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 and IDA matrices have been loaded with a broad range of transition metals, as optimized for the individual interaction. Magnetic beads are ideal for protein purification from dilute supernatants and for pull-down experiments. Zn-NTA MagBeads are the optimal matrix for the purification of zinc-finger proteins, either via his tag, or via direct interaction of the zinc-binding motif with the NTA ligand. All PureCube IMAC MagBeads are delivered as a 25% suspension.
PureCube Zn-NTA MagBeads provide:
- Ready-to-use magnetic beads
- Purification of zinc-finger proteins, with or without his tag
- Purification of his-tagged proteins with different specificity
- Zn-NTA Agaroses also available
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. 1) and an exchange of zinc bound to the active site of the protein by nickel is avoided (2). At the same time, non-tagged zinc-finger proteins can be purified using zinc IMAC matrices, simply by their affinity to this metal (1).
Fig. 1: 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. 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.
2. Block et. al. Immobilized-metal affinity chromatography (IMAC) a review. Methods Enzymol. (2009), 463:439-73.