Nickel affinity chromatography is a type of immobilized metal ion affinity chromatography (IMAC) used for the purification of proteins that have been genetically engineered to include a polyhistidine tag (His-tag). His-tags are short sequences of histidine residues (usually 6–10) that bind strongly to nickel ions. This method is widely used in protein expression and purification systems because it is simple, efficient, and provides high purity of recombinant proteins.
Principle of Nickel Affinity Chromatography
Nickel affinity chromatography exploits the affinity between histidine residues in a His-tag and Ni²⁺ ions immobilized on a resin. The nickel is coordinated with nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) groups, which are covalently attached to the resin. The His-tagged protein binds to the nickel ions through the nitrogen atoms in the imidazole ring of the histidine residues, allowing selective retention of the target protein on the column, while other proteins flow through.
Steps of Nickel Affinity Chromatography
Preparation of the Column and Buffer Solutions
Resin Preparation: Nickel affinity chromatography uses resins such as Ni-NTA (nickel-nitrilotriacetic acid) or Ni-IDA (nickel-iminodiacetic acid) agarose.
Ni-NTA provides four coordination sites for nickel ions, making it more stable compared to Ni-IDA, which has three coordination sites.
Equilibration Buffer: This buffer is typically composed of:
50 mM Tris or phosphate buffer (pH 7.4–8.0): Maintains a stable pH.
150–500 mM NaCl: Helps maintain ionic strength and stability.
10–20 mM imidazole: Added to the buffer to reduce non-specific binding of host cell proteins, but still allows His-tagged proteins to bind to the resin.
Elution Buffer: The same buffer as the equilibration buffer, but with a higher concentration of imidazole (usually 200–500 mM) to elute His-tagged proteins by competing with the histidines for binding to nickel.
Loading the Protein Sample
The cell lysate or crude protein extract containing the His-tagged protein is first clarified by centrifugation or filtration to remove insoluble debris.
The clarified lysate is then applied to the equilibrated Ni-NTA or Ni-IDA column.
His-tagged proteins bind to the nickel ions on the resin, while other proteins lacking a His-tag are washed away.
Washing the Column
Wash the column with 5–10 column volumes of wash buffer containing a low concentration of imidazole (10–30 mM) to remove weakly bound proteins and non-specific contaminants.
The imidazole concentration must be optimized depending on the protein of interest. Too little imidazole may lead to non-specific binding, while too much may cause premature elution of the target protein.
Elution of His-Tagged Protein
After washing, the His-tagged protein is eluted by increasing the concentration of imidazole in the elution buffer (200–500 mM). Imidazole competes with the histidine residues in the His-tag for binding to the nickel ions, releasing the His-tagged protein from the resin.
The eluted protein fractions are collected, and the concentration of imidazole in the elution buffer can be varied to fine-tune the elution of the target protein.
Buffer Exchange or Desalting
If required, the eluted protein can be further processed by dialysis or desalting columns to remove imidazole, which can interfere with downstream applications such as enzyme assays or protein crystallization.
The final protein is then stored in an appropriate buffer, such as phosphate-buffered saline (PBS) or Tris-HCl, depending on the application.
Detailed Protocol for Nickel Affinity Chromatography
Materials
Ni-NTA or Ni-IDA resin
Equilibration buffer: 50 mM Tris-HCl or 50 mM phosphate buffer, 150 mM NaCl, 10–20 mM imidazole, pH 7.4–8.0
Wash buffer: Same as equilibration buffer, with 20–50 mM imidazole
Elution buffer: Same as equilibration buffer, with 200–500 mM imidazole
Cell lysate containing His-tagged protein
Column: Either gravity-flow columns or FPLC-compatible columns
pH meter, Centrifuge, Filter units (0.45 µm)
Imidazole, NaCl, Tris or phosphate buffer
1. Preparation of the Resin
Equilibrate the resin: If using a pre-packed column or gravity-flow column, equilibrate the resin with 5–10 column volumes of equilibration buffer.
If using loose resin for batch purification, slurry the resin with equilibration buffer and transfer to the column, then equilibrate as above.
2. Sample Preparation
Prepare the cell lysate: Lyse the cells (bacterial, yeast, or mammalian cells) by sonication, freeze-thaw cycles, or detergent-based lysis (e.g., using Triton X-100 or NP-40).
