- Why tag a protein?
- Tech note: cobalt resin
- Purification methods overview
- Simplified purification of active, secreted his-tagged proteins
- Overview: His60
- Tech note: Capturem technology
- Tech note: Capturem large volume
- Magnetic beads
- TALON resin selection guide
- Selection guide: His60 resin
- FAQs: TALON
- Video: Capturem his maxiprep
- Video: Capturem his miniprep
- Visual protocol: Capturem his maxiprep
- Visual protocol: Capturem his miniprep
- Capturem nickel column reagent compatibility
- TALON reagent compatibility
- His60 reagent compatibility
- TALON: Native vs denaturing purification
- Protocol: denaturing purification with TALON resin, imidazole elution
- Protocol: native purification with TALON resin, imidazole elution
- Protocol: native purification with TALON resin, pH elution
His60 reagent compatibility
His60 Ni resin is a high-capacity Ni-IDA resin that permits one-step protein purification of his-tagged proteins under either native or denaturing conditions. The resin is compatible with multiple denaturants and detergents—refer to the User Manual for a complete reagent compatibility table. Purified recombinant his-tagged proteins can be eluted by the addition of 300 mM imidazole or by a reduction in pH.
|Reagents compatible with His60 Ni resins|
|Beta-mercaptoethanola||20 mM (with caution)|
|CHAPSb,c||1% (with caution)|
|DTT (dithiothreitol)d||1 mM (with caution)|
|Guanidine hydrochloridef||6 M|
|HEPESg||Up to 100 mM (with caution)|
|MOPSg||Up to 100 mM (with caution)|
|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)|
- 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.
- Detergents cannot be easily removed by buffer exchange.
- 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. Perform at least 20 column volumes of washes, preferably with low concentrations of imidazole (40 mM) to wash out any reduced metal ions.
- Ethanol may precipitate proteins, causing low yields and column clogging.
- With high concentrations, protein unfolding generally takes place. Protein refolding on-column (or after elution) is protein-dependent.
- Amine groups that are present in these buffers can interact with Ni2+ ions, diminishing the resin’s binding capacity.
- Binds to His60 Ni and competes with histidine residues in the his-tag.
- Has high absorbance at 280 nm.
- 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:|
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