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Protein Expression & Purification

Products >  Protein_Expression_and_Purification >  His-Tagged_Protein_Purification >  Nickel_Columns-Gravity_Flow

His-Tagged Recombinant Protein Purification—His60 Ni Gravity Flow Columns

His60 Ni Superflow Resin is a high-capacity Ni-IDA resin for the one-step purification of recombinant his-tagged proteins from bacterial, mammalian, and baculovirus-infected cells. His60 Ni Resin is compatible with batch/gravity flow applications, as well as with the major automated liquid chromatography systems and manual syringe processing. The resin enables fast, easy, and reproducible chromatographic separations and can be regenerated for multiple uses.

His60 Ni Gravity Columns are prepacked columns containing 1 ml resin. The His60 Ni Gravity Column Purification Kit supplies all buffers needed for protein extraction and purification, and prepacked His60 Ni Gravity Columns. These buffers are also available separately in the His60 Ni Buffer Set.

The Recombinant His-Tagged Protein Purification Principle

His60 Ni Superflow purification resin has low metal ion leakage and the highest binding capacity to date (outperforming Ni-NTA and other resins). The modified Ni-IDA chemistry makes this resin one of the best on the market in terms of capacity, metal ion leakage, and purity, when compared to other Ni-based resins such as Ni-NTA or other Ni-IDA resins.

The Recombinant His-Tagged Protein Purification Procedure

Purifying recombinant his-tagged proteins requires cell lysis/extraction, binding, washing, and elution. His60 Ni makes protein purification under either native or denaturing conditions a one-step process. The resin is compatible with multiple denaturants and detergents. Purified recombinant his-tagged proteins can be eluted by the addition of 300 mM imidazole or by a reduction in pH.

  At-A-Glance   Documents   Images & Data   Resources


  • Obtain up to 60 mg/ml of recombinant his-tagged protein
  • Low metal ion leakage in eluted his-tagged protein
  • Purify under native or denaturing conditions
  • Use for batch/gravity flow and automated FPLC purification


Purified recombinant his-tagged proteins can be used for:

  • Protein labeling
  • Immunization to produce antibodies
  • Animal studies
  • Other applications

Reagents Compatible with His60 Ni Superflow Resin

Reagent Acceptable Concentration
Beta-mercaptoethanola 20 mM (with caution)
CaCl2 5 mM
CHAPSb,c 1% (with caution)
DTT (dithiothreitol)d 1 mM (with caution)
Ethanole 20%
Glycerol 20%
Guanidine hydrochloridef 6 mM
HEPESg Up to 100 mM (with caution)
Histidineh 20 mM to inhibit nonspecific binding.
Up to 100 mM to elute his-tagged proteins.
MgCl2 4 M
MOPSg Up to 100 mM (with caution)
NP-40b,i 2%
SDSb,c 1% (with caution)
Sodium acetate Up to 100 mM (with caution)
Sodium phosphate Up to 50 mM
Trisj Up to 50 mM (with caution)
Triton-X 100b,i 1%
Tween 20 2%
Ureaf 8 M

a Use the resin immediately after equilibrating with buffers containing beta-mercaptoethanol. Otherwise, a slight change in color (yellowing of the resin) will occur. Do not store the resin in buffers containing beta-mercaptoethanol.
b Detergents cannot be easily removed by buffer exchange.
c Ionic detergents like CHAPS (3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane-sulfonate), SDS (sodium dodecyl sulfate), and sarkosyl are compatible up to 1%. However, due to their charged nature, you should anticipate interference with binding, even at low concentrations.
Since DTT is a reducing agent, low concentrations will reduce the metal ions in His60 Ni Superflow resin. Although enough of these ions may remain unaffected to allow protein purification, please use it with caution. Do at least 20 column volumes of washes, preferably with low concentrations of imidazole (40 mM) to wash out any reduced metal ions.
e Ethanol may precipitate proteins, causing low yields and column clogging.
f With high concentrations, protein unfolding generally takes place. Protein refolding on-column (or after elution) is protein-dependent.
g Amine groups that are present in these buffers can interact with Ni2+ ions, diminishing the resin’s binding capacity.
h Binds to His60 Ni and competes with histidine residues in the his tag.
i Has high absorbance at 280 nm.
j Tris coordinates weakly with metal ions, causing a decrease in capacity.

Reagents Incompatible with His60 Ni Superflow Resin

These reagents are incompatible at any concentration:

  • Arginine, glycine, and glutamine

  • EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol tetraacetic acid), and PEI
    NOTE: Using these chelating agents will strip metal ions from the resin, resulting in protein elution and a resin color change.

  • DTE (dithioerythritol)

Cat. # Product Contents Size Price Units Select
635658 His60 Ni Gravity Column Purification Kit 5 x 1 mL Columns $288.00
635657 His60 Ni Gravity Columns 5 x 1 mL Columns $78.00


Recommended Products
Cat. # Product Contents Size Price Units Select
631210 6xHis mAb-HRP Conjugate 100 uL $350.00
631212 6xHis Monoclonal Antibody (Albumin Free) 200 ug $509.00
635665 His60 Ni Buffer Set 20 Purifications $253.00
635673 ProteoGuard™ EDTA-Free Protease Inhibitor Cocktail 10 x 100 uL $140.00

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