Cost-effective nickel-NTA resin for His-tagged protein purification.

Thermo Scientific HisPur Ni-NTA Resin is a high-capacity, high-performance nickel-IMAC resin for routine affinity purification of His-tagged fusion proteins.

The specially prepared support consists of beaded agarose derivatized with the nitrilotriacetic acid (NTA) chelation moiety and loaded with divalent nickel ions (Ni2+). The immobilized metal affinity chromatography (IMAC) resin provides exceptional binding capacity and performance for recombinant His-tagged protein purification.

HisPur Ni-NTA Resin is available in several package sizes of resin slurry, three sizes of centrifuge-ready columns, complete purification kits, and two sizes of FPLC-ready chromatography cartridges. With these options, high-yield His-tag protein purification is possible at nearly any scale.

HisPur Ni-NTA Resin Highlights:

• High capacity – bind up to 60mg of 6xHis-tagged protein per milliliter of resin
• Versatile – purify proteins using native or denaturing conditions
• Compatible – use with Thermo Scientific Cell Lysis Reagents and a variety of buffer additives
• Flexible – available in multiple formats including bulk resin, spin columns and chromatography cartridges
• Cost-effective – reuse the same batch of resin at least five times
• Easy to use – pre-formulated buffers available for kit formats

Product Details:

The expression and purification of recombinant proteins is central to protein regulation, structure and function studies. The majority of recombinant proteins are expressed as fusions with short affinity tags, the most popular being the polyhistidine (6xHis) tag. The method used to purify recombinant His-tagged proteins is immobilized metal affinity chromatography (IMAC), consisting of chelating resins charged with either nickel or cobalt ions that coordinate with the histidine side chains.

HisPur Ni-NTA Resin effectively purifies high levels of overexpressed His-tagged fusion proteins from bacterial lysates, such as those that are attained with Thermo Scientific B-PER Bacterial Protein Extraction Reagents. The resin performs well in batch-binding and spin-column procedures at a variety of scales. (See Chromatography Column data.) Performance equals or exceeds popular Ni-NTA resins from other suppliers.

Thermo Scientific HisPur Ni-NTA resin is effective for purifying His-tagged proteins in batch-bind and spin-column formats with comparable results to another suppliers resin. Left panel: Bacterial lysate (10mg total protein) containing over-expressed 6xHis-annexin V was applied to 0.2mL of HisPur Ni-NTA Resin and purified by the batch-bind method. Center panel: Bacterial lysate (1.7mg total protein) containing over-expressed 6xHis-annexin V was applied to a HisPur Ni-NTA Spin Column (0.2mL) and purified by the spin method. Right panel: The same amount of lysate (1.7mg) was applied to Supplier Qs spin column and purified per the manufacturers instructions. Gels lanes were normalized to equivalent volume. M = molecular-weight marker, L = lysate load, FT = flow-through and E = elution.

 

Thermo Scientific HisPur Ni-NTA resin performs as well or better than other suppliers nickel resins. Bacterial lysate (12mg total protein) containing over-expressed 6xHis-green fluorescent protein (GFP) was applied to HisPur Ni-NTA Resin (0.2mL) and purified by the batch-bind method. The same amount of total protein was applied to Supplier Q, Supplier C, and Ni-IDA resins per the manufacturers instructions. Gel lanes were normalized to equivalent volume. M = molecular-weight marker and L = lysate load.

HisPur Ni-NTA Resin is a high-quality, stable and resilient affinity support. Tests confirm that no decrease in performance occurs after at least five repeated uses. These data indicated that the resin is highly resistant to structural degradation or nickel ion leaching during normal use.

Thermo Scientific HisPur Ni-NTA Resin can be used at least five times without losing performance. Bacterial lysate (20mg total protein) containing over-expressed 6xHis-Protein L was applied to HisPur Ni-NTA Resin (0.2mL) and purified by the batch-bind method. Before each reuse, the resin was washed with 20mM MES buffer, 0.1M NaCl; pH 5 (1mL) and followed by a water wash (1mL). Gels lanes were normalized to equivalent volume. M = MW marker, L = lysate load, FT = flow-through and E = elution.

