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. Magnetic beads are ideal for protein purification from dilute supernatants and for pull-down experiments. Cu-NTA MagBeads can be used for the purification of his-tagged proteins. They provide high yields of protein, at lower specificity than e.g. Ni- or Co-IMAC. Cu-NTA matrices have also been used for the enrichment of copper-binding proteins, in particular with plant extracts (1). PureCube Cu-NTA MagBeads are delivered as a 25% suspension.
PureCube Cu-NTA MagBeads provide:
- Ready-to-use magnetic beads
- Enrichment of copper-binding proteins
- Purification of his-tagged proteins with different specificity
- Cu-NTA Agaroses also available
Purification of phosphorylated proteins
IMAC methods, in particular Al-IDA, 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 (5). Also, magnetic beads have been useful for this kind of sample preparation (2).
||Specific binding of his tagged and copper binding proteins
||Affinity to his tagged and copper binding proteins
||Depending on the application
||Delivered as a 25 % suspension
|Metal Ion capacity
||>12 µeqv Cu2+/mL.
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. 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.