Affinity purification is a frequently used technique to isolate proteins for further research and other applications. However, there are a lot of different protein affinity tags, and if you have never performed affinity purification before, you might wonder which one you should use.

In this article we will discuss small peptide affinity tags, such as his-tags. These tags are a great choice because they usually do not interfere with the tagged-protein’s function, and allow versatility in where you tag the protein.

Three common small peptide affinity tags are his-tags, flag-tags and strep-tags. They are fused to target proteins for purification and detection of that protein. Small affinity tags are typically ~6 to 25 residues long, and bind to metal ions, small molecules or other proteins for purification.

The small size of these peptide tags provides flexibility in adding them to either the amino or carboxy termini of the protein of interest – or even within a protein’s flexible loops. Since these tags are small, they usually do not impact a protein’s structure or function. However, this point should always be experimentally verified when working with a fusion protein with a new affinity tag.

In contrast, other affinity tags, such as GST-tags, consist of entire protein domains. These large tags are frequently used when the protein of interest has limited solubility in an expression host. To improve solubility, these tags need to be added to the protein’s amino terminus. To learn more about large solubility affinity tags, check out this article!

Article Table of Contents:




General Purification Strategy

Tagging Considerations

Consider downstream uses


His-tag is a stretch of histidine residues that bind to nickel and other divalent transition metals. When fused to a protein, the his-tag can be used to purify that protein using a nickel column.

His-tags typically range from 6 to 10 histidine residues in length. 6x his-tags are frequently used, however lengthening the tag up to 10 histidine residues can increase the efficiency of the purification process.

These histidine residues interact with nickel and other divalent transition metal ions. Nickel ions are conjugated onto agarose beads, often via nitrilotriacetic acid (NTA), and used to bind his-tagged proteins.

his-tagged proteins attached to nickel beads in affinity purification

Figure 1. The target his-tagged proteins (purple) bind to nickel agarose beads. Low concentration of imidazole (blue) prevents non-target proteins from binding to the agarose beads and they are washed out.

Imidazole is the eluent for his-tags. Histidine side chains coordinate nickel through their imidazole moiety (Figures 1 and 2). Adding free imidazole in sufficient excess competes to bind with the nickel column and thereby elutes his-tags from the column. Typically, ~ 0.25 to 1 molar imidazole is used to elute his-tags from nickel columns. If you are unfamiliar with molar measurements – check out this article to learn what those are.

Imidazole should also be added at lower concentrations to the loading and wash buffers (Figure 1). This is because many endogenous proteins contain polyhistidine stretches that act like naturally occurring his-tags and weakly bind to nickel columns (Salichs et al., 2009). In fact, depending on the protein context, even a single histidine residue can be sufficient to bind to nickel columns (Hemdan et al, 1989).

The imidazole side chains of histidine residues in the His-tag (purple) bind to Ni2+-conjugated agarose beads.

Figure 2. The

imidazole side chains of histidine residues in the His-tag (purple) bind to Ni


agarose beads. Free imidazole (blue) is added in excess to elute His-tagged

proteins from the Ni

2+ beads.

Binding strength correlates with the length of polyhistidine residues (Fessenden, 2009; Guignet et al., 2004; Hemdan et al, 1989). However, most of these contaminating proteins can be separated from your his-tagged protein of interest by loading the protein mixture and washing with ~ 5 – 50 millimolar imidazole.

This is also why going with a longer his-tag, such as 10 histidine residues, and using a higher concentration of imidazole in the loading and wash steps can lead to higher purity after affinity purification.

His-tags are a widely used affinity purification technique, yet there are a few things to keep in mind when taking this approach.

First, the protein and buffer solutions cannot contain strong metal chelating agents such as EDTA or EGTA. This means if you are using protease inhibitors, make sure to use the EDTA/EGTA-free variety. Including EDTA/EGTA in your buffers will strip nickel off of your column thereby eliminating the binding site for your his-tagged protein!