Do you use an AirTag^®, Tile^®, or other Bluetooth^® tracker to keep tabs on anything? When people use these types of tracking devices, it is usually on something that is small and easy to lose (keys, phone), something that is valuable (computer, luggage), or both.

Fortunately, these types of Bluetooth trackers are specific for the single item that you stick the tracker on. Can you imagine if they weren’t and you had to search through everyone’s trackers to eventually find the specific one that you’re looking for?

There are similar trackers in the molecular world that are used to find and grab proteins. There are different versions of these trackers, but collectively they’re known as “protein tags” or “tags” for short. Tagging your target protein makes it easy to identify and isolate it in experiments and when purifying that protein, and it can also modify the properties of your protein in really interesting and useful ways.

Protein tags are proteins or peptide sequences that are genetically fused to a target protein to enable the detection and purification of the target protein. Some protein tags even improve the solubility of target proteins, making them easier to work with, or change other properties such as a protein’s localization or degradation in cells.

In this article we’ll discuss common protein tags and how they’re used to facilitate the detection and purification of target proteins.

 

In this article:

Types of protein tags

Small peptide tags

Solubility tags

Visualizing tags

Localization signal tags

Degron tags

Purifying proteins with affinity tags

Experiments using protein tags

Binding experiments

Visualization experiments

References

 

Types of protein tags

Protein tags provide useful handles for grabbing onto or visualizing your protein of interest for purification or experimental purposes. Tags can also change the visual properties of proteins, including their solubility or cellular localization. So, the type of tag you’ll want to use will depend on what specific purpose you have in mind.

There are lots of different protein tags that have distinct functions. We’ve categorized those tags into the below categories. Keep in mind that some tags will fit into more than one category.

·         Small peptide tags

·         Solubility tags

·         Visualizing tags

·         Localization tags

·         Degron tags

 

Small peptide tags

Small peptide tags include his-tags, strep-tags, and flag-tags. These are small peptides, approximately 6 - 24 amino acids long, for purifying and detecting the proteins that they are attached to (Figure 1).

Figure 1. His-tags (orange), strep-tags (purple), and flag-tags (green) are all small peptide tags added to proteins for purification or detection.

 

Small peptide tags are popular because they are usually versatile and innocuous. Meaning you can often put them on either the amino (N-) or carboxy (C-) terminus of your target protein. Also, since they’re small, they usually don’t impact a protein’s function or localization. While this is generally true, it is always prudent to confirm that the tag is well tolerated on your protein of interest.

 

Solubility tags

Compared to small peptide tags, solubility tags are relatively larger. They consist of protein domains, or even entire proteins, that are used to improve the solubility of your target protein. Frequently-used examples of solubility tags include GST-, MBP-, and SUMO-tags. It is recommended to put the solubility tag on the N-terminus of your target protein to best improve its solubility.

Solubility tags don’t always have to be kept on the protein of interest throughout purification and experiments. Sometimes using a solubility tag to keep the target protein soluble during expression and initial purification steps is sufficient, and then the solubility tag can be cleaved off and purified away.

However, it is worth keeping the solubility tag for some experiments such as GST-pulldowns which leverage the molecular interaction between GST and glutathione to investigate which proteins or other molecules that your protein of interest binds to (Figure 2).

Figure 2. Adding a GST tag (purple) to your protein of interest (dark blue) enables GST pulldowns to discover novel interacting partners (orange, green, and light blue).

 

Visualizing tags

Visualizing tags come in a couple of different options. Many of the small-peptide tags that we already discussed can be used indirectly for visualization by using another molecule that recognizes the tag. For example, a labeled antibody that recognizes His-tags (Figure 3).

Figure 3. His-tagged proteins can be visualized using a fluorophore-labeled antibody.

 

GoldBio offers antibody labeling kits that quickly and reliably add biotin or a wide variety of fluorophores to your favorite antibody. These are excellent tools if you want to generate a labeled antibody to recognize a tag.

Alternatively, there are protein tags that fluoresce themselves, or readily conjugate to small-molecule fluorophores. Green fluorescent protein (GFP) and all of its colorful variants are examples of protein tags that intrinsically fluoresce (Nienhaus & Nienhaus, 2021). Alternatively, SNAP tags readily conjugate a small molecule fluorophore for labeling (Porzberg et al, 2025). So, either GFP or SNAP tags are good choices to tag a protein with for visualization.

