Thermo Scientific HisPur Ni-NTA Magnetic Beads are high-capacity nickel-IMAC beads for affinity purification of His-tagged fusion proteins in manual or automated formats.
The blocked magnetic bead surface is derivatized with the nitrilotriacetic acid (NTA) chelation moiety and loaded with divalent nickel ions (Ni2+). The immobilized metal affinity chromatography (IMAC) beads provide high binding capacity with very low background. The HisPur Ni-NTA Magnetic Beads can be used both manually with a magnetic stand as well as with automated platforms such as the Thermo Scientific KingFisher Instruments for high-throughput needs.
- High capacity – equivalent or higher binding capacity than Ni-NTA magnetic beads from other suppliers
- Low nonspecific binding – the bead surface is pre-blocked and the protocol provides optimized buffers for purification
- Fast – protocol is completed in 1 hour
- Scalable – process microliter to milliliter sample volumes
- Versatile – purify proteins using native or denaturing conditions
- Reagent compatible – can be used with common cell lysis reagents and a variety of buffer additives
- Multiple formats – protein coupling to the beads and downstream applications can be performed both manually and on an automated platform (e.g., Thermo Scientific KingFisher Instruments)
HisPur Ni-NTA Magnetic Beads are used for small scale affinity purification as well as high-throughput screening of recombinant His-tagged proteins. The polyhistidine tag is the most popular affinity tag and typically consists of six consecutive histidine residues (6xHis). These tagged proteins are overexpressed in a number of different systems, most commonly in bacteria, and purified from cell lysates such as those prepared using B-PER Bacterial Protein Extraction Reagents. Purification of His-tagged proteins is achieved using a NTA chelate charged with nickel that coordinates with the histidine side chains. The NTA chelate contains four metal-binding sites which allow for low metal ion leaching and high binding capacity. The protocol for the HisPur Ni-NTA Magnetic Beads has been optimized to allow for high purity of the isolated His-tagged protein. Performance is equivalent to or better than Ni-NTA magnetic beads from other suppliers.
|Characteristics of Thermo Scientific HisPur Ni-NTA Magnetic Beads.
||Nickel loaded on nitrilotriacetic acid that has been covalently coupled to a blocked magnetic bead surface
||Superparamagnetic (no magnetic memory)
||12.5 mg/mL in 20% ethanol
||≥ 40μg of 6X-His-tagged GFP/mg of beads;
≥ 500μg 6X-His-tagged GFP/mL of beads
Superior performance of Thermo Scientific HisPur Ni-NTA Magnetic Beads when compared to magnetic beads from other suppliers. Bacterial lysate (100μg total protein) containing over-expressed 6xHis-GFP (Panel A) or over-expressed 6xHis-β Galactosidase (Panel B) was applied to 0.5mg of HisPur Ni-NTA Magnetic Beads, Mag Sepharose* (GE Healthcare), PureProteome* (Millipore), and Magnetic Agarose (Qiagen) Ni-NTA bead products. All samples were run in duplicate, and the beads were processed using the Thermo Scientific protocol with buffers recommended by the manufacturers. For the HisPur Ni-NTA beads the amount of imidazole in the Equilibration, Wash and Elution Buffers was 30mM, 50mM and 250mM, respectively. All three buffers contained 100mM sodium phosphate and 600mM sodium chloride. Binding was performed with all samples for 30 minutes. The beads were collected on a magnetic stand and the flow throughs were saved for analysis. The beads were then washed twice and bound protein was eluted for 2 x 15 minutes with Elution Buffer. The eluates were combined, resolved on an SDS-PAGE gel and stained with Thermo Scientific Imperial Stain. Purity analyses were performed on a Thermo Scientific myECL Imager with myImageAnalysis Software. The % purity was determined by measuring the ratio of the background-corrected 6xHis-tagged protein band of interest to the sum of all bands and multiplying by 100%.
Comparable yields and comparable % purity were observed for 6xHis-GFP with HisPur Ni-NTA and Qiagen Ni-NTA agarose magnetic beads. HisPur Ni-NTA beads provided higher yield and purity than Qiagen Ni-NTA agarose magnetic beads for purification of 6xHis-β galactosidase. Millipore and GE Ni-NTA magnetic beads gave lower purity and lower yield than Thermo Scientific beads in both purifications.
Review of His-tagged Proteins
Other Magnetic Beads
HisPur Ni-NTA Agarose, Columns and Kits
HisPur Cobalt Agarose, Columns and Kits
B-PER Bacterial Protein Extraction Reagents
Cell Lysis Reagents