What is an Epitope Tag?

Epitope tags such as V5, c-myc, and HA are biological structures, usually proteins, that are genetically engineered onto either the N or C terminus of a recombinant protein. Tags function as universal epitopes, easily detected by commercially available antibodies, and generally do not compromise the native structure or function of the protein.

Epitope Tags

What are epitope tags used for?

Tags and anti-tag antibodies are useful for many different immunoassays such as western blotting, immunoprecipitation, immunohistochemistry, immunocytochemistry, and flow cytometry. If your epitope tag ever loses antigenicity following deparaffinization and rehydration of formalin-fixed tissues in immunohistochemistry, try following our antigen retrieval protocol. Epitope tags are the most economical way to detect newly discovered proteins for which specific antibodies are not yet available or for targets that are poorly immunogenic ¹. Additionally, epitope tags are also useful for affinity purification of target proteins from cell lysates ², enhancing the solubility of target proteins ³ protecting against intracellular protease cleavage (e.g. GST) ⁴, and adding enzymatic function to a target protein ⁵.

Western Blot

V5 Epitope Tag Antibody

V5 Epitope Tag Antibody [NB600-381] - Detection of 200, 100, or 50 ng of E. coli whole cell lysate expressing a multi-tag fusion protein.

Flow Cytometry

V5 Epitope Tag Antibody

V5 Epitope Tag Antibody [MAB8926] - HEK293 human embryonic kidney cell line transfected with V5-tagged proteins.

Immunocytochemistry/Immunofluorescence

HA Epitope Tag Antibody

HA Tag Antibody [NB600-363] - Detection of HA-tagged Tubulin in CHO cells transfected with mutant beta-tubulin cDNA encoding an HA epitope tag.

Immunohistochemistry-Paraffin

c-Myc Epitope Tag Antibody

c-Myc Antibody (9E10) [NB600-302] - c-Myc was detected in immersion fixed paraffin-embedded sections of human breast cancer using anti-human mouse monoclonal antibody

Affinity Purification

Ni-NTA Affinity Purification Column

HA, His, DYKDDDDK, and V5 are commonly used epitope tags for affinity purification.

Choosing Your Epitope Tag

No single epitope tag is useful for every application --- when choosing a tag, consider how the size and charge of the tag will ultimately affect the protein’s function, solubility and downstream analysis. Multiple tags can be added to a single protein to get enhanced functionality, however, this increases the risk of losing native function, compromising the original structure, or altering protein trafficking ^6,7,8.

