His-tagged Protein Purification

The His-tag is the most widely used affinity tag due to its small size, low immunogenicity, and versatility under native or denaturing conditions, as well as in presence of detergents and many other additives. Cambio offers high-performance Ni-NTA, Ni-IDA and Co-NTA Agaroses made using BioWorks Workbeads. For purification of his-tagged proteins from cell culture supernatants or for pull-down experiments, we recommend Cambio Ni-NTA, Ni-IDA or Co-NTA MagBeads.  For pre-packed FPLC columns for his-tag purification please see the “FPLC Cartridges” tab.

Which resin should I be using for His-tag purification? 

If you are using immobilized-metal affinity chromatography (IMAC) to purify His-tagged proteins, you can choose between the nickel ion being coupled to the resin matrix via either nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA). Or you can use the cobalt ion bound to NTA resin.

When asked, most researchers cannot explain why they use one ligand over another for their favourite his-tagged target protein purification. However, it may be advantageous to explore different ligands because binding capacity of a given IMAC resin is very dependent on the ion metal used and the nature of the protein being purified.

 

In deciding on an IMAC resin for your protein purification needs consider the following questions:

 

• How pure does your target protein need to be?
• What yield of target protein do you need?
• Does your sample contain reagents like DTT or EDTA?
• What are your budgetary constraints?
• What yields do you anticipate from your expression?

 

If you seek high yields and the purity of the recovered protein is of lesser priority, an IDA-based resin might be the right choice. It is less expensive and is still a robust resin that is easily regenerated and reloaded. To minimize non-specific binding, the use of alternate metal ions such as zinc or cobalt could be explored. For applications where purity of the recovered protein is essential (e.g., for subsequent crystallization), NTA-based resins are the most optimal choice. Specificity can be further improved by loading the resin with a metal ion that exhibits highly specific binding (e.g., cobalt). Another advantage of NTA-based resins is their resilience when exposed to agents such as DTT and EDTA, which accommodates a wider range of sample buffers.

 

Cobalt NTA resin, easily recognized by its characteristic pink colour, has similar properties to the nickel variant. It can be very useful if the use of Ni-NTA resin gives sub-optimal purity of his-tagged proteins. The reason for this is that the cobalt ion has higher specificity of binding to poly-histidine stretches than the nickel ion resulting in higher purity of purified protein albeit with slightly reduced yields.  In addition Co-NTA resin is more stable in the presence of DTT and EDTA than nickel-based resins.

 

Quick his-tag purification selection guide:

Ni-IDA

• High yield but max. purity less of an issue
• Lower cost

Ni-NTA

• Maximum purity needed
• Greater stability in DTT and EDTA-containing buffers

Co-NTA

• Nickel based resin previously gave low purity of purified protein
• Greater stability in DTT and EDTA buffers

If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200