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PureCube Ni-NTA Agarose XL
The term Ni-NTA (Nickel NTA) refers to a nickel2+ ion that has been coupled to 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 rates and the protein yield. Our PureCube XL Ni-NTA agarose resins are beads with an average diameter of ~400 µm. They are used for the purification of His tagged proteins from cells. 400 µm agarose beads are a special creation by us for assays in which the flow rate is compromised by smaller beads. For higher protein yield we recommend our PureCube Ni-NTA agarose beads with a 40 µm diameter and our PureCube 100 Ni-NTA resin beads with a 100µm diameter.
1. Protein Binding Capacity
The protein binding capacity is at least 20 mg/mL, as determined by purification of 6xHis-tagged GFP protein from E.coli cleared lysates, and quantified via spectrophotometry.
2. Compatibility
PureCube Ni-NTA Agarose XL is very stable and can resist the following conditions in most situations: pH 2-14, 100% methanol, 100% ethanol, 8 M urea, 6 M guanidinium hydrochloride, 30% (v/v) acetonitrile.
3. 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.
Fig. 1: 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).
4.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. 2: 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.
5.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.
Fig. 3: 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).
Type: | Agarose |
Ligand: | NTA |
Coupled Ion: | Ni2+ |
Usage | Specific binding and purification of 6x His tagged proteins |
Specifity | Affinity to His tagged proteins |
Binding capacity | >20 mg/mL |
Bead Ligand | Ni-NTA |
Bead size | ~400 μm(XL Agarose) |
Chelator stability | Stable in buffer containing 10 mM DTT and 1 mM EDTA |
Filling quantity | Delivered as a 50 % suspension |
Required equipment |
-Lysis Buffer -Wash Buffer -Elution Buffer -Ice bath -Refrigerated centrifuge for 50 mL tube (min 10,000 x g) -50 mL centrifuge tube -Micropipettor and Micropipetting tips -Disposable gravity flow columns with capped bottom outlet, 2 ml -pH meter - End-over-end shaker -SDS-PAGE buffers, reagents and equipment Optional: Western Blot reagents and equipment |
Order number | Products | Bead size | Protein Binding Capacity | Shipment Temperature | Storage temperature | Usage | Price(yuan) |
55103 | PureCube Ni-NTA Agarose XL(10ml) | 400 | 20mg/mL | Ambient temperature | 4° C | Used for the purification of His-tagged recombinant proteins when large particle size fillers are required to process a large number of samples quickly; pH Stability: 2-14 | 1456 |
55105 | PureCube Ni-NTA Agarose XL(50ml) | 400 | 20mg/mL | Ambient temperature | 4° C | 5751 | |
55110 | PureCube Ni-NTA Agarose XL(250ml) | 400 | 20mg/mL | Ambient temperature | 4° C | 24783 | |
55112 | PureCube Ni-NTA Agarose XL(500ml) | 400 | 20mg/mL | Ambient temperature | 4° C | 44335 |
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