Thermo Scientific HisPur Ni-NTA Superflow Agarose is a nitrilotriacetic acid (NTA) modified Superflow 6 support charged with divalent nickel (Ni+2) designed for FPLC purification of poly-histidine-tagged proteins.
HisPur Ni-NTA Superflow Agarose is a highly crosslinked, durable resin that does not compress at flow rates used for medium- or large-scale FPLC purifications. The resin holds up well to a variety of chemicals and pH values, and is compatible with common clean-in-place procedures. Compared to cobalt and other ligands used for immobilized metal affinity chromatography (IMAC), nickel provides greater capacity for His-tagged protein purification. HisPur Ni-NTA Superflow Agarose exhibits a high dynamic binding capacity across a range of flow rates, making it an excellent choice for larger scale purifications.
- High capacity – binds greater than 20mg of His-tagged GFP per mL of resin at a linear flow rate of 300cm/hour
- High purity – provides greater than 80% purity of eluted fractions when used to purify from lysates
- Versatile – can be used to purify proteins under native and denaturing conditions
- Robust – highly crosslinked beads tolerate linear flow rates up to 1200cm/hour
- Compatible – maintains function in a wide variety of chemicals and pH conditions
- Cost effective – competitively priced resin is stable through multiple cycles of cleaning and reuse
- Medium- to large-scale FPLC purification of polyhistidine tagged proteins
Properties of Thermo Scientific HisPur Ni-NTA Superflow Agarose.
||Superflow 6 Resin, highly crosslinked 6% agarose
||60 to 160μm
||Recommended: 300cm/hour (binding, wash, elution)
||Metal: ≥13μmol Ni2+/mL resin
||Static: approx. 60mg 6xHis-GFP/mL resin
Dynamic‡: approx. 20mg 6xHis-GFP/mL resin
||1M acetic acid pH 2, 1% SDS pH 7, 6M guanidine-HCl, 70% ethanol for ≥1 week at 37°C; 8M urea, 10mM DTT, 5mM TCEP for ≥2 hours at 22°C
||pH 3 to 9 for ≥1 week at 4°C
pH 2 to 3 or 9 to 12 for ≥ 2 hours at 22°C
||Up to 25 times
|† Maximum Linear Flow Rate Conditions: Column Dimensions (w x h): 13mm x 38mm (5mL resin); Sample: deionized water at room temperature; Linear Flow Rate: volumetric flow rate (mL/min) x 60 (min/hr)/cross-sectional area (cm2).
‡ Dynamic Binding Conditions (10% breakthrough): Column Dimensions (w x h): 5mm x 50mm (1mL resin); Sample: 1mg/mL 6xHis-GFP 27kDa) pure protein in 20mM NaH2PO4, 300mM NaCl, 10mM imidazole; Flow Rate: 1mL/min.
Affinity chromatography is often used as a quick and easy approach for the purification of recombinant proteins. One such method of purification involves the binding of a poly-histidine tag to a divalent metal cation that has been coordinated by a chelator immobilized on a beaded support. For immobilized metal affinity chromatography (IMAC) purification of His-tagged proteins, the type of bead, chelator, and metal immobilized influences purity, yield, and flow rate performance of these purifications.
HisPur Ni-NTA Superflow Agarose is a nitrilotriacetic acid (NTA) modified Superflow 6 support charged with divalent nickel (Ni+2) designed for FPLC purification of poly-histidine-tagged protein. To demonstrate performance, 6xHis-GFP was over expressed in a 100L reactor and the cell mass was collected in multiple fractions. A portion of the biomass (240g) was lysed with 2L of lysis buffer supplemented with Thermo Scientific Halt Protease Inhibitor (Part No. 78439). The lysate was clarified and split into two fractions. The target protein was then purified using HisPur Ni-NTA Superflow Agarose and a leading competitor’s resin in equivalent 200mL packed bead columns. Total yield, recovery, and purity (>80%) were nearly identical for both resins, and more than 4 grams of target protein was purified in less than 3 hours.
|High-yield, high-purity, medium-scale purification of 6xHisTagged protein. More than 4 grams of over-expressed 6xHis-GFP were purified in 3 hours using 200mL columns containing HisPur Ni-NTA Superflow Agarose or Qiagen™ Ni-NTA Superflow. One liter of lysate was loaded at a flow rate of 20mL/min, then washed until baseline with wash buffer containing 30mM imidazole. Bound protein was eluted with buffer containing 300mM imidazole. Fractions containing purified 6xHis-GFP were pooled and quantitated using Pierce 660nm Protein Assay (Part No. 22662). Load, flow-through, wash, and eluate fractions were separated by SDS-PAGE, stained with Imperial Protein Stain (Part No. 24615) and evaluated using myImageAnalysis Software (Part No. 62237) to determine purity.
Review of His-tagged fusion protein production and purification
Other Affinity Resins for His-tagged Protein Purification
Glutathione Resins for GST-tagged Protein Purification
B-PER Bacterial Protein Extraction Reagent
Protease Inhibitor Solutions and Tablets
Slide-A-Lyzer Dialysis Flasks (250mL capacity)