Special IMAC Agaroses
Immobilized metal affinity chromatography, or IMAC, has been employed for many applications making use of its versatile interaction with aromatic nitrogens and phosphates. To this end, NTA matrices have been loaded with a broad range of transition metals, as optimized for the individual interaction. Most commonly used alternative transition metals are copper (Cu), iron (Fe), and zinc (Zn), but also gallium (Ga), aluminium (Al), and zirconium (Zr) are sometimes used.
Please note that Al-IDA, Cu-IDA, Co-IDA, Fe-IDA, and Zn-IDA Agaroses have been discontinued effective May 1, 2016. Please refer to our dedicated protocols for loading IMAC resins or contact
us for alternative options.
Applications that can be addressed with IMAC:
Purification of phosphorylated proteins and peptides (Fe, Ga, Al, Zr)
Purification of zinc-finger or copper-binding proteins (Zn, Cu)
Purification of his-tagged proteins with different specificity (Zn, Cu, Co)
MagBeads also available
Purification of phosphorylated proteins
IMAC methods, in particular Fe-NTA or Ga-NTA, have been widely used to enrich phosphoproteins and phosphopeptides as part of the sample preparation for mass spectrometry (1,3,4). Depending on the sample, and the kind of phosphorylated proteins to be analyzed, also other transition metals, such as zirconium or aluminium have been loaded on NTA or IDA matrices for enrichment (6). Also, magnetic beads have been useful for this kind of sample preparation (2).
Fig. 1: PureCube His Affinity Resins feature flexible ligand loading. Both IDA and NTA resins can be loaded with the metal ion most suitable for a given application (Fe: iron, Ni: nickel, Cu: copper, Co: cobalt; Al: aluminium
Purification of zinc-finger proteins
When purifying his-tagged zinc-finger proteins which require a bound zinc ion for activity, it is often advisable to use Zn-IMAC instead of Ni-IMAC materials. Zinc matrices provide a high specificity for his-tagged protein purification (see Fig. 3) and an exchange of zinc bound to the active site of the protein by nickel is avoided (6). At the same time, non-tagged zinc-finger proteins can be purified using zinc IMAC matrices, simply by their affinity to this metal (5). Similar approaches have been done to enrich copper-binding proteins by Cu-IMAC, in particular with plant extracts (7).
Fig. 3: Affinity and specificity of metal ions commonly used for IMAC. Loading an IMAC resin with different metal ions can adjust the affinity and specificity to optimize the purity and yield of a purified protein.
Different metal ions confer different binding affinity and specificity
Loading different metal ions to a resin results in differing affinity and specificity for a his-tagged protein. Generally, cobalt exhibits the higest binding specificity of commonly used IMAC metal ions, leading to relatively low yields but high purity. Copper, at the other end of the spectrum, has a high affinity leading to high yields but unspecific binding. In searching for the optimal resin to purify a protein, it is recommended to explore different chelating ligands (IDA or NTA) and different metal ions.
1. Albuquerque, C.P. et al. A multidimensional chromatography technology for in-depth phosphoproteome analysis. Mol Cell Proteomics (2008), 7(7), 1389-1396.
2. Herskowitz, J., et al. Phosphoproteomic analysis reveals site-specific changes in GFAP and NDGR2 phosphorylation in frontotemporal lobar degeneration. J. Proteome Res (2010), 9(12):6368-6379.
3. Yu, P. et al. Global analysis of neuronal phosphoproteome regulation by chondroitin sulfate proteoglycans. PLoS One (2013), 8,3, e59285.
4. Aryal, U.K. et al. Optimization of immobilized Gallium (III) ion affinity chromatography for selective binding and recovery of phosphopeptides from protein digests. Journal of Biomolecular Techniques (2008), 19:296-310.
5. Vorácková, I. et al. Purification of proteins containing zinc finger domains using immobilized metal ion affinity chromatography. Protein Expr. Purif. (2011) 79(1):88-95.
6. Block et. al. Immobilized-metal affinity chromatography (IMAC) a review. Methods Enzymol. (2009), 463:439-73.
7. Kung. C.C. et al. Proteomic survey of copper-binding proteins in Arabidopsis roots by immobilized metal affinity chromatography and mass spectrometry. Proteomics (2006), 6(9)2746-58.