For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome
If you continue without changing your cookie settings, we'll assume you’re happy with this.
Download our in-depth PDF guide to fusion tags. From determining the best application to identifying common issues, our guide will help you make the most of your bench time.
Affinity tags are so named because they are often used in affinity purification: a technique for purifying proteins from cell lysate. By attaching an affinity tag to your protein of interest, it can be pulled out of the solution via chemical or physical interactions with an immobilized substrate1 [Figure 1]. The exact affinity chromatography method is dependent on the specific tag used. In this section, you will learn about some of the most commonly used affinity tags.
Figure 1: This diagram shows the steps for affinity chromatography. First, lyse your sample to release the proteins. When this lysate is added to the column, the fusion tag will bind to the affinity resin. Unwanted proteins are washed through the column, and then your protein of interest can be eluted.
Molecular Weight: ~26 kDa
Size: 211 amino acids.
Tag location: C- or N- terminals
Affinity Resin: Glutathione
Applications: protein purification, protein-DNA interactions, protein-protein interactions.
Strengths: Can aid solubility and stabilize fusion protein structure.
Limitations: Relatively large tag, so it can affect the functionality of the fusion protein. Possible contamination of eluent with heat-shock proteins.
Glutathione-S-transferase (GST) is a protein consisting of 211 amino acids and has a molecular weight of ~26 kDa1. Native GST is responsible for protecting the cell against noxious compounds and oxidative stress. A major property of GST is its affinity for the tripeptide, glutathione, which can be utilized in affinity chromatography. When a solution containing GST is run through a column lined with immobilized glutathione, the GST binds to the glutathione, separating it from the rest of the solution. It is recommended to use near-neutral buffers for optimum binding of GST to the immobilized glutathione1,3. Once bound, GST can be eluted with 10 mM glutathione, which is a mild eluent and aids retention of the fused protein function2.
|Anti-GST antibody (HRP)||ab3416|
|GST Assay Kit (Colorimetric)||ab65326|
|GST-Tag Protein Expression Check Kit||ab270052|
|Glutathione Affinity Resin - Amintra®||ab270237|
Molecular Weight: 0.2–1.6 kDa. 6x-His tag is 0.8 kDa.
Size: 2–10 histidine residues
Tag location: C- or N- terminals, or internal.
Affinity Resin: transition metal ions, usually Ni2+
Applications: protein purification
Strengths: Small size so lower possibility of affecting fusion protein functionality. Doesn’t cause inclusion bodies.
Limitations: Can have significant background binding in mammalian and insect cells.
PolyHis tags are widely used for protein purification due to their small size and stable binding1,2,3,4. Although tags can range from 2–10 histidine residues, the most common His-tag is the 6x-His tag, or hexatag, which contains six histidine residues. Histidine forms coordination bonds with immobilized transition metal ions, and this property can be utilized for protein purification. Cobalt and zinc columns are available for immobilized metal affinity chromatography (IMAC), but nickel columns are usually used. As His-tags are short peptide sequences, they rarely affect the properties of the fused protein, although the optimal placement of the tag is protein specific2.
What to watch out for:
Endogenous His: As His residues are prevalent in mammalian and insect systems, there can be high background when using these systems. Background binding can be avoided by washing in 5–10 mM imidazole, although this could prematurely elute your protein of interest. Background binding should also be considered when using anti-His antibodies.
Using proteins with metal centers: It is not recommended to fuse a protein with a metal center to a His-tag as the metal could be absorbed by the affinity resin.
Anaerobic conditions: Anaerobic conditions should be avoided as it can reduce the affinity resin.
|Anti-6X His tag® antibody [4D11]||ab5000|
|Anti-6X His tag® antibody (HRP)||ab237339|
|His-Tag Protein Purification Column (Pre-packed, 5 x 1ml) - AminTrap||ab270528|
|His-Tag Protein Purification Column (Pre-packed, 5 x 5ml) - AminTrap||ab270529|
Molecular Weight: 244 Da
Affinity Resin: Avidin/Streptavidin
Applications: protein purification, surface plasmon resonance, protein expression.
Strengths: can utilize strong affinity for avidin and streptavidin.
Limitations: co-elution with other biotinylated proteins can be encountered in mammalian systems.
Biotin, also known as vitamin H, is a small molecule with a molecular weight of 244 Da. It is a commonly used molecular tool and is often conjugated to secondary antibodies as a visualization technique for ELISA and western blot assays. Biotin forms a very strong bond with both streptavidin and avidin molecules and this property can be utilized for affinity purification. As biotin is a small molecule, it is unlikely to affect protein functionality4.
Another valuable tag is the Strep-tag. As it is 8 amino acids in length (WRHPQFGG), it is also unlikely to affect protein function, and it binds reversibly to the same pocket as biotin. This means that proteins fused to the Strep-tag can be efficiently purified using streptavidin resins, and eluted using biotin in mild buffer conditions5.
What to watch out for:
Tetrameric vs monomeric affinity resins: Biotin tagged proteins can be purified using avidin-coated columns, which are available in either a tetrameric or monomeric format. The tetrameric avidin column requires strong denaturing agents, such as urea or guanidine hydrochloride, for the elution process, whereas monomeric resins enable a milder elution using a 10 mM biotin buffer.
Use in mammalian systems: Mammalian systems contain at least four biotinylated protein species that can co-elute with your protein of interest. However, it is rare to encounter non-specific binding in E. coli systems.