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Fusion tags

Related

  • TAP and Tag Cleavage
    • Affinity Tags
      • Epitope Tags
        • Fluorescent Tags
          • Detection and Applications
            • His-tags
              • HA Tags

                ​Fusion tags are known proteins or peptides that are fused onto your protein of interest.  

                Download your in-depth PDF guide to fusion tags. 

                From determining the best application to identifying common issues, our guide will help you make the most of your bench time.


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


                ​


                Sections in our guide:


                Introduction to fusion tagsExplore what fusion tags are
                Tandem affinity purification and tag cleavageDiscover how to use multiple tags and to remove tags after detection
                Affinity tagsPurifying a protein? Visit this page.
                Epitope tagsOften used for protein localization and expression
                Fluorescent tagsUseful if you need to use tags in live cells
                Detecting your protein and applicationsFusion tags can be used in many applications. Find out how they can enhance your research.


                Introduction to fusion tags

                • Introduction
                • Why should you consider fusion tags?
                • Demystifying tags: epitope, affinity and fluorescent tags
                • C vs N: which terminal?
                • Next steps
                • FAQs
                • Fusion tag antibodies


                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 [Figure 1].

                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 properties1 .


                Introduction
                Why should you consider fusion tags?
                Demystifying tags: epitope, affinity and fluorescent tags
                C vs N: which terminal?
                FAQs

                ​​

                ​

                ​​

                ​​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


                Demystifying tags: epitope, affinity and fluorescent tags


                There are three main categories of fusion tags that are used for different applications: 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 2. 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 substrates3.


                Next steps

                If you would like to discover more, see our pages on:

                • Affinity tags
                • Epitope tags
                • Fluorescent tags

                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 detection and applications page.


                FAQs


                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?

                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. 


                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.


                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.


                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.


                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.


                Fusion tag antibodies


                Affinity tags

                TagSequence/Sequence length

                His


                HHHHHH

                CBPKRRNKKNFIAVSAA
                GST211 amino acids
                Strep-tagWSHPQFEK or AWAHPQPGG


                Epitope tags

                TagSequence/Sequence length

                c-myc

                EQKLISEEDL
                HAYPYDVPDYA
                DDDK, or Flag®  (Sigma)DYKDDDK
                T7MASMTGGQQMG
                V5GKPIPNPLLGLDST
                VSV-GYTDIEMNRLGK
                Glu-GluEYMPME or EFMPME
                S-tagKETAAAKFERQHMDS


                Fluorescent tags

                TagSequence/Sequence length

                GFP

                220 amino acids
                mCherry236 amino acids



                References

                1. Chen, X., Zaro, J. & Shen, W. Fusion protein linkers: Property, design and functionality. Advanced Drug Delivery Reviews 65, 1357-1369 (2013)
                2. Palmer, E. & Freeman, T. Investigation Into the use of C- and N-terminal GFP Fusion Proteins for Subcellular Localization Studies Using Reverse Transfection Microarrays. Comparative and Functional Genomics 5, 342-353 (2004).
                3. Snapp, E. Fluorescent proteins: a cell biologist's user guide. Trends in Cell Biology 19, 649-655 (2009).
                4. Costa, S., Almeida, A., Castro, A. & Domingues, L. Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system. Frontiers in Microbiology 5, (2014)
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