 

Localization signal tags

So far, we’ve been talking about tags that we engineer into proteins to give them particular properties that we desire. But the next couple of tags are actually sequences that are already present in many natural proteins.

Localization tags are included in many natural proteins that tell the protein what cellular compartment it should go. This is like a shipping label that is used to show where a package should be sent to.

For example, there are nuclear import signals for proteins to be sent to the nucleus, and nuclear export tags for proteins that need to go to the cytoplasm. There are also localization tags for organelles such as mitochondria and endoplasmic reticulum (Neji et al, 2015).

While these signals are naturally encoded in proteins, scientists can rewire these tags in order to study how cellular location impacts a protein’s function. For example, if a protein naturally cycles back and forth between the cytoplasm and nucleus, a scientist could scramble either the nuclear import or export sequence to see how trapping the protein in the cytoplasm or nucleus, respectively, impacts its function (Figure 4).

Figure 4. Some proteins (green oval) naturally have nuclear import (blue star) and export (purple triangle) tags. When the protein has both tags, it will cycle back and forth between the nucleus and cytoplasm (left). If one of the tags is removed the protein will stay in the cytoplasm (middle) or nucleus (right) only.

 

Degron tags

The last kind of tag that we’ll discuss is a degron tag. These tags are like marking that a protein should be sent to the cellular garbage can and get broken down. Degrons are bound by proteins called E3 ligases that add a chain of ubiquitin proteins to the original target protein. Then, the “ubiquitinated” target protein is sent to the proteasome, a protein complex that will degrade the target protein into small peptides (Micel et al, 2013).

Just like cellular localization tags, degron tags are present in many natural proteins, and can also be rewired so that scientists can modify the abundance of a protein in cells or even entire organisms (Hernández-Morán et al, 2024). For example, adding an extra degron tag would deplete that protein, whereas scrambling an existing degron tag would allow the protein to build up (Figure 5).

Figure 5. Many natural proteins have a degron tag (left), and you can modify their cellular abundance by deleting that tag (middle) or adding an extra degron tag (right).

 

Localization tags and degron tags are critical elements that contribute to proper protein function. It is no surprise, then, that mutations in these tags which alter their function are frequently found in various human diseases.

A recent study investigated over 3,000 protein mutations found in several diseases including rare heritable diseases and many different types of cancer. They found that approximately one-sixth of these disease-causing mutations resulted in mislocalization of the protein that the mutation resides in (Lacoste et al, 2024). Their results suggest that protein mislocalization contributes to a substantial fraction of human diseases. 

Drastic changes in the abundance of specific proteins are also responsible for a variety of diseases. For example, mutations that hide the degron in oncoproteins result in a buildup of these proteins, which help them drive cancer formation and progression (Micel et al, 2013; Vitari et al, 2011).

These are just a couple of examples of how naturally-occurring protein tags contribute the appropriate localization and abundance of proteins, and how disease can result when these processes go haywire.

 

Purifying proteins with affinity tags

Purified proteins are powerhouse reagents in molecular biology and biochemical experiments (a few of which we cover in the next section). Affinity tags are really useful tools to enable protein purification.

His-tags, for example, bind to nickel ions, so nickel agarose beads are used to purify his-tagged proteins (Figure 6).

 

Figure 6. Nickel agarose beads bind to his-tagged proteins and are used for their purification and in binding experiments.

 

GST stands for Glutathione S-Transferase and is a protein that binds to the small molecule glutathione. So, glutathione-conjugated beads are used for purifying GST-tagged proteins (Figure 7).

Figure 7. Glutathione agarose beads bind to GST-tagged proteins and are used in their purification.

 

 

Experiments using protein tags

Experiments with protein tags usually come in two varieties:

·         Binding to the tag to isolate the protein of interest and interacting molecules

·         Using the tag to visualize the protein of interest

Protein tags aren’t necessarily required for these types of experiments. If you already have a validated antibody for your protein of interest, then you could perform these experiments with that antibody. However, tags provide a bit of a shortcut as the reagents used to perform these experiments with protein tags are already well validated and widely used.

Also, note that localization and degron tags are a bit different in this regard. Scientists usually wouldn’t perform these experiments using these tags, per se, but rather would visualize the protein of interest to see where it localizes or how much of it there is, with and without the localization or degron tag.

 

Binding experiments

Binding experiments use the interaction between a protein tag and another protein or small molecule to isolate the tagged protein of interest and any other molecules that it is interacting with.