HisPur Ni-NTA Resin and HisPur Cobalt Resin are alternative forms of IMAC, the former using nickel and the latter using cobalt as the chelated metal ion responsible for His-tag binding. Ni-NTA resins have been the most common IMAC resin choice for 6xHis-tag protein purifications because of the four metal-binding sites on the chelate, which enables high-protein binding and low-metal ion leaching. Cobalt binds less strongly and therefore more discriminantly, enabling greater purity but usually less yield in His-tagged protein purification.

Table panel: Elution fractions were analyzed for protein content using the Coomassie Plus (Bradford) Protein Assay Kit (Product #23236). Purity was determined by analyzing the gel with densitometry software. Sample Resin Yield Purity 6xHis AIF2 (73kDa) HisPur Ni-NTA 0.5mg 32% Ni-IDA 0.5mg 25% HisPur Cobalt 0.4mg 49% 6xHis GFP (28kDa) HisPur Ni-NTA 0.8mg 90% Ni-IDA 0.6mg 52% HisPur Cobalt 0.7mg 91%

HisPur Ni-NTA and HisPur Cobalt Resins maximize yield and purity, respectively. Gel panels: Bacterial lysate containing over-expressed 6xHis-AIF2 (6mg total protein) or 6xHis-GFP (4mg total protein) was applied to HisPur Ni-NTA Resin (0.2mL) and purified by the batch-bind method. The same amount of total protein was applied to Ni-IDA and HisPur Cobalt Resins and purified as described in the instructions. Gels lanes were normalized to equivalent volume. M = molecular-weight marker, L = lysate load and FT = flow-through.

Related Products and Resources:

HisPur Cobalt Resins and Kits – for high-purity His-tagged protein purification
B-PER Bacterial Protein Extraction Reagents – for high-yield extraction from bacteria
Protein Methods Library: His-tagged Proteins

Available Formats:

• Resin Slurries– crosslinked 6% beaded agarose; 10mL, 100mL, 500mL bottles
• Spin Columns – 0.2mL (microcentrifuge), 1mL, 3mL columns
• Purification Kits – complete kits in all three column sizes
• Chromatography Cartridges – compatible with automated liquid chromatography systems and with syringe processing (learn more about our Chromatography Cartridges)
• Bulk and Custom – let us know your needs for bulk HisPur Ni-NTA Resin.

  Product #	Description	Pkg. Size
  88221	HisPur Ni-NTA Resin Support: Crosslinked 6% beaded agarose Supplied: 50% slurry in 20% ethanol	10mL
  88222	HisPur Ni-NTA Resin	100mL
  88223	HisPur Ni-NTA Resin	500mL
  88224	HisPur Ni-NTA Spin Columns, 0.2mL	25 columns
  88225	HisPur Ni-NTA Spin Columns, 1mL	5 columns
  88226	HisPur Ni-NTA Spin Columns, 3mL	5 columns
  88227	HisPur Ni-NTA Spin Purification Kit, 0.2mL Kit contents: HisPur Ni-NTA Spin Columns, 0.2mL, 25 columns Phosphate-buffered Saline (10X), 30mL Imidazole (2M), 8mL Collection Tubes, 80 tubes Column Plugs, 25 plugs	25-column kit
  88228	HisPur Ni-NTA Spin Purification Kit, 1mL Kit contents: HisPur Ni-NTA Spin Columns, 1mL, 5 columns Phosphate-buffered Saline (10X), 30mL Imidazole (2M), 8mL Collection Tubes, 80 tubes Column Plugs, 25 plugs	5-column kit
  88229	HisPur Ni-NTA Spin Purification Kit, 3mL Kit contents: HisPur Ni-NTA Spin Columns, 3mL, 5 columns Phosphate-buffered Saline (10X), 30mL Imidazole (2M), 25mL Collection Tubes, 80 tubes Column Plugs, 25 plugs	5-column kit
  90098	HisPur Ni-NTA Chromatography Cartridges, 1mL Learn more about Pierce Chromatography Cartridges.	5 cartridges
  90099	HisPur Ni-NTA Chromatography Cartridges, 5mL Learn more about Pierce Chromatography Cartridges.	2 cartridges