- 1 Chromatography
- 1.1 Affinity Chromatography
- 1.2 Ion Exchange Chromatography
- 1.3 Hydrophobic Interaction Chromatography
- 1.4 Gel Exclusion Chromatography (also termed Size Exclusion Chromatography or Gel Filtration)
- 2 Purifying untagged proteins
- 3 Notes
Immobilized Metal Ion Affinity Chromatography (IMAC)
Immobilized metal ion affinity chromatography (IMAC) is based on the specific coordinate covalent binding of amino acids to metal ions. This technique works by allowing proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions, such as Co2+ , Ni2+ , Cu2+ , and Zn2+ . Most naturally occurring proteins do not have an affinity for metal ions and recombinant DNA techniques are used to introduce this property into a protein of interest. Typically an N- or C-terminal oligohistidine tag of 6-12 histidine residues in length is introduced into the protein sequence. In its most common form, IMAC involves binding of a His6-tagged (or simply "His-tagged") protein to a resin charged with Ni2+ ions. Specificity and affinity of the His-tagged protein binding can be increased charging the resin with Co2+, Cu2+ , or Zn2+.
IDA, NTA and other proprietary chelators
Specific resins/manufacturers: Ni-NTA (Qiagen), Talon (Clontech), Ni sepharose FF (GE Healthcare/Amersham), Ni MCC (Bioline), His-select (Sigma-Aldrich)
Different metal ions
- Competitive elution with imidazole or (less commonly histidine), increasing competitor concentration in either a step-wise or gradient manner.
- Elution at low pH (typically eluting directly into a solution of higher pH buffer to restore pH to an optimum value).
My protein precipitates following elution, what should I do?
- Use a different buffer system, e.g. Tris, HEPES, other Good buffers. Optimum binding occurs at pH 7.5-8.0.
- Following elution chelate any leached Ni2+ ions using EDTA.
- Rapidly remove imidazole - dilution, desalting.
- Put a desalting column in line with the IMAC column to buffer exchange into a more stabilising buffer.
- Switch to a resin requiring lower imidazole concentrations for elution, e.g. Talon and His-select.
- Maintain a reducing environment - TCEP is compatible with all IMAC resins, and low concentrations of 2-ME and DTT can be used with some resins if care is taken. DTT can also be added post-elution, but take care to first add EDTA to sequester any leached metal ions.
- Maintain a high ionic strength, e.g. use a NaCl concentration of 500 mM.
- Elute with histidine.
- Use a different metal ion.
- Add 5-10% glycerol to all buffers.
More desperate measures involve other additives such as:
- A small amount of detergent
- 0.5 M LiCl (a chaotropic agent to reduce hydrophobic interactions)
- NDSB compounds (non-detergent sulfobetaines; see the Anatrace or Sigma catalogs)
- L-arginine to reduce non-specific aggregation
- 0.25-0.5 M trimethylaminoxide.
Ion Exchange Chromatography
Ion exchange (IEX) chromatography separates proteins by charge. IEX is a good first step for the purification of proteins from crude lysates. Protein solutions should be in a low ionic strength buffer to allow protein to stick to the column. Elution is normally accomplished by increasing the concentration of NaCl. For anion exchange, e.g. Q-sepharose, 20 mM Tris-Cl, pH 8.0, 10 uM EDTA is suitable for most proteins. For cation exchange, e.g. SP-sepharose, 20 mM sodium phosphate, pH 6.5, 10 uM EDTA is a reasonable choice. For optimization of loading and elution conditions, bind protein to column, wash out unbound proteins, and run a gradient of 0-1 M NaCl. The final purification protocol should utilize the maximum NaCl concentration that allows complete binding of the target protein during the loading phase, and the minimum NaCl concentration that allows for complete elution of the target protein during the elution phase. The column should be stripped of protein after use by passing buffer with 1 M NaCl through it. A 2.6 x 10 cm column of high-capacity IEX medium (e.g., Q-sepharose) is sufficient to process crude lysate from 1-4 L of bacterial culture.
Hydrophobic Interaction Chromatography
Hydrophobic interaction (HIC) chromatography is a powerful and often overlooked method of protein purification. This method separates proteins by surface hydrophobicity. HIC is an especially good followup step to IEX, as it requires high salt concentrations for protein binding. Protein solutions should typically be brought to 1.0 M ammonium sulfate in an appropriate buffer. (This can be easily accomplished by slowly adding solid salt to an IEX eluate or crude lysate.) Most proteins will readily stick to an HIC column under these conditions. Choices of chromatographic medium include, in order of increasing hydrophobicity, butylsepharose, octylsepharose, and phenylsepharose. Butylsepharose is a good first choice, as the more hydrophobic media can be too "sticky" for many protein. Elution of protein is accomplished by lowering the ammonium sulfate concentration. For optimization of elution conditions, run a 1-0 M gradient of ammonium sulfate. The final purification protocol should utilize the minimum ammonium sulfate concentration that allows complete binding of the target protein during the loading phase, and the maximum ammonium sulfate concentration that allows for complete elution of the protein during the elution phase. The column should be stripped of protein after use by passing a low ionic strength buffer with no salt through it. A 1.6 x 10 cm HIC column is sufficient to process crude lysate from and IEX pool derived from 1-4 L of bacterial culture.
Gel Exclusion Chromatography (also termed Size Exclusion Chromatography or Gel Filtration)
Gel exclusion chromatography (GEC) separates proteins by size (volume). GEC is an especially good followup step to IEX or HIC, as it can desalt protein preparations, and is typically used as a "polishing" step near the end of a purification. For maximum resolution, a large column should be used (2.6 x 60 cm is typical) and protein should be concentrated to <4% of the total column volume, and flow rates should be as slow as practical. A typical loading for a 2.6 x 60 cm Superdex 200 column is no more than 2.0 mL, and a typical flow rate is 1 mL/min. It is advisable to include 100 mM NaCl or another salt to suppress non-specific binding of protein to the chromatographic medium
Purifying untagged proteins
A typical strategy for purifying untagged proteins is IEX-HIC-GEC, in that order. These three steps are sufficient to produce homogeneous protein from nearly any overexpression system that can produce recombinant protein at >2% of total cellular protein. Variant proteins can typically be purified using a protocol identical to the wild-type, but occasionally single amino acid mutations can produce wildly different chromatographic behavior.