Clarify the lysate: Centrifuge the lysate at high speed (e.g., 15,000 x g for 15–20 minutes) to remove debris. Filter the supernatant through a 0.45 µm filter to avoid clogging the column.
Check the pH: Ensure that the pH of the lysate is close to neutral (pH 7.4–8.0) for optimal binding.
3. Loading the Sample
Apply the clarified lysate to the equilibrated Ni-NTA or Ni-IDA column.
Flow rate: For gravity-flow columns, use a slow flow rate (~1 drop/second) to allow maximum interaction between His-tagged proteins and the resin. For FPLC, use a recommended flow rate depending on column capacity.
Collect the flow-through (non-binding fraction) and retain for analysis to check if any target protein is lost.
4. Washing the Column
Wash the column with 5–10 column volumes of wash buffer containing 20–50 mM imidazole. This removes non-specifically bound proteins while retaining His-tagged proteins on the column.
Collect the wash fractions for analysis (e.g., by SDS-PAGE) to monitor the removal of contaminants.
5. Elution
Elute the bound His-tagged protein using elution buffer containing a higher concentration of imidazole (200–500 mM).
Collect fractions during elution and monitor the protein elution using UV absorbance (e.g., at 280 nm) or by performing SDS-PAGE to identify the fractions containing the target protein.
If the protein does not elute efficiently, increase the imidazole concentration or extend the elution time.
6. Analysis and Storage
Analyze the purity of the eluted fractions by SDS-PAGE or Western blotting.
If needed, pool the fractions containing the target protein and concentrate or dialyze the protein to remove imidazole or exchange the buffer for downstream applications.
Troubleshooting Tips for Nickel Affinity Chromatography
Low Yield
Cause: Improper binding of the His-tagged protein to the column.
Solution: Check that the pH of the buffer is in the optimal range (pH 7.4–8.0). Ensure that the His-tag is not blocked (e.g., ensure it’s not cleaved off or inaccessible).
Poor Purity
Cause: Non-specific proteins binding to the column.
Solution: Increase the imidazole concentration in the wash buffer (e.g., from 10 mM to 30 mM) to reduce non-specific binding without affecting His-tagged protein binding.
Protein Doesn’t Elute
Cause: Strong binding of the His-tagged protein to the nickel resin.
Solution: Increase the imidazole concentration in the elution buffer (up to 500 mM). Alternatively, you can use EDTA (ethylenediaminetetraacetic acid) to strip the nickel from the resin, but this may co-elute unwanted proteins or damage the resin for reuse.
Protein Precipitation
Cause: Imidazole concentration or pH change during elution.
Solution: Include a stabilizer in the buffer (e.g., glycerol or reducing agents like DTT) to prevent precipitation. Avoid rapid pH changes or high imidazole concentrations that might destabilize the protein.
Advantages of Nickel Affinity Chromatography
- High Specificity: The interaction between His-tags and Ni²⁺ ions is highly specific, leading to selective binding of His-tagged proteins.
- Simplicity: The procedure is straightforward and relatively quick, allowing for rapid purification.
- Scalability: Nickel affinity chromatography can be scaled from small laboratory volumes to industrial-scale protein purification.
- Reusability: Ni-NTA resin can be regenerated and reused multiple times, making it cost-effective.
Disadvantages
- Non-Specific Binding: Low concentrations of imidazole in the buffer may lead to non-specific binding of other proteins.
- Imidazole Contamination: High concentrations of imidazole in the elution buffer can interfere with downstream applications, requiring additional steps for removal.
- Tag Dependence: The method requires the presence of a His-tag, which may not always be practical for native protein purification.
Applications
Recombinant Protein Purification: Used extensively for purifying His-tagged proteins expressed in bacterial, yeast, or mammalian systems.
Enzyme Assays: Purified proteins can be used for biochemical assays and enzymatic activity studies.
Structural Biology: High-purity His-tagged proteins are often required for X-ray crystallography or NMR studies.
Immunoprecipitation: Used to isolate His-tagged proteins from complex mixtures for further analysis.
Nickel affinity chromatography is a widely-used technique for the purification of recombinant His-tagged proteins due to its high efficiency, specificity, and versatility.