can help to assess how the fusion protein interacts with known substrates.

References

1. Palmer, E. & Freeman, T. Investigation into the use of C- and N-terminal GFP fusion proteins for subcellular localization studies using reverse transfection microarrays.  Comp. Funct. Genomics  5, 342–353 (2004).

2. Snapp, E. Fluorescent proteins: a cell biologist's user guide.  Trends Cell Biol.  19, 649–655 (2009).

3. Chen, X., Zaro, J. & Shen, W. Fusion protein linkers: property, design and functionality.  Adv. Drug Deliv. Rev.  65, 1357–1369 (2013).