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 |