The development of the Myc tag stemmed from the need for a small, recognizable epitope that could be fused to proteins, enabling researchers to detect and purify these target proteins using anti-Myc antibodies.
This article mainly introduces the Myc tag, including its definition, functions, applications, and comparison with other epitope tags.
A Myc tag, also called c-Myc-tag, is an epitope tag coded by the gene fragment corresponding to the C-terminal amino acids Glu410-Leu419 of the human c-Myc protein [1]. Myc tag DNA sequence is EQKLISEEDL (or -Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu-). Its molecular weight is about 1.202 kDa. A Myc tag can be co-expressed with a protein at either its C- or N-terminus using recombinant DNA technology.
Although the Myc tag can be fused to either the C-terminus or N-terminus of a protein, it is not advisable to place it directly behind the signal peptide of a secretory protein, as it can disrupt translocation into the secretory pathway.
The properties that small size (little interference with the protein's native structure and function), high specificity for anti-Myc antibodies, as well as versatility in various organisms and expression systems of Myc tag make it an important tool in protein detection, purification, and localization, as well as protein-protein interaction studies and protein function.
Figure 1. Myc antibody detects Myc-tagged protein through WB
Myc tag’s functions support a wide array of applications in molecular biology and biochemistry, facilitating protein research by providing reliable tools for identifying, visualizing, isolating, and studying proteins and their interactions [2].
Applications | Description |
---|---|
Western Blotting | Detects and quantifies Myc-tagged proteins using specific anti-Myc antibodies, providing information on protein expression levels and size. |
Immunofluorescence | Visualizes the localization of Myc-tagged proteins within cells using fluorescently labeled anti-Myc antibodies, aiding in subcellular localization studies [2,3]. |
Affinity Chromatography | Purifies Myc-tagged proteins from cell lysates or other mixtures using anti-Myc antibody columns, allowing for efficient and selective protein isolation and purification. |
Co-Immunoprecipitation (Co-IP) | Studies protein-protein interactions by immunoprecipitating Myc-tagged proteins along with their binding partners using anti-Myc antibodies [2,3]. |
Pull-Down Assays | Identifies and analyzes proteins that interact with Myc-tagged proteins, facilitating the study of protein binding partners and interaction networks. |
Enzyme-Linked Immunosorbent Assay (ELISA) | Quantifies Myc-tagged proteins in various samples using anti-Myc antibodies, providing a sensitive method for protein quantification. |
Reporter Assays | Examines gene expression regulation by using Myc-tagged transcription factors or other regulatory proteins, enabling the study of their impact on reporter gene activity. |
Functional Studies | Assesses the functional effects of overexpression or knockdown in various experimental systems; studies the effects of specific mutations on protein function, stability, and interactions in mutational analyses by tagging mutant versions of a protein with the Myc tag. |
Dual Tagging Strategies | Combines Myc tag with other tags (e.g., His, FLAG) for dual detection and purification, enhancing experimental flexibility and validation. |
Note: The elution of Myc-tagged recombinant protein needs low pH conditions, which can affect protein stability and function. So Myc tag is often used for detection and less for purification.
In addition to Myc tag, there are many other epitope tags, including FLAG, hemagglutinin (HA), 6*His, GST, and V5 tags. The following table shows their comparison.
Epitope Tag | Amino Acid Sequence | Molecular Weight | Advantages | Disadvantages | Common Applications |
---|---|---|---|---|---|
Myc | EQKLISEEDL | ~1.202 kDa | - Small size - Highly specific antibodies - Minimal interference with protein function |
- Moderate detection sensitivity compared to some tags | - Protein detection (WB, immunofluorescence) - Protein purification - Protein localization - Protein-protein interaction studies |
FLAG | DYKDDDDK | ~1.01 kDa | - Small size - High affinity antibodies - Suitable for tandem tagging - Contains an enterokinase cleavage site |
- May affect protein folding/function in some cases | - Protein detection - Affinity purification - Co-immunoprecipitation - Protein crystallization - Immunoblotting |
HA | YPYDVPDYA | ~1.13 kDa | - Small size - Highly specific antibodies - Well-characterized |
- Slightly less sensitive than FLAG or His tags | - Protein detection - Immunoprecipitation - Protein-protein interaction studies |
6*His | HHHHHH | ~0.84 kDa | - Small size - Can be used for metal affinity chromatography (IMAC) - Highly efficient purification |
- May require denaturing conditions for elution - Can affect protein solubility |
- Protein purification - Protein detection - Immobilized metal affinity chromatography (IMAC) |
GST | ~220 amino acids | ~26 kDa | - Enhances solubility - Allows for affinity purification - Can improve protein expression |
- Large size may affect protein function - Requires cleavage for removal |
- Protein detection - Affinity purification - Protein-protein interaction studies - Increasing protein solubility |
V5 | GKPIPNPLLGLDST | ~1.40 kDa | - Small size - Specific antibodies available - Rarely affects protein function |
- May show cross-reactivity in mammalian systems - Detection sensitivity varies |
- Protein detection - Immunoprecipitation - Protein-protein interaction studies |
In short, the Myc tag is highly effective for protein detection and localization due to its small size and the availability of specific antibodies. His and GST tags are generally preferred for protein purification due to their higher binding affinities and more efficient elution conditions. Each tag has its unique advantages and is chosen based on the specific requirements of the experiment.
CUSABIO provides many products that aid in Myc-associated research, including Myc-tagged recombinant proteins and Myc tag-specific antibodies.
Myc tag is a small peptide tag that can be fused to proteins for their recognition and detection through Myc antibodies. Myc tag has been successfully applied in many biological assays, including WB, immunoprecipitation (IP), immunofluorescence, pull-down, ELISA, and flow cytometry.
References
[1] Evan GI, Lewis GK, Ramsay G, Bishop JM (1985). Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product [J]. Molecular and Cellular Biology. 5 (12): 3610–6.
[2] Hilpert K, Hansen G, et al. Anti-c-myc antibody 9E10: epitope key positions and variability characterized using peptide spot synthesis on cellulose [J]. Protein Eng. 2001 Oct;14(10):803-6.
[3] Fan H, Villegas C, Chan AK, Wright JA. Myc-epitope tagged proteins detected with the 9E10 antibody in immunofluorescence and immunoprecipitation assays but not in western blot analysis [J]. Biochem Cell Biol. 1998;76(1):125-8.
Comments
Leave a Comment