Protein binding: Proteins containing one or more 6xHis affinity tags, located at either the amino and/or carboxyl terminus of the protein, can bind to the Ni-NTA groups on the matrix with an affinity far greater than that of antibody−antigen or enzyme−substrate interactions. Binding of the 6xHis tag does not depend on the three-dimensional structure of the protein. Even when the tag is not completely accessible it will bind as long as more than two histidine residues are available to interact with the nickel ion; in general, the smaller the number of accessible histidine residues, the weaker the binding will be. Untagged proteins that have histidine residues in close proximity on their surface may also bind to Ni-NTA, but in most cases this interaction will be much weaker than the binding of the 6xHis tag. Any host proteins that bind nonspecifically to the NTA resin itself can be easily washed away under relatively stringent conditions that do not affect the binding of 6xHis-tagged proteins. Binding can be carried out in a batch or column mode ( Supplementary section ). If the concentration of 6xHis-tagged proteins is low, or if they are expressed at low levels, or secreted into the media, the proteins should be bound to Ni-NTA in a batch procedure, and under conditions in which background proteins do not compete for the binding sites, i.e. at a slightly reduced pH or in the presence of low imidazole concentrations (10−20 mM). At low expression levels under native conditions, binding can be optimized for every protein by adjusting the imidazole concentration and/or pH of the lysis buffer. If high levels of background proteins are still present, equilibrating the Ni-NTA matrix with lysis buffer containing 10−20 mM imidazole prior to binding is recommended. The matrix is thus "shielded", and non-specific binding of proteins that weakly interact is significantly reduced.
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Supplementary section : Proteins may be purified on Ni-NTA resins in either a batch or a column procedure. The batch procedure entails binding the protein to the Ni-NTA resin in solution and then packing the protein−resin complex into a column for the washing and elution steps. This strategy promotes efficient binding of the 6xHis-tagged protein especially when the 6xHis tag is not fully accessible or when the protein in the lysate is present at a very low concentration. In the column procedure, the Ni-NTA resin is first packed into the column and equilibrated with the lysis buffer. The cell lysate is then slowly applied to the column. Washing and elution steps are identical in the batch and column procedure. |
Wash: Endogenous proteins with histidine residues that interact with the Ni-NTA groups can be washed out of the matrix with stringent conditions achieved by adding imidazole at a 10−50 mM concentration.
Protein elution : The histidine residues in the 6xHis tag have a pKa of approximately 6.0 and will become protonated if the pH is reduced (pH 4.5−5.3). Under these conditions the 6xHis-tagged protein can no longer bind to the nickel ions and will dissociate from the Ni-NTA resin. Similarly, if the imidazole concentration is increased to 100−250 mM, the 6xHis-tagged proteins will also dissociate because they can no longer compete for binding sites on the Ni-NTA resin. Elution conditions are highly reproducible, but must be determined for each 6xHis-tagged protein being purified. Reagents such as EDTA or EGTA chelate the nickel ions and remove them from the NTA groups. This causes the 6xHis-tagged protein to elute as a protein−metal complex. NTA resins that have lost their nickel ions become white in color and must be recharged if they are to be reused. Whereas all elution methods (imidazole, pH, and EDTA) are equally effective, imidazole is mildest and is recommended under native conditions, when the protein would be damaged by a reduction in pH, or when the presence of metal ions in the eluate may have an adverse effect on the purified protein (Supplementary section).
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