An affinity purification protocol is a method to isolate and purify a specific protein from a complex mixture using affinity chromatography. The following is a typical protocol for affinity purification, often used for His-tagged recombinant proteins via immobilized metal ion affinity chromatography (IMAC). You can adapt this protocol to different tags or ligands as needed.
Affinity Purification Protocol (His-Tag Example)
Materials Needed
- Affinity Chromatography Column (e.g., Ni-NTA or Co-NTA resin for His-tag purification)
- Binding Buffer (e.g., 50 mM sodium phosphate, 300 mM NaCl, pH 7.4)
- Wash Buffer (similar to the binding buffer, with a low concentration of imidazole to remove weakly bound proteins, e.g., 10-20 mM imidazole)
- Elution Buffer (e.g., binding buffer with 250-500 mM imidazole to elute the His-tagged protein)
- Regeneration Buffer (optional, if you plan to reuse the column)
- Sample (Lysate) containing the His-tagged protein
- Centrifuge for clearing the lysate
- pH meter or pH strips to adjust buffer pH
- Syringe/Peristaltic Pump or Chromatography System for applying the lysate to the column
Procedure
- Preparation of Lysate
Cell Lysis: Harvest the cells expressing the recombinant His-tagged protein. Lyse the cells using one of the following methods:
Sonication: Disrupt the cells with sonication in a lysis buffer (e.g., binding buffer + protease inhibitors).
Chemical Lysis: Use a lysis buffer containing detergents or lysozyme.
Centrifugation: Centrifuge the lysate at 10,000–15,000 × g for 15–30 minutes at 4°C to remove cellular debris. Collect the supernatant containing the target protein.
- Equilibrate the Column
Wash the affinity column with 5–10 column volumes (CVs) of binding buffer to equilibrate the resin and remove impurities. Ensure the pH and conditions of the buffer match the protein’s pH to ensure optimal binding.
- Apply the Lysate
Load the Lysate: Slowly apply the cleared lysate to the column. You can either use gravity flow or a pump system. Allow enough time for the His-tagged protein to bind to the Ni-NTA or Co-NTA resin through its affinity for the metal ions.
- Wash the Column
After loading, wash the column with 10–20 CVs of wash buffer (e.g., binding buffer with 10–20 mM imidazole) to remove non-specifically bound proteins and impurities. Monitor the flow-through using UV absorbance or collect fractions for analysis via SDS-PAGE.
- Elute the Protein
Elute the bound His-tagged protein using the elution buffer, typically containing 250–500 mM imidazole. You may apply 5–10 CVs of elution buffer and collect the eluate in fractions.
Check for Protein: Measure the protein concentration of the fractions using a UV spectrophotometer at 280 nm, or analyze fractions by SDS-PAGE.
- Regenerate the Column (Optional)
If reusing the column, wash it with regeneration buffer (e.g., 100 mM EDTA or low pH buffer) to remove bound metal ions or proteins. Then recharge the column with the appropriate metal ion (e.g., 50 mM NiSO₄) and equilibrate with binding buffer for the next use.
- Protein Analysis and Storage
Pool the pure target protein fractions based on SDS-PAGE analysis or spectrophotometric measurement.
Dialysis: If necessary, dialyze the protein to remove imidazole or adjust the buffer conditions for downstream applications.
Storage: Store the purified protein at 4°C for short-term use or at –80°C for long-term storage, ideally in a buffer containing stabilizers like glycerol or sucrose.
Buffers for His-Tag Affinity Purification (IMAC Example)
- Lysis/Binding Buffer:
50 mM Sodium phosphate (pH 7.4)
300 mM NaCl
10 mM imidazole (to reduce non-specific binding)
- Wash Buffer:
50 mM Sodium phosphate (pH 7.4)
300 mM NaCl
20–30 mM imidazole (for washing non-specifically bound proteins)
- Elution Buffer:
50 mM Sodium phosphate (pH 7.4)
300 mM NaCl
250–500 mM imidazole (for eluting the His-tagged protein)
- Regeneration Buffer (optional):
100 mM EDTA (pH 8.0) or low pH buffer for stripping the resin of metal ions
50 mM metal ion (e.g., NiSO₄) to recharge the resin
Notes and Tips
Optimize Imidazole Concentration: Use a low concentration of imidazole (e.g., 10–20 mM) in the binding and wash buffers to prevent non-specific binding but allow the His-tagged protein to bind. Higher imidazole concentrations are used for elution.
Monitor Elution: Collect small fractions (0.5–1 mL) during elution, and monitor for protein using absorbance at 280 nm or a Bradford assay.
Maintain Cold Conditions: Perform the purification steps at 4°C to prevent protein degradation, especially if the target protein is temperature-sensitive.
This protocol can be adapted for other affinity systems, such as GST, MBP, or antibody-antigen affinity purification, by changing the ligand and optimizing the binding and elution conditions.