Epitope Tags	Length (Sequence) (kDa)	Alternate Names	Source	Notes
HA	YPYDVPDYA	Hemagglutinin	Amino acids 98-106 of Human influenza hemagglutin which is a surface glycoprotein required for the infectivity of the human virus	Strong immunoreactive epitope and mild elution conditions makes it a popular epitope tag to purify tagged proteins ¹⁰; useful in mammalian expression systems ⁹; not suitable for detection or purification of proteins from apoptotic cells since it is cleaved by Caspase-3 and / or Caspase-7 after its sequence DVPD, causing it to lose its immunoreactivity ¹¹
HIS	H-H-H-H-H-H	6 His 6X-His HHHHHH tag polyHistidine hexa-histidine tag	Synthetic	Most common purification tag; Purification via Nickel, Cobalt, Copper, or Zinc column (Find Nickel and Cobalt Resin here); Regenerable affinity matrix ⁹
FLAG ^TM	DYKDDDDK	Ty 1 Tag DDK tag DDDDK tag DYKDDDDK tag xxxDDDDK epitope tag Enterokinase Cleavage Site tag ECS tag Ty 1	Synthetic	Hydrophilic epitope tag introduced to purify fusion proteins ¹⁰; Contains internal Enterokinase cleavage site
AU1	DTYRYI		Major capsid protein of bovine papillomavirus-1 (BPV-1)	Not recommended for affinity purification as protein properties may get altered during low pH elution step ⁹
AU5	TDFYLK		Major capsid protein of bovine paillomavirus-1 (BPV-1)	Not recommended for affinity purification as protein properties may get altered during low pH elution step ⁹
Myc	EQKLISEEDL		Amino acid residues 410-419 of Human c-Myc	Not recommended for affinity purification as protein properties may get altered during low pH elution step ⁹
Glu-Glu	EYMPME		Amino acid residues 314-319 of middle T antigen of mouse polyomavirus	Not recommended for affinity purification as protein properties may get altered during low pH or 30°C elution step ⁹
OLLAS	SGFANELGPRLMGK	OLLA-2 E.coli OmpF Linker and mouse Langerin fusion Sequence	14 amino acid sequence residing in the junction between E. coli OmpF protein (OFL/OmpF linker) and mLangerin extracellular domain.	The OLLAS epitope tag is detectable at approximately 100-fold lower levels than similar commercially available tags
T7	MASMTGGQQMG		11 amino acid N-terminus of Bacteriophage T7 gene 10	May help increase expression; Not recommended for affinity purification as protein properties may get altered during low pH elution step ⁹
V5	GKPIPNPLLGLDST	Parainfluenza virus 5 V/P tag Paramyxovirus SV5 tag	Amino acid residues 95 to 108 of RNA polymerase alpha subunit of simion virus 5	Some cross reactivity may occur when using mammalian expression systems; Recommended for affinity purification in combination with His-tag ⁹
VSV-G	YTDIEMNRLGK	Vesicular stomatitis virus glycoprotein tag	11 amino acid C-terminus of the Vesicular Stomatitis viral glycoprotein	Not recommended for affinity purification as protein properties may get altered during low pH elution step ⁹
E-Tag	GAPVPYPDPLEPR		Bone hormone osteocalcin produced by osteoblasts
S-Tag	KETAAAKFERQHMDS	S 7 Epitope tag	15 amino acid sequence at N-terminus of RNase A	Not recommended for affinity purification as protein properties may get altered during low pH elution step ⁹
Avi	CGLNDIFEAQKIEWHE		Synthetic	Allows for either the in-vivo or in-vitro enzymatic biotinylation by the biotin ligase BirA from E. coli; May lead to decreased solubility ¹²
HSV	SQPELAPEDPED		Herpes Simplex Virus tag	C-terminal placement only; Not recommended for affinity purification as protein properties may get altered during low pH elution step ⁹
KT3	KPPTPPPEPET		Simian Virus 40 (SV40) large T-antigen	Not recommended for affinity purification as protein properties may get altered during low pH elution step ⁹
TK15	(R/K)TV(I/L)HGESNSA(I/L)(I/L)(I/L)GPR		Xenopus Orc1p	No cross-reactivity with mammalian or bacterial proteins has been detected; Used for immunoaffinity purification ¹⁶
Strep-tag II	WSHPQFEK or AWAHPQPGG		Synthetic	Regenerable affinity matrix; compatible with purifications requiring anaerobic conditions ⁹
Beta-Galactosidase	116 kDa		Enzyme coded by lac z gene in the lac operon of E. coli	Metalloenzyme that splits lactose into glucose and galactose; Allows for enzymatic protein quantification assays; May protect from proteolytic activity; May decrease solubility; Tag may form tetramers in solution ⁹
Maltose Binding Protein	42.5 kDa	MBP	Maltose/maltodextrin system of E. coli	Amylose purification column compatible with non-ionizing detergents and high salt but not reducing agents; Large size; N-terminal tagging may reduce translation efficiency; ⁹; Enhanced protein production ¹³
Calmodulin Binding Protein (CBP)	4 kDa		Cytoplasm of all Eukaryotic cells	No cross-reaction with any endogenous E.coli proteins; High yield affinity Calmodulin column purification; Not useful for purification from eukaryotic cells; N-terminus tagging may reduce translation efficiency ⁹
Green Fluorescent Protein (GFP)	27 kDa		Jellyfish Aequorea Victoria	Major excitation peak at a wavelength of 395 nm and a minor one at 475 nm; emission peak is at 509 nm; Detectable in living organisms without antibody; Can sometimes be non-specifically directed to nucleus; Useful to monitor protein-protein interactions by FRET; Very large; Dimerization may occur ⁹; Useful in protein-folding assays ⁵
Glutathione S Transferase (GST)	27 kDa		Eukaryotes and Prokaryotes	Affinity purification via relatively reusable glutathione conjugated columns; purification under native conditions only; Highly antigenic; may cause insolubility; may dimerize ⁹
mCherry	28 kDa	DsRED Red Fluorescent Protein RFP	Disc corals of Discosoma species	Useful to monitor protein-protein interactions by FRET; Excitation maximum at 587 nm and an emission maximum at 610 nm
Fc-Fusion Proteins		Fc chimeric fusion proteins Fc-Ig's Fc-tag protein Ig-based Chimeric fusion protein	Fc domain of IgG Antibody	The Fc domain can improve the solubility and stability of the tagged protein both in vitro and in vivo; Easy cost-effective purification by protein-G/A affinity chromatography ¹⁴
Thioredoxin	12 kDa	Trx TRDX	Found in all organisms; Encoded by TXN and TXN2 genes in humans	Can enhance the solubility of tagged protein ¹⁵
Peptide tags in grey.			Fusion Proteins in blue.

