Immobilized metal affinity chromatography (IMAC) is most frequently used for the purification of polyhistidine (His-) tagged proteins. This technique is based on the interaction between certain exposed protein residues (preferentially histidines) with transition metal cations (Cu2+, Ni2+, Zn2+, Co2+). The transition metal itself is immobilized to a cross-linked agarose matrix via a chelating group such as iminodiacetic acid (IDA)[1] or nitrilotriacetic acid (NTA).

Successful purification experiments of His-tagged proteins strongly depend on the particular amino acid sequence, the protein conformation and the microenvironment and location of the His-tag (C- or N-terminal). Furthermore, undesired co-purification of non-specific host cell proteins is often observed, especially in the case of E. coli expression systems.

Alternative to common solution strategies e.g. the increase of salt or detergent concentration or the variation of the His-tag location optimization of the purification strategy can easily be achieved by

  • variation of metal ion density since a lower amount of metal ions may minimize non-specific protein binding[2-4].
  • changing the metal ion itself[2,3].
 
Affinity Chromatography - His-tagged Proteins Affinity vs. Specificity towards metal ions

Based on the HSAB concept, IDA-immobilized Cu2+, Ni2+, Zn2+ and Co2+ ions exhibit varying affinities & specificities toward histidines[2,3]. While Ni2+ ions show a high affinity but low specificity, Co2+-ions are more specific but with reduced binding affinity and are therefore, an option to reduce non-specific protein binding.
 

Nickel and Cobalt NTA Magnetic Agarose Beads

Ferrimagnetic agarose beads suitable for fast & efficient small-scale purification of polyhistidine (His) tagged proteins.
 

Nickel and Cobalt IDA Magnetic Agarose Beads

Ferrimagnetic agarose beads suitable for fast & efficient small-scale purification of polyhistidine (His) tagged proteins.
 

Nickel NTA Agaroses, Fast Flow

6 % cross-linked agarose suitable for FPLC, gravity flow and batch purification by a one-step procedure from crude lysates both under denaturing and non-denaturing conditions
 

Nickel and Cobalt IDA Agaroses, Fast Flow

High-performance 6 % cross-linked agarose suitable for FPLC, gravity flow and batch purification of polyhistidine tagged proteins.
 

Nickel, Zinc, Cobalt, Copper and Metal Free IDA Agaroses, Gravity Flow

6 % cross-linked agarose material suitable for fine-tuning of purification strategies through flexible choice of the metal ion and the metal loading density.
 


Selected References:

[1] Porath et al. (1975) Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258:598.

[2] Gaberc-Porekar et al. (2001) Perspectives of immobilized-metal affinity chromatography. J. Biochem. Biophys. Methods 49:335.

[3] Ueda et al. (2003) Current and prospective applications of metal ion–protein binding. Journal of Chromatography 988:1.

[4] Liesiene et al. (1997) Immobilized metal affinity chromatography of human growth hormone-effect of ligand density. Journal of Chromatography 764:27.