Fusion Tags

See how fusion tags can aid you in highlighting your protein of interest.

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Fusion tags guide

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Quick check: use our decision tree to determine which tag you need

Introduction

There are many reasons to highlight a protein of interest: perhaps you want to observe its cellular localization or purify it for crystallization. Although there are commercially available antibodies for many proteins, sometimes it can be difficult to create a specific antibody against a target. To overcome these problems, scientists have developed an extensive molecular toolbox of fusion tags.

A fusion tag is a known protein or peptide that is fused onto your protein of interest. As these tags are well characterized there is a wide range of top-performing antibodies available, enabling easy detection of a specific protein for a variety of applications. Attaching the known sequence to your protein is most commonly achieved by using recombinant DNA, where the DNA of your protein of interest is incorporated into a plasmid containing the fusion tag sequence. When this plasmid is expressed, the fusion tag will be attached to the protein

Apart from which tag to use, the linker sequence should also be considered carefully. The linker sequence joins your protein to the tag and is important to ensure correct protein folding and function. Linker sequences can be rigid, flexible or cleavable and, like fusions tags, each sequence has distinctive perspective¹

Figure 1: First, insert both the DNA of your protein of interest and fusion tag into a plasmid. This is then transcribed and translated to create your protein of interest attached to the fusion tag.

Why should you consider fusion tags?

Advantages

Can isolate a protein of interest without a specific antibody
Cleavage of the tag after purification is sometimes possible
Multiple tags can be attached to the same protein, which increases versatility
Avoids antibody interference in immunoprecipitation
Fluorescent tags can be used to visualize proteins in live cells
Multiple tags are available to suit individual applications

Disadvantages

Some tags can affect the functionality of the protein
Optimal tag placement can take several attempts, leading to increased experimental cost

We aim to guide you through potential strengths and limitations of the most commonly used tags, but, unfortunately, there is no definitive answer for which tag or fusion location will be best for your experiment. We recommend using this guide to choose a starting point depending on your target and application. Watch out for our Top Tips, which will provide insight into frequent stumbling blocks.

Ask yourself key questions

What is the purpose of using a fusion tag, for example, is it to increase solubility or to use in affinity purification?
Will the size of your tag affect your intended application?
Do you need a high yield of protein? Larger tags can be better for structural studies, while smaller tags are better for physiological interactions.
Is there a preference for the tag location?
Will your application tolerate denaturing reagents?

If you do not achieve optimal results at first, try varying the location of your tag, change or add a protease site, modify the linker sequence, or attach multiple tags to take advantage of the differing properties. Additionally, novel tags are constantly being developed. These new tags are sometimes established for a specific purpose or they aim to build upon specific properties of existing tags.

This guide aims to show you how versatile fusion tags can be and how they can help you achieve your mission faster.

Next steps

If you are considering utilizing more than one application or need to remove your tag, read more about tandem affinity purification and tag cleavage.
If you would like to learn more about how to detect your tag, visit our d etection and applications page.

FAQs

1. Is there a specific tag I should be using?
Unfortunately, there is no definitive answer for which tag or fusion location will be best for your experiment. We recommend using the information in our guide to choose a starting point depending on your target and application4.

Ask yourself key questions:

What is the purpose of using a fusion tag, for example, is it to increase solubility or to use in affinity purification?
Will the size of your tag affect your intended application?
Do you need a high yield of protein? Larger tags can be better for structural studies, while smaller tags are better for physiological interactions.
Is there a preference for the tag location?
Will your application tolerate denaturing reagents?

2. I need to view my protein in live cells. Which tag should I use?
If you want to view your protein in live cells, use fluorescent tags. There are a wide variety of fluorescent tags available, which enables the detection of multiple proteins if desired. View our page on fluorescent tags to find out more.

3. I want to see where my protein is localized. Which tag should I use?
High-quality primary antibodies are available against many tags but epitope tags are often used. Read our page on epitope tags to find out more.

4. What tag is best for purification?
Although many tags are available for purification, affinity tags are most commonly used. Read our page on affinity tags to find out which one best suits your application.

5. What should I do if my tag is not working?
If you do not achieve optimal results at first, try varying the location of your tag, change or add a protease site, modify the linker sequence or attach multiple tags to take advantage of each tag’s properties. Additionally, novel tags are constantly being developed. These new tags are sometimes established for a specific purpose or they aim to build upon specific properties of existing tags.

6. Where can I find the molecular weight of common tags?
You can compare the molecular weights of common affinity, epitope and fluorescent tags using our graph.