View All Epitope Tags

Cleavage Sites

Tags are removable by proteolytic enzymes that cleave the tag from the protein of interest, and generally the cleavage site needs to be engineered between the tag and the protein. Consider utilizing these commonly used proteases in your experiments: (Note: // denotes cleavage site)

Protease	Recognition Site
TEV Protease	Glu-Asn-Leu-Tyr-Phe-Gln-Gly//Ser
Thrombin	Leu-Val-Pro-Arg//Gly-Ser
Factor Xa	Ile-(Glu/Asp)-Gly-Arg// Occasionally Gly-Arg//
Enteropeptidase/Enterokinase	Asp-Asp-Asp-Asp-Lys//

References
1. BioTechniques, Vol. 44, No. 5, Mini-Review: Epitope Tagging; Bill Brizzard; Published online: 16 May 2018, https://doi.org/10.2144/000112841
2, Kimple ME, Brill AL, Pasker RL. Overview of affinity tags for protein purification. Curr Protoc Protein Sci. 2013;73:Unit 9.9. Published 2013 Sep 24. doi:10.1002/0471140864.ps0909s73
3. Walls D., Loughran S.T. (2011) Tagging Recombinant Proteins to Enhance Solubility and Aid Purification. In: Walls D., Loughran S. (eds) Protein Chromatography. Methods in Molecular Biology (Methods and Protocols), vol 681. Humana Press
4. Terpe, K. Appl Microbiol Biotechnol (2003) 60: 523. https://doi.org/10.1007/s00253-002-1158-6
5. Nat Biotechnol. 1999 Jul;17(7):691-5.Rapid protein-folding assay using green fluorescent protein. Waldo GS1, Standish BM, Berendzen J, Terwilliger TC.
6. Sabourin, M. , Tuzon, C. T., Fisher, T. S. and Zakian, V. A. (2007), A flexible protein linker improves the function of epitope‐tagged proteins in Saccharomyces cerevisiae. Yeast, 24: 39-45. doi:10.1002/yea.1431
7. Zordan RE, Beliveau BJ, Trow JA, Craig NL, Cormack BP. Avoiding the ends: internal epitope tagging of proteins using transposon Tn7. Genetics. 2015;200(1):47-58.
8. Maue, R. A. (2007), Understanding ion channel biology using epitope tags: Progress, pitfalls, and promise. J. Cell. Physiol., 213: 618-625. doi:10.1002/jcp.21259
9. Kimple ME, Brill AL, Pasker RL. Overview of affinity tags for protein purification. Curr Protoc Protein Sci. 2013;73:Unit 9.9. Published 2013 Sep 24. doi:10.1002/0471140864.ps0909s73
10. Zhao X, Li G, Liang S. Several affinity tags commonly used in chromatographic purification. J Anal Methods Chem. 2013;2013:581093.
11. Nat Methods. 2007 Feb;4(2):107-8. The HA tag is cleaved and loses immunoreactivity during apoptosis. Schembri L, Dalibart R, Tomasello F, Legembre P, Ichas F, De Giorgi F
12. Mukherjee S, Ura M, Hoey RJ, Kossiakoff AA. A New Versatile Immobilization Tag Based on the Ultra High Affinity and Reversibility of the Calmodulin-Calmodulin Binding Peptide Interaction. J Mol Biol. 2015;427(16):2707-25.
13. Reuten R, Nikodemus D, Oliveira MB, et al. Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems. PLoS One. 2016;11(3):e0152386. Published 2016 Mar 30. doi:10.1371/journal.pone.0152386
14. Exp Cell Res. 2011 May 15;317(9):1261-9. doi: 10.1016/j.yexcr.2011.02.013. Epub 2011 Mar 1. Introduction to current and future protein therapeutics: a protein engineering perspective. Carter PJ.
15. J Biol Chem. 1995 Oct 27;270(43):25328-31. Increase of solubility of foreign proteins in Escherichia coli by coproduction of the bacterial thioredoxin. Yasukawa T1, Kanei-Ishii C, Maekawa T, Fujimoto J, Yamamoto T, Ishii S.
16. J Biol Chem. 1998 Dec 4;273(49):32421-9. The Orc4p and Orc5p subunits of the Xenopus and human origin recognition complex are related to Orc1p and Cdc6p. Tugal T1, Zou-Yang XH, Gavin K, Pappin D, Canas B, Kobayashi R, Hunt T, Stillman B.

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