FAQ-49
What are the compatibilities of different reagents with Ni-NTA matrices?
Compatibility of reagents with Ni-NTA matrices
| Reagent | Effect | Comments |
| Buffer reagents | ||
| Tris, HEPES, MOPS | Buffers with secondary or tertiary amines will reduce nickel ions |
Up to 100 mM has been used successfully in some cases Sodium phosphate or phosphate-citrate buffer is recommended |
| Chelating reagents | ||
| EDTA, EGTA | Strip nickel ions from resin | Up to 1 mM has been used successfully in some cases, but care must be taken |
| Sulfhydril reagents | ||
| beta-mercaptoethanol | Prevents disulfide cross-linkages | Up to 20 mM |
| DTT, DTE | Low concentrations will reduce nickel ions | A maximum of 1 mM may be reduce nickel ions used, but beta-mercaptoethanol is recommended |
| Detergents | ||
| Nonionic detergents (Triton, Tween, NP-40, etc.) | Removes background proteins and nucleic acids | Up to 2% can be used |
| Cationic detergents | Up to 1% can be used | |
| CHAPS | Up to 1% can be used | |
| Anionic detergents (SDS, sarkosyl) | Not recommended, but up to 0.3% has been used success-fully in some cases | |
| Denaturants | Solubilize proteins | |
| GuHCl | Up to 6 M | |
| Urea | Up to 8 M | |
| Amino acids | ||
| Glycine | Not recommended | |
| Glutamine | Not recommended | |
| Arginine | Not recommended | |
| Histidine | Binds to Ni-NTA and competes with histidine residues in the 6xHis tag | Can be used at low concentrations (20 mM) to inhibit non-specific binding and, at higher concentrations (>100 mM), to elute the 6xHis-tagged protein from the Ni-NTA matrix |
| Other additives | ||
| NaCl | Prevents ionic interactions | Up to 2 M can be used, at least 300 mM should be used |
| MgCl2 | Up to 4 M | |
| CaCl2 | Up to 5 mM | |
| Glycerol | Prevents hydrophobic interaction between proteins | Up to 50% |
| Ethanol | Prevents hydrophobic interactions between proteins | Up to 20% |
| Imidazole | Binds to Ni-NTA and competes with histidine residues in the 6xHis tag | Can be used at low concentrations (20 mM) to inhibit non-specific binding and, at higher concentrations (>100 mM), to elute the 6xHis-tagged |
| Sodium bicarbonate | Not recommended | |
|
Hemoglobin
Ammonium
Citrate |
Not recommended
Not recommended
Up to 60mM has been used successfully |
Related products
Ni-NTA Fast Start Kit
The Ni-NTA Fast Start Kit provides everything needed for fast, efficient purification of His-tagged proteins from cleared E.coli lysates, including prefilled Ni-NTA columns. Buffers supplied in the kit enable proteins to be purified either under native or denaturing conditions. The kit also contains an Anti-His antibody for detection of expressed His-tagged proteins.
Ni-NTA Superflow
Ni-NTA Superflow is an affinity chromatography matrix for purifying recombinant proteins carrying a His tag.
Histidine residues in the His tag bind to the vacant positions in the coordination sphere of the immobilized nickel ions with high specificity and affinity. Cleared cell lysates are loaded onto the matrices. His-tagged proteins are bound, and other proteins pass through the matrix. After washing, His-tagged proteins are eluted in buffer under native or denaturing conditions.
Ni-NTA Spin System
Ni-NTA silica combines Ni-NTA with a macroporous silica support material optimized to suppress nonspecific hydrophobic interactions. Ni-NTA Spin Columns (His-protein purification spin columns) in the Ni-NTA Spin Kit and available separately provide Ni-NTA silica in a convenient microspin format for easy preparation of multiple samples in parallel. They provide a simple method for functional screening of engineered proteins, selection of clones expressing full-length translation products and comparison of expression levels. Each spin column can purify up to 300 µg of His-tagged protein. Like all Ni-NTA matrices, Ni-NTA spin columns can be used for one-step protein purification under native or denaturing conditions. The Ni-NTA Spin Kit is a complete kit for spin purification of His-tagged proteins. It can be automated on the QIAcube Connect (see image "QIAcube Connect").
Ni-NTA Agarose
Ni-NTA Agarose is an affinity chromatography matrix for purifying recombinant proteins carrying a His tag. Histidine residues in the His tag bind to the vacant positions in the coordination sphere of the immobilized nickel ions with high specificity and affinity. Cleared cell lysates are loaded onto the matrices. His-tagged proteins are bound, and other proteins pass through the matrix. After washing, His-tagged proteins are eluted in buffer under native or denaturing conditions