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(New page: ==Chromatography== ===Affinity Chromatography=== ====Immobilized Metal Ion Affinity Chromatography (IMAC)==== =====Methodology===== Immobilized metal ion affinity chromatography (IMAC...) |
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My protein precipitates following elution, what should I do? | 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. | *Use a different buffer system, e.g. Tris, HEPES, other Good buffers<ref>Good, N.E. et al. Hydrogen ion buffers for biological research. Biochemistry 5, 467-77 (1966).[http://dx.doi.org/10.1021/bi00866a011]</ref><ref>Ferguson, W.J. et al. Hydrogen ion buffers for biological research. Analytical biochemistry 104, 300-10 (1980).[http://dx.doi.org/10.1016/0003-2697%2880%2990079-2]</ref>. Optimum binding occurs at pH 7.5-8.0. | ||
*Following elution chelate any leached Ni<sup>2+</sup> ions using EDTA. | *Following elution chelate any leached Ni<sup>2+</sup> ions using EDTA. | ||
*Rapidly remove imidazole - dilution, desalting. | *Rapidly remove imidazole - dilution, desalting. | ||
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*L-arginine to reduce non-specific aggregation | *L-arginine to reduce non-specific aggregation | ||
*0.25-0.5 M trimethylaminoxide. | *0.25-0.5 M trimethylaminoxide. | ||
==Notes== | |||
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Revision as of 13:18, 19 February 2008
Chromatography
Affinity Chromatography
Immobilized Metal Ion Affinity Chromatography (IMAC)
Methodology
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 cobalt, nickel, copper, and zinc. 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.
Resin types
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
Elution methods
- 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).
Troubleshooting
My protein precipitates following elution, what should I do?
- Use a different buffer system, e.g. Tris, HEPES, other Good buffers[1][2]. 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.