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Epitope tags header

Epitope tags

Related

  • Fusion Tags
    • TAP and Tag Cleavage
      • Affinity Tags
        • Fluorescent Tags
          • Detection and Applications
            • Overview of c-myc
              • HA-tags
                • His-tags

                  Epitope tags are widely used in applications such as ELISA, Western blot, and immunoprecipitation.

                  ​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.

                  Contents

                  • What are epitope tags?
                  • c-myc
                  • Human influenza hemagglutinin (HA)
                  • DDDK (FLAG®, Sigma)
                  • V5


                  What are epitope tags?


                  Epitope tags, such as c-myc and HA, are short sequences that are fused onto proteins and are frequently used in antibody-based assays. Epitope tags are generally shorter in length than affinity tags and are, therefore, less likely to affect protein function1.

                  Although they can be used for affinity purification, the columns are based upon immobilized antibodies, which are usually more costly or not as efficient as columns for affinity tags1. However, due to their specificity for their respective primary antibodies, epitope tags are a useful tool for the detection of fusion proteins1.

                  Epitope tags are widely used in cell culture and immunoprecipitation (IP), including protein complex immunoprecipitation (co-IP)2 [Figure 1]. 

                  Discover commonly used epitope tags, recommended applications, and what to watch out for below.
                  ​


                  Introduction to epitope tags
                  c-myc
                  Human influenza hemagglutinin (HA)
                  DDDK (FLAG®, Sigma)
                  V5Introduction to epitope tags
                  c-myc
                  Human influenza hemagglutinin (HA)
                  DDDK (FLAG®, Sigma)
                  V5

                  ​

                  ​​​Figure 1: This diagram shows the steps for co-immunoprecipitation. First lyse your sample to release the proteins. When this lysate is added to the tube, along with antibodies against the fusion tag, the antibody will recognize the fusion tag. The antibodies are then bound to Protein A or G coupled beads, which will pull out your protein of interest, along with any proteins that are complexed with it. 

                  ​​

                  c-myc


                  Molecular Weight: 1.2 kDa

                  Size: 10 amino acids (EQKLISEEDL)

                  Tag location: C- or N- terminals

                  Applications: Western blot, immunoprecipitation, flow cytometry. Can be used in affinity purification.

                  Affinity Resin: divinyl sulphone-activated agarose

                  Strengths: Can be placed at either N- or C-terminal. c-myc tagged proteins can be crystallized successfully.

                  Limitations: Elution for affinity chromatography is at low pH, which could affect fusion protein functionality. 


                  c-myc overview

                  Human c-myc is expressed at low levels in proliferating cells and plays a key role in human oncogenesis. The c-myc tag is derived from the c-terminal of the c-myc gene and it can be effectively recognized by antibodies. As such, it is a widely used tag for the detection of proteins in applications such as western blot, immunoprecipitation, and flow cytometry. Purified c-myc tagged proteins have been successfully crystallized3.


                  c-myc: what to watch out for

                  Fusing to a secretory signal: Although the c-myc tag can be placed at either the C- or N- terminals, it is not recommended to fuse the tag to secretory signals as it can impact translocation to the secretory pathway.  

                  Affinity purification: While the c-myc tag can be used for protein purification, it is rarely used for this purpose. This is due to the low pH required for elution, which could negatively affect protein functionality. 


                  ProductAb ID

                  Anti-Myc tag antibody [9E10] - ChIP Grade


                  ab32

                  Recombinant Anti-Myc tag antibody [9E10]

                  ab206486

                  Anti-Myc tag antibody [9E10] (HRP)

                  ab62928  


                  Human influenza hemagglutinin (HA)


                  Molecular Weight: 1.1 kDa

                  Size: 9 amino acids (YPYDVPDYA)

                  Tag Location: C- or N- terminals

                  Affinity Resin: Anti-HA antibody immobilized onto agarose beads

                  Applications: ELISA, western blot, immunoprecipitation and immunofluorescence, protein purification

                  Strengths: Unlikely to affect protein functionality

                  Limitations: Not recommended for use in apoptotic cells


                  HA overview

                  The human influenza hemagglutinin (HA) tag is derived from the HA glycoprotein, which is found on the surface of influenza viruses and is responsible for the infectivity of the virus. As the HA-tag is a small peptide tag, it rarely affects protein function, which means it is a valuable tool for the detection of proteins via ELISA, western blot, immunoprecipitation, and immunofluorescence. Anti-HA antibodies can also be immobilized on agarose beads for protein purification. 

                  HA: what to watch out for

                  Affinity purification: It is not recommended to use HA-tags for proteins deriving from apoptotic cells. The HA-tag can be cleaved by Caspases 3 and 7, which results in loss of immunoreactivity.


                  ProductAb ID

                  Anti-HA tag antibody - ChIP Grade


                  ab9110

                  Anti-HA tag antibody [HA.C5]

                  ab18181

                  Anti-HA tag antibody (HRP)ab1190
                  Anti-HA Affinity Resin - Amintra®
                  ​
                  ab270603


                  DDDK (FLAG®, Sigma)


                  Molecular Weight: 1.01 kDa

                  Size: 8 amino acids (DYKDDDDK).

                  Tag Location: C- or N- terminals, or internal.

                  Affinity Resin: Immobilized DDDK antibodies.

                  Applications: ELISA, western blot, protein purification, protein crystallization.

                  Strengths: Not likely to affect the functionality of fusion protein. Contains an internal cleavage site.

                  Limitations: Affinity resin is not as stable as others and can be expensive.


                  DDDK overview

                  The DDDK, or FLAG®, tag is the only patented tag (Sigma). It is more hydrophilic than other epitope tags, with the result that it is less likely to affect the functionality of the protein to which it is fused. If necessary, the DDDK tag includes an enterokinase cleavage sequence so it can be easily removed from the protein of interest3. 


                  DDDK: what to watch out for

                  Affinity purification: Although columns comprised of immobilized anti-DDDK antibodies can be effective, they are also more expensive than other types of affinity columns.


                  V5


                  Molecular Weight: 0.95–1.4 kDa

                  Size: 9–14 amino acids (IPNPLLGLD or GKPIPNPLLGLDST)

                  Tag Location: C- or N- terminals

                  Applications: Western blot, ELISA, flow cytometry, protein visualization, ChIP, immunoprecipitation

                  Strengths: Available in two peptide lengths.

                  Limitations: Potential cross-reactivity in mammalian systems.


                  V5 overview

                  The V5 tag is derived from the P and V proteins of the paramyxovirus simian virus 5. There are two sizes of the V5 tag ranging from 9–14 amino acids, although the longer tag is usually used. Using the V5-tag in combination with a His-tag is sometimes recommended.


                  V5: what to watch out for

                  Cross-reactivity: If you are using a mammalian expression system, there is a chance of cross-reactivity.


                  ProductAb ID

                  Anti-V5 tag antibody

                  ab9116
                  Anti-V5 tag antibody [SV5-Pk1]
                  ​
                  ab27671
                  Anti-V5 tag antibody (HRP)ab1325



                  References

                  1. Kimple, M., Brill, A. & Pasker, R. Overview of Affinity Tags for Protein Purification. Current Protocols in Protein Science 73, (2013).
                  2. Brizzard, B. Epitope tagging. BioTechniques 44, 693-695 (2008).
                  3. Terpe, K. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Applied Microbiology and Biotechnology 60, 523-533 (2003).
                  4. Jarvik, J. & Telmer, C. EPITOPE TAGGING. Annual Review of Genetics 32, 601-618 (1998).
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