Affinity Tags

More Information

What is an affinity tag?

An affinity tag, also referred to as an epitope tag, is a short polypeptide or protein that is added to the target protein. Even in the absence of antibodies against the target protein, the target protein can be detected or purified if there is an antibody that recognizes the affinity tag. In general, an affinity tag is added N-terminal, C-terminal, or inside of the target protein.

• Advantages

  • Can detect or purify the target protein even in the absence of antibodies against the target protein
  • Can distinguish the target protein from endogenous proteins
  • Does not cross-react with proteins similar to the target protein
  • Can improve protein solubility (depending on the type of affinity tag)

• Disadvantages

  • Conformation or activity of the target protein may be changed by the addition of the affinity tag.
  • Insertion of an affinity tag sequence by genetic engineering is required.
  • When expressed on plasmids or the like, it is overexpressed as compared with endogenous proteins.

Affinity tag comparison table

The characteristics, advantages, and disadvantages of major affinity tags are summarized below.

Affinity tag	PA	DYKDDDDK (FLAG®)*1	6×His	HA	V5
Source	Human podoplanin	Artificial sequence	Artificial sequence	Influenza virus Hemagglutinin	Simian virus V protein
Sequence	GVAMPGAEDDVV	DYKDDDDK	HHHHHH	YPYDVPDYA	GKPIPNPLLGLDST
Number of residues	12	8	6	9	14
Molecular weight(kDa)	1.2	1.0	0.8	1.1	1.4
Antibody binding force*2 (Kd(M))	4.9×10-10	2.8×10-8	1.0×10-5~6	2.8×10-9	-
Elution method	Peptide competitive elution Acid elution	Peptide competitive elution Acid elution	Imidazole competitive elution Chelator elution, etc.	Peptide competitive elution Acid elution	Peptide competitive elution Acid elution
Resin reuse method	3M MgCl2 + MES (pH6.0)	Tris-HCl + Glycine Buffer (pH2 to 3.5)	0.5M Imidazole (pH7.4)	Tris-HCl + Glycine Buffer (pH2 to 3.5)	Tris-HCl + Glycine Buffer (pH2 to 3.5)
Advantage	●Very high affinity between tag and antibody ●Highly specific tag and antibody (vs. DYKDDDDK*2) ●Can regenerate resins under neutral conditions ●Can also be inserted into a membrane protein because antibodies can recognize the loop structure of the tag	●More likely to be located close to the surface of the fusion protein because it is hydrophilic ●Can detect 100 fmol of protein with 3×FLAG ●No crossing of antibodies in most mammalian cells or bacterial cell lysates ●Can be cleaved by enterokinases	●Can also purify proteins denatured with urea or guanidine hydrochloride ●Negligible impact on recombinant proteins due to the small size of the tag ●Relatively low cost ●Can be refolded while being bound to the carrier	●Negligible impact on recombinant proteins due to the small size of the tag ●More likely to be located close to the surface of the fusion protein because it is hydrophilic	●Negligible impact on recombinant proteins due to the small size of the tag
Disadvantage	●Endogenous podoplanin detected in cells expressing human podoplanin (HEK293, COS-7)	●Relatively high cost	●Non-specific adsorption on histidine-rich regions of the host ●Cannot be used for samples containing reducing agents, oxidizing agents, or chelators	●Cleaved by apoptosis-activated caspase 3/7, losing immunoreactivity*3

Affinity tag	c-Myc	GFP	GST	MBP
Source	Human c-Myc	A.victoria GFP	S.japonicum Glutathione-S-transferase	E.coli Maltose Binding Protein
Sequence	EQKLISEEDL	-	-	-
Number of residues	10	238	218	370
Molecular weight(kDa)	1.2	27.0	26.0	43.0
Antibody binding force*2 (Kd(M))	2.2×10-9	-	1.0×10-5~6	-
Elution method	Peptide competitive elution Acid elution	Acid elution	Glutathione competitive elution	Maltose competitive elution
Resin reuse method	Tris-HCl + Glycine Buffer (pH2 to 3.5)	-	Glutathione, Tris-HCl + Glycine Buffer (pH2 to 3.5)	-
Advantage	●Negligible impact on recombinant proteins due to the small size of the tag ●More likely to be located close to the surface of the fusion protein because it is hydrophilic	●GFP fluorescence observable	●Increased solubility of the expressed protein ●High specificity due to enzyme-substrate reaction (when glutathione is used as the ligand)	●Purification under very mild conditions due to competitive elution with maltose ●Increased solubility of the expressed protein
Disadvantage	●Endogenous c-Myc also detected	●High molecular weight with potential to inhibit protein structure or function ●Inability of antibodies to recognize the tag under denaturing conditions, precluding purification	●High molecular weight with potential to inhibit protein structure or function ●Inability of antibodies to recognize the tag under denaturing conditions, precluding purification	●High molecular weight with potential to inhibit protein structure or function ●Inability of antibodies to recognize the tag under denaturing conditions, precluding purification

• 1 FLAG^® is a registered trademark of Sigma-Aldrich.
• 2 The data are from our internal study and should not be construed as a guarantee of the performance of each affinity tag.
• 3 Schembri, L., et al.: Nature methods, 4(2), 107(2007).

References

Jarvik, J. W., & Telmer, C. A.: Annual review of genetics, 32 (1), 601 (1998).

"Protein experiment handbook" ed. by Takenawa, T., Yodosha, Japan, (2003). (Japanese).

"Protein experiment note” ed. by Okada, M. and Miyazaki, K., Yodosha, Japan, (2011). (Japanese).

"Protein expression protocol that can be selected according to purpose” ed. by Nagata, K. and Okuwaki, M., Yodosha, Japan, (2011). (Japanese).