Purification Resins
The term Ni-NTA (Nickel NTA) refers to a nickel2+ ion that has been coupled Nitrilotriacetic acid (NTA). Ni-NTA can then be coupled to agarose resin or magnetic beads for IMAC (Immobilized Metal Chelate Affinity Chromatography). This is a purification method to obtain functional His tagged protein. The size of the used agarose resin beads or magnetic beads influences the flow rated and the protein yield.

Overview of Cube Biotech's Ni-NTA products for His tag protein purification.


Usage Specific binding and purification of 6x his-tagged proteins
Specifity Affinity to His-tagged proteins
Binding capacity >80 mg/mL
Chelator stability Stable in buffer containing 10 mM DTT and 1 mM EDTA
Bead Ligand Ni-NTA

Ni-NTA products by Cube Biotech were used in the following publications:

Different metal ions confer different binding affinity and specificity

Fig. 1: Depending on which metal ion is coupled to NTA the balance between protein yield and purification specificity shifts. Nickel is a very balanced choice between the two extremes.
His tag purification is based on ionic interactions of a the coupled metal ion and the Poly His tag. Therefore other metal ions can also fullfill the function of Nickel. Figure 1 shows the most frequently used metal ions for His tag purification. Depending on the used metal ion the balance between the specificity and the affinity of the metal ion to the His tag shifts. Nickel is a balanced choice between both extremes. Copper has the highest affinity, but Cobalt is the choice for the most pure His tag purifications.

Fig. 2: Over 20% more yield obtained with PureCube Ni-NTA Agarose. SDS-PAGE of GFP expressed in E. coli and purified in gravity columns with PureCube Ni-NTA Agarose and Ni-NTA resin from Competitor Q. 80 mg/mL protein yield was obtained with PureCube Ni-NTA Agarose (E1–E4, Cube) compared to 65 and 48 mg/mL, respectively, with the widely used alternative resins G and Q (E1–E4, Competitor G / Competitor Q).

High yield and purity

Our unique production process yields a Ni-NTA Agarose that exhibits a protein binding capacity >20% higher than that of two leading competitor products. Figure 1 shows the SDS-PAGE of GFP expressed in E. coli and purified in gravity colums with PureCube Ni-NTA Agarose and the Ni-NTA resin from Competitor G and Competitor Q. The protein yield in 4 elutions (E1-E4, Cube) was 80 mg/mL, compared to 65 and 48 mg/mL obtained with the alternative resins (E1-E4, Competitor G, Competitor Q). Similar results (10-18% higher binding capacity; data not shown here) were obtained comparing the purification of JNK1 (Kinase, 48 kDa) on PureCube Ni-NTA and the Ni-NTA of leading providers.

Ni-NTA_DTT & EDTA Stability
Fig. 3: NTA is robust in the presence of reducing and chelating agents. GFP-His was purified on gravity columns containing PureCube Ni-NTA Agarose after exposing the resin for 1 h to 3 concentrations of DTT or EDTA. NTA exhibits a shallow decay rate in binding capacity.

Superior DTT and EDTA stability

PureCube Ni-NTA Agarose is very robust in the presence of DTT and EDTA. In a stability test, PureCube Ni-NTA Agarose was exposed to increasing concentrations of DTT or EDTA for 1 h. Thereafter, the resins were used to purify E. coli-expressed GFP-His in gravity columns. The binding capacity of the resin decreased in the presence of both DTT and EDTA but the decay rate was shallow. In presence of DTT, PureCube Ni-NTA Agarose lost on average 8% binding capacity with each increase in DTT concentration, resulting in an overall decay of 22% at 10 mM. Even at 1.5 mM EDTA, the resin still exihibits 54% of its maximum binding capacity (Fig. 2).

Fig. 4: PureCube Ni-NTA Agarose is robust against oxidation and regenerable. PureCube Ni-NTA Agarose was exposed to 5mM DTT for 1 h (A). After demonstrating that it could still bind GFP (B), the resin was washed, stripped (C), and reloaded with Ni2+ (D) following standard Cube protocol (see Cube Protocols & Datasheets).

Robust against oxidation and regenerable

PureCube Ni-NTA Agarose retains its color and function after exposure to as much as 10 mM DTT. Figure 3 shows a photo series of the resin after a 1 h exposure to 5 mM DTT. Unlike other resins, PureCube Ni-NTA Agarose did not turn brown (A). The resin was still able to bind GFP (B), with a measured binding capacity of 65 mg/mL (see Fig. 2). The resin could then be regenerated by stripping the NTA, turning the resin white (C), and reloading it with nickel ions (D). The protocol for regenerating PureCube Ni-NTA Agarose can be downloaded.

Frequently asked questions

Can I get the datasheet for the Ni-NTA resin?
What are the reasons for non specific binding?
Some histidine rich proteins can also bind to nickel. But washing with NaOH after elution of your protein of interest removes unspecific bound proteins from your resin.
I want to use high concentration of EDTA and DTT. Is it possible to use Ni-NTA from Cube Biotech?
No it is not recommended because nickel is reduced with DTT or dissolved with EDTA. If you want to use high concentrations of EDTA and DTT you should use our Indigo resin.
How is the capacity at high flow rates?
Due to the high ligand density of PURE Cube Ni-NTA agarose the material shows good performance even at high flow rates.
After using DTT my resin turned orange. How to regenerate it?
Read our detailed protocol for more information.
How much resin do I have to use?
That is depending on your expression level.


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