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:
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.
Quick his-tag purification selection guide:
Ni-IDA
Ni-NTA
Co-NTA
If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200
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有趣的事实:
1986年,我们的创始董事彼得·迪恩(Peter Dean)与马丁·埃文斯(Martin Evans,现为教授)合作,开发了第一台使用专利“智能加热块(IHB)”技术进行PCR反应的机器,用于实时温度控制