Abstract: Ni column is a purification column material frequently used in protein purification and belongs to an immobilized metal ion affinity chromatography(IMAC). Invented by Porath etc in 1975, Ni column has been continuously improved till now.
Keywords: Nickel Column Protein Purification, Ni Column Purification, Nickel Column Purification
1. Nickel Column Protein Purification Protocol
By negative-electronic element(O, N) on electron donor ligand interacting with metal ions like carboxyl or amiano, transition metals form stable metal chelates. In IMAC, a Ni2+ has six coordination sites fixed on adsorbent by the metal chelate containing 3/4/5 electron donor groups(coordination sites). Other unoccupied coordination sites are exposed in the solution and can specifically bind with histidine, cysteine, the side chain of tryptophan or histidine tagged proteins.
Figure 1. Interaction between neighboring residues in the 6xHis tag and Ni-NTA matrix.
2. Nickel Column Affinity Chromatography
Common chelate ligands of IMAC are TED(carboxymethyl ethylenediamine), NTA(nitrilotriacetic acid) and IDA(iminodiacetic acid). They have 3/4/5 coordination sites binding with Ni2+. Redundant sites binding with histidine tag are 3/2/1. Thus, the binding ability of IDA with his tag protein is relatively strong and the specificity is weak. The situation of TED is opposite. In the actual application, we usually select the NTA type with suitable load and specificity.
Figure 2. Comparison between Ni-IDA and Ni-NTA.
3. Application of Ni Column Purification
Usually, competitive elution is used for eluting Ni column. First, wash off impure proteins and loosely bound proteins by imidazole with low concentration. Then, elute the target protein with suitable concentration. The elution order is relevant to the binding ability of proteins.
Figure 3. Comparison between NTA-Ni and Ned-Ni.
In the application of Ni column, reductants and chelators shouldn't exist in the buffer to prevent the exfoliation of Ni2+ from reduction. If a lot of impure proteins are accumulated in the column material due to the long-term use, the regeneration can be completed by chelating Ni2+ with EDTA, washing column material with NaOH and rechelating Ni ion. When not in use, the Ni column should be stored in 20% ethanol to prevent the growth of bacteria.
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REFERENCES
[1] Recombinant proteins attached to a nickel-NTA column: use in affinity purification of antibodies, PMID: 7980919.
[2] 6xHis-Ni-NTA chromatography as a superior technique in recombinant protein expression/purification, PMID: 7921034.