Epitope tags

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

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• 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.
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​​​_{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.} 

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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. 

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.