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Epitope tags are widely used in applications such as ELISA, Western blot, and immunoprecipitation.
From determining the best application to identifying common issues, our guide will help you make the most of your bench time.
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.
Discover commonly used epitope tags, recommended applications, and what to watch out for below.
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.
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.
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.
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.
Anti-Myc tag antibody [9E10] - ChIP Grade
|Recombinant Anti-Myc tag antibody [9E10]|
|Anti-Myc tag antibody [9E10] (HRP)|
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
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.
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.
Anti-HA tag antibody - ChIP Grade
|Anti-HA tag antibody [HA.C5]|
|Anti-HA tag antibody (HRP)||ab1190|
|Anti-HA Affinity Resin - Amintra® |
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.
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.
Affinity purification: Although columns comprised of immobilized anti-DDDK antibodies can be effective, they are also more expensive than other types of affinity columns.
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.
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.
Cross-reactivity: If you are using a mammalian expression system, there is a chance of cross-reactivity.
Anti-V5 tag antibody
|Anti-V5 tag antibody [SV5-Pk1]|
|Anti-V5 tag antibody (HRP)||ab1325|