GST pulldowns, which we discussed above, are probably the most common form of this type of experiment, but you can perform analogous isolation experiments with other protein tags including his-tags, flag-tags, and strep-tags (Figure 8).

Figure 8. Similar to GST pulldowns, nickel agarose (left), streptavidin agarose (middle), and aFLAG agarose (right), are used to detect interactions with his-tagged, strep-tagged, and flag-tagged target proteins, respectively.

 

Visualization experiments

Tags are also used to visualize target proteins, either in cells or in test tube (in vitro) experiments. These experiments use light generating molecules that either bind to one of the protein tags, such as the fluorophore-labeled antibody in Figure 3. Or the protein tags generate light themselves, like GFP.

Either way, these experiments are really useful and can tell you a lot of different things about your target protein. You can determine where in a cell a protein localizes to. They can determine if two different proteins are found in the same area of a cell. And they can be used to quantitatively determine how many proteins are in your cells or test tube (Currie et al, 2023; Xing et al, 2020).     

 

That’s all about protein tags and how they’re used in research. If you are interested in a particular type of tag, check out our articles below to learn more. Also, if you are ready to work with protein tags in the lab, check out the reliable and affordable research reagents below that will get you on your way to experimental success!

 

References

Currie, S. L., Xing, W., Muhlrad, D., Decker, C. J., Parker, R., & Rosen, M. K. (2023). Quantitative reconstitution of yeast RNA processing bodies. Proceedings of the National Academy of Sciences of the United States of America, 120(14), e2214064120. https://doi.org/10.1073/pnas.2214064120

Hernández-Morán, B. A., Taylor, G., Lorente-Macías, Á., & Wood, A. J. (2024). Degron tagging for rapid protein degradation in mice. Disease models & mechanisms, 17(4), dmm050613. https://doi.org/10.1242/dmm.050613

Lacoste, J., Haghighi, M., Haider, S., Reno, C., Lin, Z. Y., Segal, D., Qian, W. W., Xiong, X., Teelucksingh, T., Miglietta, E., Shafqat-Abbasi, H., Ryder, P. V., Senft, R., Cimini, B. A., Murray, R. R., Nyirakanani, C., Hao, T., McClain, G. G., Roth, F. P., Calderwood, M. A., … Taipale, M. (2024). Pervasive mislocalization of pathogenic coding variants underlying human disorders. Cell, 187(23), 6725–6741.e13. https://doi.org/10.1016/j.cell.2024.09.003

Micel, L. N., Tentler, J. J., Smith, P. G., & Eckhardt, G. S. (2013). Role of ubiquitin ligases and the proteasome in oncogenesis: novel targets for anticancer therapies. Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 31(9), 1231–1238. https://doi.org/10.1200/JCO.2012.44.0958

Nienhaus, K., & Nienhaus, G. U. (2021). Fluorescent proteins of the EosFP clade: intriguing marker tools with multiple photoactivation modes for advanced microscopy. RSC chemical biology, 2(3), 796–814. https://doi.org/10.1039/d1cb00014d

Negi, S., Pandey, S., Srinivasan, S. M., Mohammed, A., & Guda, C. (2015). LocSigDB: a database of protein localization signals. Database : the journal of biological databases and curation, 2015, bav003. https://doi.org/10.1093/database/bav003

Porzberg, N., Gries, K., & Johnsson, K. (2025). Exploiting Covalent Chemical Labeling with Self-Labeling Proteins. Annual review of biochemistry, 94(1), 29–58. https://doi.org/10.1146/annurev-biochem-030222-121016

Vitari, A. C., Leong, K. G., Newton, K., Yee, C., O'Rourke, K., Liu, J., Phu, L., Vij, R., Ferrando, R., Couto, S. S., Mohan, S., Pandita, A., Hongo, J. A., Arnott, D., Wertz, I. E., Gao, W. Q., French, D. M., & Dixit, V. M. (2011). COP1 is a tumour suppressor that causes degradation of ETS transcription factors. Nature, 474(7351), 403–406. https://doi.org/10.1038/nature10005

Xing, W., Muhlrad, D., Parker, R., & Rosen, M. K. (2020). A quantitative inventory of yeast P body proteins reveals principles of composition and specificity. eLife, 9, e56525. https://doi.org/10.7554/eLife.56525

 

 

 

Tags

his-tag Materials-and-Methods protein protein labeling protein purification Protein-Research-Expression-Purification

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